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PubMed1984

A randomized trial comparing smoking cessation programs utilizing behaviour modification, health education or hypnosis.

The purpose of this study was to determine the efficacy of hypnosis, health education, and behaviour modification programs for cigarette smoking cessation. A randomized clinical trial comparing these three programs and a control group was conducted in 168 volunteers. Follow-up data three weeks after completion was available in 140 subjects. Each program showed significant reductions in reported cigarette consumption and serum thiocyanate levels, an indicator of long-term cigarette consumption, compared to entry and to the control group. However, there were no significant differences between the hypnosis, health education, or behaviour modification groups with respect to the proportion who reported quitting smoking, the number cigarettes smoked or change in serum thiocyanate levels. Reported cigarette consumption ascertained six months later again showed no significant differences between these three approaches. Factors such as subject age, age at starting cigarette smoking, educational level, marital status, spouse or partners smoking did not identify subgroups with differences between treatment responses. Thus, hypnosis, health education, and behaviour modification are each effective programs for changing cigarette smoking and each is equally effective in this regard.

Adult↗
PubMed1989

Modification of the radiation response of pig skin by manipulation of tissue oxygen tension using anesthetics and administration of BW12C.

The importance of tissue oxygen tension on radiosensitivity was studied by examining modifications in the incidence of moist desquamation in pig skin after irradiation with strontium-90 plaques. The effects were analyzed using quantal dose-response data and comparisons were made using ED50 values for moist desquamation. Under standard anesthetic conditions of 2% halothane, approximately 70% oxygen, and approximately 30% nitrous oxide, the ED50 value (+/- SE) for moist desquamation was 27.32 +/- 0.52 Gy with no significant variation in radiosensitivity between dorsal, lateral, and ventral skin sites on the flank. Irradiation with 2% halothane and air increased the ED50 to 31.25 +/- 0.94 Gy, primarily due to an increased radioresistance of the dorsal sites. When combined with BW12C, a drug which binds oxygen selectively to hemoglobin and hence reduced the oxygen availability to tissues, a further increase in the ED50 values was observed. This was approximately 39 Gy with BW12C concentrations of 30 mg/kg and 50 mg/kg b.w. of BW12C, indicating a dose modification factor (DMF) of approximately 1.26. However, when animals were breathing the standard gas mixture, this DMF was reduced to 1.15 for 30 mg/kg of BW12C, indicating that a higher level of oxygen partly counteracted the effects of the drug in these studies with BW12C. The greatest variability in radiosensitivity was seen in the dorsal fields. This suggested complex physiological adaptation, a phenomenon that might also explain the absence of any modification of the radiation response when 100 mg/kg of BW12C was used.

Aldehydes↗
PubMed1978

Cloning of restriction and modification genes in E. coli: the HbaII system from Haemophilus haemolyticus.

The genes for a Class II restriction-modification system (HhaII) from Haemophilus haemolyticus have been cloned in Escherichia coli. The vector used for cloning was plasmid pBR322 which confers resistance to tetracycline and ampicillin and contains a single endonuclease R-PstI site, (formula: see text), in the ampicillin gene. The procedure developed by Bolivar et al. (1977) was used to form DNA recombinants. H. haemolyticus DNA was cleaved with PstI endonuclease and poly(dC) extensions were added to the 3'-OH termini using terminal deoxynucleotidyl transferase. Circular pBR322 DNA was cleaved to linear molecules with PstI endonuclease and poly(dG) extensions were added to the 3'-OH termini, thus regenerating the PstI cleavage site sequences. Recombinant molecules, formed by annealing the two DNAs, were used to transfect a restriction and modification-deficient strain of E. coli (HB101 r-m-recA). Tetracycline-resistant clones were tested for acquisition of restriction phenotype (as measured by growth on plates seeded with phage lambdacI-0). A single phage-resistant clone was found. The recombinant plasmid, pD110, isolated from this clone, had acquired 3 kilobases of additional DNA which could be excised with PstI endonuclease. In addition to the restriction function, cells carrying the plasmid expressed the HhaII modification function. Both activities have been partially purified by single-stranded DNA-agarose chromatography. The cloned HhaII restriction activity yields cleavage patterns identical to HinfI. A restriction map of the cloned DNA segment is presented.

