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PubMed1980

Deoxyribonucleic acid restriction and modification systems in Salmonella: chromosomally located systems of different serotypes.

With the use of four different phages, Salmonella strains representing 85 different serotypes were examined to determine their restriction-modification phenotype. They fell into one of three groups on this basis: group 1, those which lacked the common LT system; group 2, those in which only the LT system could be recognized; and group 3. those which possessed the LT system and at least one other system shown with some serotypes to be closely linked to serB. The specificity of the serB-linked restriction-modification system was unique for each serotype, but different strains of the same serotype expressed the same specificity. Two of the systems were shown to behave in genetic crosses as functional alleles of the S. typhimurium SB system. It is possible that these serB-linked restriction-modification systems constitute a large multiallelic series of genes extending throughout the Salmonella genus and Escherichia coli. We suggest that the division of the Salmonella into the three restriction-modification groups may be significant in defining a "biological grouping" of the different serotypes within the genus which may ultimately be useful in describing the Salmonella species. From the genetic relatedness between the genes of some of the Salmonella restriction-modification systems with those of the E. coli systems, we deduce that the restriction endonuclases produced by the Salmonella serB-linked systems are of type 1. Determination of the nucleotide sequences of the recognition sites of the restriction endonucleases of selected Salmonella systems should further our understanding of specificity with these enzymes.

Chromosome Mapping↗
PubMed1995

An anticodon sequence mutant of Escherichia coli initiator tRNA: possible importance of a newly acquired base modification next to the anticodon on its activity in initiation.

Initiator tRNAs from eubacteria and chloroplasts lack a base modification next to the anticodon. This is in contrast to virtually all other tRNAs from these sources. We show that a mutant Escherichia coli initiator tRNA which has an anticodon sequence change from CAU to CUA now has a 2-methylthio-N6-(delta 2-isopentenyl)adenosine (ms2i6A) modification, produced by posttranscriptional modification of A, next to the anticodon. This newly acquired base modification may be important for the function of the mutant tRNA in initiation. In a miaA mutant strain of E. coli defective in biosynthesis of ms2i6A, the mutant initiator tRNA is 10- to 12-fold less active in initiation. The mutant tRNA is aminoacylated and formylated normally in the miaA strain. Thus, the absence of the base modification affects the activity of the mutant tRNA at a step subsequent to its formylation.

Anticodon↗
PubMed1979

Unusual modification of bacteriophage Mu DNA.

Bacteriophage Mu DNA was labeled after induction in the presence of [2-(3)H]adenine or [8-(3)H]adenine. Both Mu mom(+).dam(+) DNA and Mu mom(-).dam(+) DNA have similar N(6)-methyladenine (MeAde) contents, as well as similar frequencies of MeAde nearest neighbors. Both DNAs are sensitive to in vitro cleavage by R.DpnI but resistant to cleavage by R.DpnII. These results indicate that the mom(+) protein does not alter the sequence specificity of the host dam(+) methylase to produce MeAde at new sites. However, we have discovered a new modified base, denoted A(x), in Mu mom(+).dam(+) DNA; approximately 15% of the adenine residues are modified to A(x). Although the precise nature of the modification is not yet defined, analysis by electrophoresis and chromatography indicates that the N(6)-amino group is not the site of modification, and that the added moiety contains a free carboxyl group. A(x) is not present in Mu mom(+).dam(+) or Mu mom(-).dam(+) phage DNA or in cellular DNA from uninduced Mu mom(+).dam(+) lysogens. These results suggest that expression of the dam(+) and mom(+) genes are required for the A(x) modification and that this modification is responsible for protecting Mu DNA against certain restriction nucleases. Mu mom(+).dam(-) DNA and Mu mom(-).dam(-) DNA contain a very low level of MeAde (ca. 1 MeAde per 5,000 adenine residues). Since the only nearest neighbor to MeAde appears to be cytosine, we suggest that the methylated sequence is 5'... C-A(*)-C... 3' and that this methylation is mediated by the EcoK modification enzyme.

