Low-density lipoprotein induced actin cytoskeleton reorganization in endothelial cells: mechanisms of action.

The inhibitory effects of the specific NADPH oxidase inhibitor, apocynin, and non-specific NADPH oxidase inhibitors, nordihydroguaiaretic acid (NDGA) and SKF525A, on the disruption of dense peripheral bands and formation of stress fibers in cultured human umbilical vein endothelial cells exposed to atherogenic low-density lipoprotein (LDL) levels has been investigated. Endothelial cells (EC) in vitro and in vivo exposed to high LDL-cholesterol levels have cytoskeletal remodeling with stress fiber formation and loss of dense peripheral bands. Cultured EC incubated with exogenously applied hydrogen peroxide (H2O2: 1 mM) have cytoskeletal structural changes much similar to those observed with high LDL exposure. Previous studies have 1) demonstrated that exposure to atherogenic LDL levels causes heightened EC H2O2 production, 2) identified the reactive oxygen species source, NADPH oxidase, in EC, and 3) shown that the specific NADPH oxidase inhibitor, apocynin, and non-specific NADPH oxidase inhibitors, NDGA and SKF525A, suppress H2O2 production increases in high LDL-perturbed EC. In the present study, the cytoskeletal structure of EC exposed to 330 mg/dl LDL-cholesterol, and incubated with or without apocynin, NDGA and SKF525A, was examined. Each of these compounds promoted the retention of dense peripheral bands and minimized stress fiber formation. These findings are consistent with NADPH oxidase and it's reactive oxygen species byproducts modulating the cytoskeleton reorganization observed in high LDL-induced EC perturbation.

An important 2'-OH group for an RNA-protein interaction.

We have investigated the role of 2'-OH groups in the specific interaction between the acceptor stem of Escherichia coli tRNA(Cys) and cysteine-tRNA synthetase. This interaction provides for the high aminoacylation specificity observed for cysteine-tRNA synthetase. A synthetic RNA microhelix that recapitulates the sequence of the acceptor stem was used as a substrate and variants containing systematic replacement of the 2'-OH by 2'-deoxy or 2'-O-methyl groups were tested. Except for position U73, all substitutions had little effect on aminoacylation. Interestingly, the deoxy substitution at position U73 had no effect on aminoacylation, but the 2'-O-methyl substitution decreased aminoacylation by 10-fold and addition of the even bulkier 2'-O-propyl group decreased aminoacylation by another 2-fold. The lack of an effect by the deoxy substitution suggests that the hydrogen bonding potential of the 2'-OH at position U73 is unimportant for aminoacylation. The decrease in activity upon alkyl substitution suggests that the 2'-OH group instead provides a monitor of the steric environment during the RNA-synthetase interaction. The steric role was confirmed in the context of a reconstituted tRNA and is consistent with the observation that the U73 base is the single most important determinant for aminoacylation and therefore is a site that is likely to be in close contact with cysteine-tRNA synthetase. A steric role is supported by an NMR-based structural model of the acceptor stem, together with biochemical studies of a closely related microhelix. This role suggests that the U73 binding site for cysteine-tRNA synthetase is sterically optimized to accommodate a 2'-OH group in the backbone, but that the hydroxyl group itself is not involved in specific hydrogen bonding interactions.

Endothelial cell oxidant production: effect of NADPH oxidase inhibitors.

The effects of known leukocyte NADPH oxidase inhibitors on general cellular oxidant production in cultured human endothelial cells (EC) has been investigated. EC were stimulated with 10 nM phorbol 12-myristate 13-acetate and cellular oxidant production measured in the presence and absence of inhibitors that act on various substituents of the oxidase complex and its activation pathways. The effects of the cytosolic oxidase subunit translocation inhibitors, catechols (3,4-dihydroxybenzaldehyde, caffeic acid, and protocatechuic acid), ortho-methoxy-substituted catechols (apocynin, vanillin, and 4-nitroguaiacol), and quinone, 1,4-naphthoquinone; flavoprotein inhibitors, diphenylene iodonium and quinacrine; haem ligands, imidazole and pyridine; directly acting thiol reagents, disulfiram and penicillamine; NADPH analogue, Cibacron Blue; redox active inhibitors, quercetin and esculetin; intracellular calcium antagonist, TMB-8; and calmodulin antagonists, W-7 and trifluoperazine, were determined. All compounds reduced oxidant production in stimulated EC. These findings add to previous observations suggesting the presence of a functionally active NADPH oxidase in EC. Identifying the major cellular reactive oxygen species source in perturbed EC will provide new insights into our understanding of endothelial dysfunction, which has been hypothesized to be a major contributing factor in the pathogenesis of atherosclerosis.