DNA Restriction Enzymes↗
PubMed1990

Modification of the anticodon wobble position of tRNA(Ala) in vitro does not require 5' or 3' processing.

Maturation of eukaryotic tRNA molecules requires nuclear processing as well as nuclear and cytoplasmic modification of specific nucleotides. Nucleotide modifications within the anticodon are found in the majority of all tRNAs and are among the last maturation events to occur in vivo. We show that 5' and 3' processing of SP6 polymerase-generated transcripts are not necessary for the in vitro modification of A----I in the anticodon of tRNA(Ala).

Animals↗
PubMed1995

Cloning of a pair of genes encoding isoschizomeric restriction endonucleases from Bacillus species: the BspEI and BspMII restriction and modification systems.

The respective genes (R-M) encoding restriction and modification systems from two Bacillus species which recognise the same nucleotide sequence, 5'-TCCGGA, have been cloned and expressed in Escherichia coli. The BspEI R-M genes were cloned on a 3.6-kb HindIII fragment, whereas the BspMII R-M genes were cloned on three contiguous HindIII fragments totalling 9.8 kb. Upon thermal induction, E. coli carrying the bspEIR clones under the control of the phage lambda PL promoter, express high levels of R.BspEI (10(6) units/g wet cell paste). The bspMIIR gene, on the other hand, is only poorly expressed (about 4 x 10(3) units/g wet cell paste) following induction. Although the enzymes of both R-M systems recognize the same sequence and the restriction endonucleases (ENases) cleave DNA at the same position, the modification specified by the methyltransferases (MTases) differ. The internal cytosine is the site of M.BspMII modification (TCmeCGGA), whereas the external cytosine is modified by M.BspEI (TmeCCGGA). The two R-M systems probably evolved independently.

Bacillus↗
PubMedDate not supplied

Exogenous and endogenous DNA modifications as monitored by 32P-postlabeling: relationships to cancer and aging.

32P-postlabeling analysis, a highly sensitive method for the detection and measurement of covalent carcinogen-DNA adducts and other DNA modifications, does not require radioactive test substances and, therefore, can be applied to DNA of mammals, including humans exposed to low doses of environmental or occupational genotoxicants. The basic procedure entails the enzymatic incorporation of 32P-label into hydrolysis products of DNA, followed by chromatographic mapping and autoradiography of the 32P-labeled digestion products and quantitative scintillation spectrometry. Microgram amounts of DNA are analyzed: Thus the assay is suited for limited amounts of cells or tissues. Various versions of the assay afford different sensitivities of adduct detection. A single aromatic or bulky/hydrophobic adduct in 10(8)-10(10) nucleotides can be detected and measured (corresponding to 0.3-30 amol adduct/micrograms DNA or 0.1-10 nmol adduct/mol DNA-P). In animal models, the assay has been successfully applied to a variety of mutagenic (genotoxic) as well as nonmutagenic carcinogens. In humans, DNA specimens from cigarette smokers, iron foundry workers, and coke oven workers whose total aromatic adduct levels ranged from 1 adduct in 10(6)-10(8) DNA nucleotides have been examined by 32P-postlabeling. The assay also detects DNA modifications--Indigenous (I)-compounds--that increase with age in untreated animals. I-compound profiles and levels are highly species-, strain-, sex-, and tissue-specific, and also depend on diet composition. Caloric restriction, a highly efficient method for improving resistance to carcinogenesis and extending life span, increased rather than decreased I-compound levels in various tissues of male rats. Nonmutagenic hepatocarcinogens reduced levels of I-compounds in the target organ. Because of the specificity of this effect, reduction of I-compound levels appears to represent a novel biomarker for the action of nonmutagenic carcinogens. DNA from various hepatomas was found largely devoid of I-compounds. The results support a possible antineoplastic and antiaging role of these DNA modifications.