Adenine↗
PubMed1995

Analysis of the posttranslational modifications of the influenza virus M2 protein.

The sites of posttranslational modifications of the influenza A virus M2 protein were examined, and the effect of these modifications on the M2 protein ion channel activity was analyzed. Cysteine residues 17 and 19 in the M2 protein ectodomain form disulfide bonds. The cytoplasmic tail is posttranslationally modified by palmitoylation, and mutagenic studies support the view that cysteine residue 50 is the site for fatty acylation. In addition, the cytoplasmic tail of the M2 protein was found to be posttranslationally modified by the addition of phosphate to specific serine residues. Site-directed mutagenesis of serine residues in the M2 protein cytoplasmic tail, combined with phosphoamino acid analysis, indicated that serine residue 64 is the predominant site for phosphorylation but that serine residues 82, 89, and 93 were also phosphorylated but to much lesser extents. Disulfide-bond formation, palmitoylation, and phosphorylation occurred on M2 protein expressed in mammalian cells infected with influenza virus, in mammalian cells in which the M2 protein was expressed from DNA expression vectors, and when the M2 protein was expressed in oocytes of Xenopus laevis. The membrane currents of oocytes of Xenopus laevis expressing wild-type and site-specifically altered forms of the M2 protein, to ablate posttranslational modifications, indicated that none of the posttranslational modifications significantly affected the ion channel activity of the M2 protein in oocytes. Therefore, these data do not indicate a functional role for posttranslational modifications of the M2 protein in its ion channel activity.

Amino Acid Sequence↗
PubMed1999

Viral immediate-early proteins abrogate the modification by SUMO-1 of PML and Sp100 proteins, correlating with nuclear body disruption.

PML nuclear bodies (NBs) are subnuclear structures whose integrity is compromised in certain human diseases, including leukemia and neurodegenerative disorders. Infection by a number of DNA viruses similarly triggers the reorganization of these structures, suggesting an important role for the NBs in the viral infection process. While expression of the adenovirus E4 ORF3 protein leads to only a moderate redistribution of PML to filamentous structures, the herpes simplex virus (HSV) ICP0 protein and the cytomegalovirus (CMV) IE1 protein both induce a complete disruption of the NB structure. Recently, we and others have shown that the NB proteins PML and Sp100 are posttranslationally modified by covalent linkage with the ubiquitin-related SUMO-1 protein and that this modification may promote the assembly of these structures. Here we show that the HSV ICP0 and CMV IE1 proteins specifically abrogate the SUMO-1 modification of PML and Sp100, whereas the adenovirus E4 ORF3 protein does not affect this process. The potential of ICP0 and IE1 to alter SUMO-1 modification is directly linked to their capacity to disassemble NBs, thus strengthening the role for SUMO-1 conjugation in maintenance of the structural integrity of the NBs. This observation supports a model in which ICP0 and IE1 disrupt the NBs either by preventing the formation or by degrading of the SUMO-1-modified PML and Sp100 protein species. Finally, we show that the IE1 protein itself is a substrate for SUMO-1 modification, thus representing the first viral protein found to undergo this new type of posttranslational modification.

Antigens, Nuclear↗
PubMed1997

Chemical modifications of Bacillus subtilis tryptophanyl-tRNA synthetase.

A concerted conformational change in Bacillus subtilis tryptophanyl-tRNA synthetase (TrpRS) was evident from previous fluorescence on the quenching of the single Trp residue Trp-92 in the 4FTrp-AMP complexed enzyme. In this study, chemical modifications of the B. subtilis TrpRS were employed to further characterize this conformational change, with the single Trp residue serving as a marker for monitoring the change. Modifications of the enzyme by means of the Trp-specific agent N-bromosuccinimide (NBS) or 3-bromo-3-methyl-2-(2-nitrophenylmercapto)-3H-indole (BNPS-skatole) inactivated the enzyme in accord with the essential role of Trp-92, as identified previously by site-directed mutagenesis. ATP sensitized TrpRS toward inactivation by NBS and BNPS-skatole, which suggested a conformational change that resulted in greater accessibility of Trp-92 toward modifications. In contrast, the cognate tRNATrp substrate exerted a specific protective effect against inactivation by both of the reagents, indicating that the TrpRS-tRNATrp interaction reduces the accessibility of Trp-92 under our experimental conditions. By comparison, modification of sulfhydryl groups by means of iodoacetamide did not reduce TrpRS activity. Observations on Trp-specific modification and substrate protection effects are discussed in the context of the Bacillus stearothermophilus TrpRS crystal structure.