Identification of a functional leukocyte-type NADPH oxidase in human endothelial cells :a potential atherogenic source of reactive oxygen species.

Cultured human endothelial cells (EC) exposed to atherogenic low-density lipoprotein levels have increased reactive oxygen species (ROS) generation. The enzyme responsible for this ROS production elevation is unknown. We have examined for the presence of a functional leukocyte-type NADPH oxidase in EC to elucidate whether this enzyme could be the ROS source. The plasma membrane fraction of disrupted EC showed a reduced-minus-oxidized difference spectra with absorption peaks identical to those observed in the spectra of the leukocyte NADPH oxidase component, cytochrome b558. Western-blot analysis, using anti-gp91 -phox. anti -p22-phox. anti -p47-phox. and anti -p67-phox antibodies, demonstrated the protein expression of NADPH oxidase subunits in EC. Reverse transcriptase-polymerase chain reaction (RT-PCR) showed the mRNA expression of gp91-phox, p22-phox, p47-phox, and p67-phox in EC. Sonicates from unstimulated EC produced no measurable superoxide; whereas, exogenously applied arachidonic acid activated superoxide generation in a manner that was dependent upon the presence of NADPH and both membrane and cytosolic fractions combined. Apocynin, a specific leukocyte NADPH oxidase inhibitor, was shown by Western-blot analysis of membrane and cytoplasmic fractions to inhibit the translocation of p47-phox to the membrane of stimulated EC. These findings support the presence of a functionally active leukocyte-type NADPH oxidase in EC. NADPH oxidase could be the major cellular ROS source in EC perturbation, which has been hypothesized to be a major contributing factor in the pathogenesis of atherosclerosis.

An examination of coaxial stacking of helical stems in a pseudoknot motif: the gene 32 messenger RNA pseudoknot of bacteriophage T2.

The RNA pseudoknot located at the 5' end of the gene 32 messenger RNA of bacteriophage T2 contains two A-form helical stems connected by two loops, in an H-type pseudoknot topology. A combination of multidimensional NMR methods and isotope labeling were used to investigate the pseudoknot structure, resulting in a more detailed structural model than provided by earlier homonuclear NMR studies. Of particular significance, the interface between the stacked helical stems within the pseudoknot motif is described in detail. The two stems are stacked in a coaxial manner, with an approximately 18 degrees rotation of stem1 relative to stem2 about an axis that is parallel to the helical axis. This rotation serves to relieve what would otherwise be a relatively close phosphate-phosphate contact at the junction of the two stems, while preserving the stabilizing effects of base stacking. The ability of the NMR data to determine pseudoknot bending was critically assessed. The data were found to be a modestly precise indicator of pseudoknot bending, with the angle between the helical axes of stem1 and stem2 being in the range of 15+/-15 degrees. Pseudoknot models with bend angles within this range are equally consistent with the data, since they differ by only small amounts in the relatively short-range interproton distances from which the structure was derived. The gene 32 messenger RNA pseudoknot was compared with other RNA structures with coaxial or near-coaxial stacked helical stems.

Embalming fluid-soaked marijuana: new high or new guise for PCP?

A growing trend of smoking marijuana soaked in what is purported to be embalming fluid has been reported in the literature since the mid-1980s. This article describes several cases of intoxication, gives regional epidemiological data on this phenomenon, and includes current nomenclature. The authors also analyze a sample of fluid said to be embalming fluid and discover PCP (phencyclidine) and multiple congeners and by-products of PCP manufacture. The implications of this finding are discussed, and the hypothesis that most embalming fluid-soaked marijuana likely contains PCP is considered.