Aging↗
PubMedDate not supplied

Synthesis, modifications, and turnover of proteins during aging.

Slowing down of bulk protein synthesis is one of the most commonly observed biochemical changes during aging. The implications and consequences of slower rates of protein synthesis are manifold, including a decrease in the availability of enzymes for the maintenance, repair, and normal metabolic functioning of the cell, an inefficient removal of inactive, abnormal, and damaged macromolecules in the cell, the inefficiency of the intracellular and intercellular signalling pathways, and a decrease in the production and secretion of hormones, antibodies, neurotransmitters, and the components of the extracellular matrix. Age-related changes in the activity, specificity, and stability of a large number of proteins have been reported. However, the molecular mechanisms responsible for such alterations are still poorly understood. Studies on various components of the protein synthetic machinery have revealed a decline in the efficiency and accuracy of ribosomes, an increase in the levels of rRNA and tRNA, and a decrease in the amounts and activities of elongation factors. Because posttranslational modifications of proteins determine their activity and stability, alterations in the extent and level of various modifications such as phosphorylation, methylation, ADP-ribosylation, oxidation, glycation, and conformational changes during aging are being studied. Changes in the regulation of protein synthesis, posttranslational modifications, and protein turnover are crucial determinants of age-related decline in the maintenance, repair, and survival of the organism.

Adenosine Diphosphate Ribose↗
PubMed1986

Influence of chemical modification on enzyme inactivation kinetics and stability.

Enzymes are placed in different categories depending on the effect of chemical modification on their inactivation kinetics and residual activity. This is done using a series-type mechanism involving degraded but stable enzyme states. The major distinction in the three basic categories is the effect of modification on residual activity. Each category is further sub-divided depending on the effect of modification on the values of the deactivation rate constants. The classification provides for a framework for comparison of a wide variety of enzyme deactivation data. Structure-function relations are provided wherever possible.

Journal Article↗
PubMed1992

Modifications of synaptosomal plasma membrane protein composition in various brain regions during aging.

The age-dependent modifications of synaptosomal plasma membrane protein composition in three different rat brain regions (cerebral cortex, cerebellum and striatum) at various ages (4, 12 and 24 months) were studied. The proteins were separated by gel-electrophoresis and the quantity of the different polypeptides was determined densitometrically from the stained gels. In the three brain regions examined several age-related modifications in the amount of the synaptosomal plasma membrane proteins were observed. In particular a significant decrease in the content of some synaptosomal plasma membrane proteins at 24 months of age was found. The age-related modifications in the protein composition of synaptosomal plasma membrane may cause changes in many brain functions, such as neurotransmission, ionic transport and enzyme activities. Particularly interesting is the decrease of a protein with 18 kDa mol. wt. This protein has been identified as calmodulin by immunoblotting assay. The decrease in the amount of this protein may be correlated to the impairment of several Ca(2+)-requiring processes in the aging brain.

Aging↗
PubMed1987

Phase variation in Bordetella pertussis is accompanied by changes in DNA modification.

Pathogenic strains of Bordetella pertussis tend to undergo a phase variation process when propagated in vitro. The phase variants do not express part or all of the virulence factors of the pathogenic strain and are phenotypically stable. We have previously shown that variation involves a non-reversible, non-random process. In an attempt to characterize the molecular changes accompanying phase variation, chromosomal DNA, isolated from B. pertussis and its variants, was digested with a variety of restriction enzymes followed by agarose gel electrophoresis. While variant DNA was digested by all tested enzymes, pathogenic strain DNA was not digested by part of the enzymes, thus suggesting modification of the DNA at specific sites. DNA isolated from reversible, growth medium induced variants, demonstrated sensitivity to digestion identical to that of spontaneous, stable variants. Analysis of the restriction sequences of all the enzymes, which did not digest DNA from pathogenic strains, failed to reveal any common sequence known to be affected by methylation. HPLC and nearest-neighbor analysis showed a 2-fold increase in the level of DNA methylation in the pathogenic strain. It was concluded that (a) the chromosomal DNA in virulent strains of B. pertussis is protected against enzymatic digestion by an as yet unknown modification and (b) variation in B. pertussis may be caused by changes in the modification of the DNA rather than by mutation.