Bacillus subtilis↗
PubMed2006

Chromatin modifications by methylation and ubiquitination: implications in the regulation of gene expression.

It is more evident now than ever that nucleosomes can transmit epigenetic information from one cell generation to the next. It has been demonstrated during the past decade that the posttranslational modifications of histone proteins within the chromosome impact chromatin structure, gene transcription, and epigenetic information. Multiple modifications decorate each histone tail within the nucleosome, including some amino acids that can be modified in several different ways. Covalent modifications of histone tails known thus far include acetylation, phosphorylation, sumoylation, ubiquitination, and methylation. A large body of experimental evidence compiled during the past several years has demonstrated the impact of histone acetylation on transcriptional control. Although histone modification by methylation and ubiquitination was discovered long ago, it was only recently that functional roles for these modifications in transcriptional regulation began to surface. Highlighted in this review are the recent biochemical, molecular, cellular, and physiological functions of histone methylation and ubiquitination involved in the regulation of gene expression as determined by a combination of enzymological, structural, and genetic methodologies.

Amino Acid Sequence↗
PubMed1999

Nonoxidative modifications of lipoproteins in atherogenesis.

The key initiating event in atherosclerosis is the retention of plasma lipoproteins in the subendothelial matrix. Subsequently, a series of biological responses to this retained material leads to specific molecular and cellular processes that promote lesion formation. There is considerable evidence that many of these biological responses, notably macrophage cholesteryl ester loading (foam cell formation), require subendothelial modification of the retained lipoproteins. Oxidation of lipoproteins is one such modification that likely occurs in vivo and promotes certain atherogenic events, but oxidation cannot explain all aspects of atherogenesis, including certain elements of macrophage foam cell formation. For this reason, there has been renewed interest in other modifications of lipoproteins that may be important in atherogenesis. This review addresses five such lipoprotein modifications, namely aggregation, glycation, immune complex formation, proteoglycan complex formation, and conversion to cholesterol-rich liposomes. The focus is on the evidence that these modifications occur in atherosclerotic lesions and on the potential role of these modified lipoproteins in atherogenesis, with an emphasis on macrophage foam cell formation.

Antigen-Antibody Complex↗
PubMedDate not supplied

Decrease in reactive amino groups during oxidation or endothelial cell modification of LDL. Correlation with changes in receptor-mediated catabolism.

The monocyte/macrophage appears to be the precursor of many of the lipid-laden cells in atherosclerotic lesions, but the mechanism by which these cells accumulate cholesterol to become foam cells remains unclear. We have previously reported that cultured endothelial cells can modify low density lipoprotein (LDL) in a manner that leads to rapid uptake by the acetyl LDL receptor of macrophages. This modification involves free radical-induced peroxidation of LDL and is accompanied by many changes in the physicochemical properties of LDL including increased electrophoretic mobility, increased density, decreased content of esterified cholesterol, hydrolysis of phosphatidylcholine, and fragmentation of apolipoprotein B. Under conditions highly favorable to oxidation, a similar modification can occur even in the absence of cells. In the present studies, oxidation of LDL simply by exposure to 5 microM Cu++ resulted in a modification that was indistinguishable from that produced by endothelial cells. Moreover, it was demonstrated that LDL oxidation by either method is accompanied by a marked decreased in amino group reactivity, comparable to that seen with the chemical modifications of LDL that lead to recognition by the acetyl LDL receptor. Inhibitors of proteolytic enzymes did not reduce fragmentation of apolipoprotein B during oxidation. The rate of catabolism of intravenously injected oxidized LDL in guinea pigs was very rapid, and over 80% of the degradation occurred in the liver. The studies demonstrate that all of the changes associated with endothelial cell modification of LDL can be attributed to oxidation. The cells can, however, promote oxidation under conditions where it would otherwise occur very slowly.(ABSTRACT TRUNCATED AT 250 WORDS)