Base Sequence↗
PubMed1989

Oxidative modification of lens crystallins by H2O2 and chelated iron.

Crystallins are the soluble structural proteins that constitute approximately 90% of the dry mass of the eye lens. The present study attempts to elucidate possible mechanisms whereby the H2O2 present in the eye could contribute to the oxidative modification of lens crystallins. The data indicate that exposure of solutions of crystallins to H2O2 and EDTA-chelated iron leads to covalent crosslinking of polypeptides, loss of intrinsic protein fluorescence, and the generation of a novel fluorophor emitting in the 420 nm range. These changes closely mimic oxidative modifications that occur in lens proteins in vivo. Exposure of the proteins to H2O2 in the absence of chelated iron failed to generate detectable levels of these modifications. These findings are contrasted with earlier studies of lenses in organ culture where H2O2 alone produced marked damage while the further addition of chelated iron protected the lenses from oxidation.

Animals↗
PubMed1995

Endogenous ubiquinol prevents protein modification accompanying lipid peroxidation in beef heart submitochondrial particles.

This article is a study of the relationship between lipid peroxidation and protein modification in beef heart submitochondrial particles, and the protective effect of endogenous ubiquinol (reduced coenzyme Q) against these effects. ADP-Fe3+ and ascorbate were used to initiate lipid peroxidation and protein modification, which were monitored by measuring TBARS and protein carbonylation, respectively. Endogenous ubiquinone was reduced by the addition of succinate and antimycin. The parameters investigated included extraction and reincorporation of ubiquinone, and comparison of the effect of ubiquinol with those of various antioxidant compounds and enzymes, as well as the iron chelator EDTA. Under all conditions employed there was a close correlation between lipid peroxidation and protein carbonylation, and the inhibition of these effects by endogenous ubiquinol. SDS-PAGE analysis revealed a differential effect on individual protein components and its prevention by ubiquinol. Conceivable mechanisms behind the observed oxidative modifications of membrane phospholipids and proteins and of the role of ubiquinol in preventing these effects are considered.

Animals↗
PubMed1995

Oxidative modification of fibrinogen inhibits thrombin-catalyzed clot formation.

Plasma fibrinogen plays a central role in controlling hemostasis. In an earlier report, we found that fibrinogen is oxidized when whole plasma is treated with a metal-catalyzed oxidation system. These studies show that oxidative modification of purified human fibrinogen leads to an exposure-dependent loss of thrombin-induced clot formation. Inhibition of clotting occurred when either metal-catalyzed oxidation or gamma-irradiation was employed to generate oxidizing radicals. Both systems caused covalent modification of fibrinogen, assessed by measuring incorporation of protein carbonyls. Thrombin-catalyzed fibrinopeptide release was normal in irradiated fibrinogen and was only slightly diminished in protein exposed to metal-catalyzed oxidation, indicating that the inhibition of clotting activity was due to impaired fibrin monomer polymerization. Thus, oxidative modification of normal fibrinogen causes dysfibrinogenemia and constitutes a novel mechanism for inhibition of thrombosis.

Ascorbic Acid↗
PubMed1996

Cupric ion/ascorbate/hydrogen peroxide-induced DNA damage: DNA-bound copper ion primarily induces base modifications.

The kinetics of frank DNA strand breaks and DNA base modifications produced by Cu(II)/ascorbate/H2O2 were simultaneously determined in purified human genomic DNA in vitro. Modified bases were determined by cleavage with Escherichia coli enzymes Nth protein (modified pyrimidines) and Fpg protein (modified purines). Single-stranded lesion frequency before (frank strand breaks) and after (modified bases) Nth or Fpg protein digestion was quantified by neutral glyoxal gel electrophoresis. Dialysis of EDTA-treated genomic DNA purified by standard proteinase K digestion/phenol extraction was necessary to remove low molecular weight species, probably transition metal ions and metal ion chelators, which supported frank strand breaks in the presence of ascorbate + H2O2 without supplemental copper ions. We then established a kinetic model of the DNA-damaging reactions caused by Cu(II) + ascorbate + H2O2. The principal new assumption in our model was that DNA base modifications were caused exclusively by DNA-bound Cu(I) and frank strand breaks by non-DNA-bound Cu(I). The model was simulated by computer using published rate constants. The computer simulation quantitatively predicted: (1) the rate of H2O2 degradation, which was measured using an H2O2-sensitive electrode, (2) the linearity of accumulation of DNA strand breaks and modified bases over the reaction period, (3) the rate of modified base accumulation, and (4) the dependence of modified base and frank strand production on initial Cu(II) concentration. The simulation significantly overestimated the rate of frank strand break accumulation, suggesting either that the ultimate oxidizing species that attacks the sugar-phosphate backbone is a less-reactive species than the hydroxyl radical used in the model and/or an unidentified hydroxyl radical-scavenging species was present in the reactions. Our experimental data are consistent with a model of copper ion-DNA interaction in which DNA-bound Cu(I) primarily mediates DNA base modifications and nonbound Cu(I) primarily mediates frank strand break production.

Ascorbic Acid↗
PubMed1993

Absence of modifications of the pharmacological properties of the GABAA receptor complex during aging, as assessed in 3- and 24-month-old rat cerebral cortex.

We have investigated the possible modifications of the pharmacological properties of Type I and Type II benzodiazepine binding sites of the gamma-aminobutyric acidA (GABAA) receptor complex in cortical membranes from 3- and 24-month-old Wistar or Fischer rats. No major changes were found in the binding parameters of [3H]zolpidem (a Type I-specific ligand) or [3H]flunitrazepam (a non-selective benzodiazepine). Neither the Kd values nor the Bmax for either ligand were modified during aging in cortical membranes from Wistar or Fischer rats. Consequently, the proportion of Type I binding sites was also unmodified in aged cortical membranes. The absence of modifications of Type I and Type II binding sites was also confirmed by Cl 218,872 displacement of [3H]flunitrazepam binding in aged cortical membranes from Wistar rats. Furthermore, the [3H]muscimol binding and the allosteric interactions between Type I or total benzodiazepine binding sites and GABA binding sites also remained unaltered with aging in cortical membranes from Wistar rats. Moreover, the pattern and proportion of the [3H]flunitrazepam photoaffinity labeled peptides were also unmodified by aging. These results demonstrate the absence of modifications in Type I or total benzodiazepine binding sites of the GABAA receptor complex from adult and aged cortical membranes in Fischer or Wistar rats.

Affinity Labels↗
PubMed1992

Activation of 15-lipoxygenase by low density lipoprotein in vascular endothelial cells. Relationship to the oxidative modification of low density lipoprotein.