Amines↗
PubMedDate not supplied

Modification of low density lipoprotein with 4-hydroxynonenal induces uptake by macrophages.

There is indirect evidence that the oxidation of low density lipoprotein (LDL) may be involved in the development of atherosclerosis. Modification of LDL by oxidation may lead to its unregulated uptake by intimal macrophages to form foam cells. Because of the complexity of events occurring during LDL oxidation, we have tested whether LDL modified directly with 4-hydroxynonenal (HNE), a major propagation product formed during lipid peroxidation and known to be present in oxidized LDL, could bring about lipid loading of macrophages. Modification was accomplished by incubating LDL with various concentrations of HNE up to 7.5 mM. When LDL was derivatized with lower concentrations of HNE, concentration-dependent increases were observed in the covalent binding of HNE to apolipoprotein B (apo B), the blockage of the epsilon-amino groups on lysine residues of apo B, and the relative electrophoretic mobility of LDL. Decreases were observed in degradation of the modified LDL by the J774 cell line, mouse peritoneal macrophages, and smooth muscle cells. Modification of LDL by incubation with the higher concentrations of HNE resulted in LDL aggregation. This modification was associated with marked increases in the macrophage degradation of LDL. Degradation of aggregated HNE-modified LDL increased linearly with incubation time, leading to lipid loading of these cells as observed by oil red O staining and cholesterol accumulation. Uptake appeared to occur by phagocytosis, since cytochalasin D, an inhibitor of phagocytosis, quantitatively inhibited uptake and degradation of labeled HNE LDL. Uptake did not appear to be mediated by either the LDL receptor or the scavenger receptor, since competition with excess amounts of LDL or acetyl LDL failed to inhibit degradation of labeled, aggregated HNE LDL. Saturation of degradation of HNE LDL by macrophages could be attributed, in part, to steric hindrance, since both excess HNE LDL and other particulate ligands could inhibit this degradation. These studies suggest that interaction of LDL with HNE formed during lipid peroxidation could be responsible for structural modifications leading to unregulated uptake of the lipoprotein by tissue macrophages. This could partially explain lipid loading or foam cell formation in atherosclerosis.

Aldehydes↗
PubMed2003

His electrogram alternans reveal dual atrioventricular nodal pathway conduction during atrial fibrillation: the role of slow-pathway modification.

BACKGROUND: Traditional tools to study dual-pathway atrioventricular nodal (AVN) electrophysiology are not applicable in subjects with permanent atrial fibrillation (AF). The presence of fast-pathway (FP) and slow-pathway (SP) wavefronts and their possible modification remain uncertain in this condition. We demonstrated previously that His electrogram (HE) alternans can determine whether the FP or the SP reaches the His bundle on a beat-by-beat basis. We have now applied this novel index to monitor dual-pathway conduction and the effects of SP modification during AF. METHODS AND RESULTS: In 12 rabbit AVN preparations, HE alternans were confirmed during a standard A(1)A(2) pacing protocol. During AF, in 9 of the 12 hearts, HE alternans indicated the presence of dual pathways. Successful SP modification guided by the HE alternans eliminated the SP, resulting in a predominantly FP conduction during AF in all hearts. This increased the average His-His interval (204+/-14 versus 276+/-51 ms, P<0.001). Morphological studies revealed that SP modification damaged only the posterior extension of the AVN. CONCLUSIONS: We have demonstrated for the first time in rabbits that HE alternans permit "visualization" of dual-pathway electrophysiology and confirmed the presence of both FP and SP wavefronts during AF. This novel index has been used in a selective SP ablation that resulted in a significant slowing of the ventricular rate. HE alternans provide a new insight into the mechanisms of AVN conduction and could guide AVN modification for ventricular rate control in AF clinically.