Oxidatively-modified low density lipoprotein (LDL) is thought to play a significant role in the formation of lipid-laden macrophages, the primary cellular component of atherosclerotic fatty lesions. Recently, lipoxygenases have been implicated as a major enzymatic pathway involved in rabbit endothelial cell-mediated LDL modification. We investigated the effect of LDL on porcine aortic endothelial cell (PAEC) and human umbilical vein (HUVEC) and aortic endothelial cell (HAEC) lipoxygenase activity. By thin layer chromatography, we observed that human LDL stimulated the metabolism of radiolabeled arachidonic acid to 12 + 15-hydroxyeicosatetraenoic acid (HETE) in indomethacin-treated PAEC. Furthermore, radiolabeled linoleic acid, a specific substrate for the 15-lipoxygenase, was metabolized to its respective product 13-hydroxyoctadecadienoic acid (13-HODE) in the presence of LDL. Increased product formation in both studies was inhibited by the lipoxygenase blockers nordihydroguaiaretic acid (NDGA) and RG 6866. 15-HETE was confirmed as the predominant HETE product in LDL-treated cells by high performance liquid chromatography. Both porcine- and human-derived LDL stimulated the CL release of 15-HETE from cells as determined by radioimmunoassay. Release of immunoreactive 15-HETE was inhibited by NDGA, RG 6866, and 5,8,11,14-eicosatetraynoic acid (ETYA) but not by the selective 5-lipoxygenase inhibitor RG 5901. These lipoxygenase inhibitors had similar effects on the modification of LDL. Our results suggest that the oxidative modification of LDL by endothelial cells may be mediated in part through activation of 15-lipoxygenase.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1995

Differential effects of endothelial cell fatty acid modification on the sensitivity of their membrane phospholipids to peroxidation.

In order to study the relationship between the fatty acid (FA) composition of human umbilical vein endothelial cells (HUVEC) and their susceptibility to oxidative stress, we modified their FA composition by long-term culturing in media supplemented with a saturated, monounsaturated, or polyunsaturated FA. Sensitivity of the cellular phospholipids to peroxidation was monitored by measuring conjugated diene formation and decrease of polyunsaturated FAs induced by CuSO4 and H2O2 in liposomes prepared from the respective phospholipid extracts. The extent of phospholipid peroxidation was found to increase with increasing content of polyunsaturated FAs. In addition, the sensitivity of individual polyunsaturated FAs to peroxidation was directly proportional to the number of double bonds present. However, no unequivocal relationship was observed between conjugated diene formation and the phospholipid unsaturation index (an indicator for the combined effect of number of double bonds and polyunsaturated FA contents of the membrane phospholipids). The results suggest that long-term FA modification of endothelial cells differentially alters the sensitivity of their membrane phospholipids to peroxidation: long-term modification with oleic acid may protect against lipid peroxidation, whereas linoleic acid may increase sensitivity to peroxidation. In contrast to what might be expected, long-term modification of endothelial cells with eicosapentaenoic or docosahexaenoic acid does not increase the sensitivity of phospholipids to peroxidation.

Cells, Cultured↗
PubMed2005

Obtaining high sequence coverage in matrix-assisted laser desorption time-of-flight mass spectrometry for studies of protein modification: analysis of human serum albumin as a model.

Several approaches were explored for obtaining high sequence coverage in protein modification studies performed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Human serum albumin (HSA, 66.5kDa) was used as a model protein for this work. Experimental factors considered in this study included the type of matrix used for MALDI-TOF MS, the protein digestion method, and the use of fractionation for peptide digests prior to MALDI-TOF MS analysis. A mixture of alpha-cyano-4-hydroxycinnamic acid and 2,5-dihydroxybenzoic acid was employed as the final matrix for HSA. When used with a tryptic digest, this gave unique information on only half of the peptides in the primary structure of HSA. However, the combined use of three enzyme digests based on trypsin, endoproteinase Lys-C, and endoproteinase Glu-C increased this sequence coverage to 72.8%. The use of a ZipTip column to fractionate peptides in these digests prior to analysis increased the sequence coverage to 97.4%. These conditions made it possible to examine unique peptides from nearly all of the structure of HSA and to identify specific modifications to this protein (e.g., glycation sites). For instance, Lys199 was confirmed as a glycation site on normal HSA, whereas Lys536 and Lys389 were identified as additional modification sites on minimally glycated HSA.

Amino Acid Sequence↗