Animals↗
PubMed1994

Control of rapid ventricular response by radiofrequency catheter modification of the atrioventricular node in patients with medically refractory atrial fibrillation.

BACKGROUND: Pharmacological control of rapid ventricular response to atrial fibrillation may be difficult in some patients. Alternative treatments, including curative surgery or atrioventricular (AV) node ablation with pacemaker implantation, have significant potential morbidity. In view of evidence that dual AV nodal physiology may exist in a significant percentage of the population, even in those without AV nodal reentrant tachycardia, we postulated that control of ventricular response might be achieved by radiofrequency (RF) catheter ablation in the region of the AV nodal slow pathway with its short refractory period. METHODS AND RESULTS: Ten patients underwent attempted AV node modification using a 4-mm-tipped electrode catheter positioned in the middle or posterior septum, between the His bundle and coronary sinus ostium on the tricuspid valve annulus. RF energy was applied at 16 to 30 W for up to 60 seconds, until average ventricular response fell below 100 beats per minute. Reduction of maximal ventricular response below 120 beats per minute was confirmed with atropine 1 mg IV. If required, additional ablations were performed progressively more posteriorly up to the coronary sinus ostium. Patients with successful AV node modification were discharged off AV node-blocking drugs and followed in the clinic at regular intervals. Twenty-four-hour ambulatory ECG recordings and/or treadmill stress tests were obtained before and after ablation for statistical comparison of maximum ventricular rate. Resting average ventricular rate was determined during electrophysiology study before and after ablation. In 7 of 10 patients (70%), maximum ventricular rate was reduced from a mean of 164 +/- 12 to 123 +/- 16 beats per minute (P < .01) and average ventricular rate from a mean of 128 +/- 11 to 83 +/- 10 beats per minute after ablation. Mean minimum ventricular rate was 54 +/- 11 beats per minute after ablation. These 7 patients have remained symptom free from rapid ventricular response for a mean of 14 +/- 8 months (range, 1 to 22). Three remain off all AV node-blocking drugs, 3 remain on digoxin alone, which was previously ineffective, and 1 remains on a beta-blocker for angina. In the 3 patients who did not respond to AV node modification, complete AV node ablation and permanent pacemaker implantation was performed in 2 and DC cardioversion after amiodarone loading was performed in 1. CONCLUSIONS: RF catheter modification of AV node conduction is effective in controlling rapid ventricular response to atrial fibrillation in a significant percentage of medically refractory patients. A possible mechanism of RF modification of AV node conduction is AV nodal slow pathway ablation. Large-scale clinical trials will be needed to determine the overall efficacy and safety of this technique.

Adult↗
PubMed1995

Radiofrequency catheter modification of sinus pacemaker function guided by intracardiac echocardiography.

BACKGROUND: The sinus P wave arises from a pacemaker complex distributed along the crista terminalis. We investigated the feasibility of modification of sinus pacemaker function using graded applications of radiofrequency energy along the crista terminalis in dogs to achieve sinus rate control. METHODS AND RESULTS: Modification of sinus pacemaker function (30 +/- 5% reduction in intrinsic heart rate with retention of a normal P-wave axis) was performed in 11 dogs (group 1). Total sinus pacemaker ablation (> 50% reduction in intrinsic heart rate with development of a low ectopic atrial or a junctional rhythm) was performed in 4 dogs (group 2). Intracardiac echocardiography was used to identify the crista terminalis as an anatomic marker of sinus node location. Sinus pacemaker modification caused a significant decrease in intrinsic heart rate (31% reduction, P < .001), heart rate responsiveness to isoproterenol (30% reduction, P < .0001), and average (20% reduction, P = .0002) and maximal (22% reduction, P = .0007) heart rates during 24-hour Holter monitoring. In 6 of the 11 animals, the targeted rate reduction of 30 +/- 5% was accurately achieved (mean, 31.6 +/- 4.3%; P < .001), and in the other 5, significant reduction of intrinsic heart rate was achieved but with greater variation (28.0 +/- 17.3%, P < .005). Corrected sinus node recovery time was not prolonged. After modification, earliest activation was mapped to the crista terminalis inferior to the lesion in all animals. In long-term follow-up (3.7 +/- 1.0 months), effects were maintained. After total sinus pacemaker ablation, junctional and low atrial escape pacemakers were unstable. CONCLUSIONS: This study demonstrates the feasibility of modification of sinus pacemaker function for sinus rate control using catheter-based radiofrequency ablation guided by intracardiac echocardiography. This can be done while pacemaker stability and attenuated responsiveness to autonomic influences are preserved. Intracardiac echocardiography accurately defined the crista terminalis and provided a reliable means to anatomically localize catheter position in relation to the sinus node.

Animals↗
PubMed2005

Protein modifications by 4-hydroxynonenal and 4-hydroxyhexenal in light-exposed rat retina.

PURPOSE: 4-Hydroxynonenal (4-HNE) and 4-hydroxyhexenal (4-HHE) are reactive aldehydes derived from the nonenzymatic oxidation of n-6 and n-3 polyunsaturated fatty acids, respectively. Increasing evidence suggests that protein modifications by reactive aldehydes are involved in various diseases. The present study was undertaken to test whether protein modifications by 4-HNE and 4-HHE increase in retinal tissues after exposure of rats to damaging levels of light. METHODS: Albino rats were exposed to 1 or 5 klux white fluorescent light for 3 hours and, at various times thereafter, the levels and localizations of aldehyde-modified proteins in retinas were assessed by densitometric analysis of semiquantitative Western dot blots and by immunohistochemistry, using 4-HNE- and 4-HHE-specific antibodies. In some rats, the protective antioxidant phenyl-N-tert-butylnitrone (PBN) was injected (50 mg/kg) before exposure to light. To assess retinal damage, outer nuclear layer (ONL) thickness was measured on hematoxylin-eosin (H&E)-stained sections, and apoptosis was semiquantitatively analyzed by TUNEL staining. RESULTS: By dot blot analysis, 4-HNE- and 4-HHE-modified proteins were significantly increased in retina (both by 1.7-fold) and RPE fraction (1.5- and 1.8-fold, respectively) after 5-klux exposure. In retina, increases in 4-HNE- and 4-HHE-modified proteins were more prominent at 3 hours than at 24 hours or 48 hours after exposure to light. In rod outer segments, only 4-HHE-modified proteins increased significantly (1.4-fold). Retinal thinning, TUNEL staining in ONL, 4-HNE-, and 4-HHE protein modifications were all found in the same retinal regions. PBN treatment inhibited the light-induced increase of 4-HNE and 4-HHE modified proteins in retina and RPE fractions. CONCLUSIONS: Exposure to intense light increases 4-HNE and 4-HHE protein modifications in the retina, suggesting that free radical initiated, nonenzymatic reactions are involved in this process. These modifications may be early events that precede photoreceptor cell apoptosis.

Aldehydes↗
PubMed1984

Iron and copper promote modification of low density lipoprotein by human arterial smooth muscle cells in culture.

Modification of low density lipoproteins by human arterial smooth muscle cells was characterized by increased electrophoretic mobility and increased content of malondialdehyde-like oxidation products reactive with thiobarbituric acid. Lipoprotein modification was promoted by micromolar concentrations of iron or copper in the culture medium and was metal ion concentration- and time-dependent. The ability of diverse media to promote smooth muscle cell-mediated low density lipoprotein modification correlated with their iron concentration. Therefore, metal ion concentration of culture media contributes substantially to low density lipoprotein modification in vitro. Human monocyte-derived macrophages took up and esterified the cholesterol from modified low density lipoprotein more extensively than from native low density lipoprotein. Metal ion-mediated modification of low density lipoprotein may be a contributing factor to the pathogenesis of arteriosclerosis.

Cells, Cultured↗
PubMed1986

Probucol inhibits oxidative modification of low density lipoprotein.

Previous studies have established that low density lipoprotein (LDL) incubated with endothelial cells (EC) undergoes extensive oxidative modification in structure and that the modified LDL is specifically recognized by the acetyl LDL receptor of the macrophage. Thus, in principle, EC-modified LDL could contribute to foam cell formation during atherogenesis. Oxidatively modified LDL is also potentially toxic to EC. The present studies show that addition of probucol during the incubation of LDL with EC prevents the increase in the electrophoretic mobility, the increase in peroxides, and the increase in subsequent susceptibility to macrophage degradation. It has also been shown that oxidation of LDL catalyzed by cupric ion induces many of the same changes occurring during EC modification. Addition of probucol (5 microM) also prevented this copper-catalyzed modification of LDL. Most importantly, samples of LDL isolated from plasma of hypercholesterolemic patients under treatment with conventional dosages of probucol were shown to be highly resistant to oxidative modification either by incubation with endothelial cells or by cupric ion in the absence of cells. The findings suggest the hypothetical but intriguing possibility that probucol, in addition to its recognized effects on plasma LDL levels, may inhibit atherogenesis by limiting oxidative LDL modification and thus foam cell formation and/or EC injury. Other compounds with antioxidant properties might behave similarly.

Animals↗
PubMed2001

Hyperglycemia inhibits endothelial nitric oxide synthase activity by posttranslational modification at the Akt site.

Endothelial nitric oxide synthase (eNOS) is activated by phosphorylation of serine 1177 by the protein kinase Akt/PKB. Since hyperglycemia-induced mitochondrial superoxide overproduction increases O-linked N-acetylglucosamine modification and decreases O-linked phosphorylation of the transcription factor Sp1, the effect of hyperglycemia and the hexosamine pathway on eNOS was evaluated. In bovine aortic endothelial cells, hyperglycemia inhibited eNOS activity 67%, and treatment with glucosamine had a similar effect. Hyperglycemia-associated inhibition of eNOS was accompanied by a twofold increase in O-linked N-acetylglucosamine modification of eNOS and a reciprocal decrease in O-linked serine phosphorylation at residue 1177. Both the inhibition of eNOS and the changes in its post-translational modifications were reversed by antisense inhibition of glutamine:fructose-6-phosphate amidotransferase, the rate-limiting enzyme of the hexosamine pathway, or by blocking mitochondrial superoxide overproduction with uncoupling protein-1 (UCP-1) or manganese superoxide dismutase (MnSOD). Immunoblot analysis of cells expressing myc-tagged wild-type human eNOS confirmed the reciprocal increase in O-linked N-acetylglucosamine and decrease in O-linked serine 1177 phosphorylation in response to hyperglycemia. In contrast, when myc-tagged human eNOS carried a mutation at the Akt phosphorylation site (Ser1177), O-linked N-acetylglucosamine modification was unchanged by hyperglycemia and phospho-eNOS was undetectable. Similar changes in eNOS activity and covalent modification were found in aortae from diabetic animals. Chronic impairment of eNOS activity by this mechanism may partly explain the accelerated atherosclerosis of diabetes.

Acetylglucosamine↗
PubMed2006

Genetic modification and genetic determinism.

In this article we examine four objections to the genetic modification of human beings: the freedom argument, the giftedness argument, the authenticity argument, and the uniqueness argument. We then demonstrate that each of these arguments against genetic modification assumes a strong version of genetic determinism. Since these strong deterministic assumptions are false, the arguments against genetic modification, which assume and depend upon these assumptions, are therefore unsound. Serious discussion of the morality of genetic modification, and the development of sound science policy, should be driven by arguments that address the actual consequences of genetic modification for individuals and society, not by ones propped up by false or misleading biological assumptions.

Genetic Determinism↗