Thrombin stimulated reactive oxygen species production in cultured human endothelial cells.

In order to study the major cellular source of reactive oxygen species (ROS) in perturbed human endothelial cells (EC), the effect of thrombin, a phospholipase A2 activator, on cultured EC ROS generation has been investigated. EC were incubated with 0.1-1 unit/ml thrombin and cellular superoxide anion (O(-)2) release and hydrogen peroxide (H2O2) production measured. Thrombin exposure caused an elevation in EC O(-)2 release and H2O2 production. The effects of protein kinase C, arachidonic acid metabolism, NADPH oxidase, and phospholipase A2 inhibitors on thrombin-induced EC H2O2 production were examined. EC were exposed to 0.5 unit/ml thrombin and cellular H2O2 production measured in the presence and absence of the protein kinase C inhibitor, H-7; arachidonic acid metabolism inhibitors, indomethacin, nordihydroguaiaretic acid, and SKF525A; NADPH oxidase inhibitor, apocynin; and phospholipase A2 inhibitor, 4-bromophenacyl bromide. All inhibitors, with the exception of H-7 and indomethacin, suppressed thrombin-induced EC H2O2 production. The pattern of effects of these metabolic antagonists on thrombin-induced EC ROS production is similar to that previously reported on ROS production in EC exposed to high low-density lipoprotein levels, and in stimulated leukocytes. These findings further implicate NADPH oxidase as a major ROS source in EC.

Base-pairings within the RNA pseudoknot associated with the simian retrovirus-1 gag-pro frameshift site.

Frameshift and readthrough sites within retroviral messenger RNAs are often followed by nucleotide sequences that have the potential to form pseudoknot structures. In the work presented here, NMR methods were used to characterize the base-pairings and structural features of the RNA pseudoknot downstream of the gag-pro frameshift site of simian retrovirus type-1 (SRV-1) and a functional mutant of the SRV-1 pseudoknot. Evidence is presented that these pseudoknots contain two A-form helical stems of six base-pairs each, connected by two loops, in a classic H-type pseudoknot topology. A particularly interesting feature is that the shorter of the two connecting loops, loop 1, consists of only a single adenosine nucleotide that spans the major groove of stem 2. In this respect, the frameshift-associated pseudoknots are structurally similar to the pseudoknot within the gene 32 mRNA of bacteriophage T2, previously characterized by NMR methods. Despite having similar nucleotide sequences, the solvent exchange rates of the imino protons at the junction of the helical stems in the wild-type and mutant frameshifting pseudoknots differ from each other and from the bacteriophage T2 pseudoknot. The implications of this finding are discussed.

Low-density lipoprotein stimulated peroxide production and endocytosis in cultured human endothelial cells: mechanisms of action.

The effects of arachidonic acid metabolism and NADPH oxidase inhibitor on the hydrogen peroxide (H2O2) generation and endocytotic activity of cultured human endothelial cells (EC) exposed to atherogenic low-density lipoprotein (LDL) levels have been investigated. EC were incubated with 240 mg/dl LDL cholesterol and cellular H2O2 production and endocytotic activity measured in the presence and absence of the arachidonic acid metabolism inhibitors, indomethacin, nordihydroguaiaretic acid, and SKF525A, and NADPH oxidase inhibitor, apocynin. All inhibitors, with the exception of indomethacin, markedly reduced high LDL-induced increases in EC H2O2 generation and endocytotic activity. EC exposed to exogenously applied arachidonic acid had cellular functional changes similar to those induced by high LDL concentrations. EC incubated with 1-25 uM arachidonic acid had increased H2O2 production and heightened endocytotic activity. Likewise, EC pre-loaded with [3H]arachidonic acid when exposed to increasing LDL levels (90-330 mg/dl cholesterol) had a dose-dependent rise in cytosolic [3H]arachidonic acid. The phospholipase A2 inhibitors, 4-bromophenacyl bromide and 7,7-dimethyleicosadienoic acid, markedly inhibited H2O2 production in EC exposed to 240 mg/dl LDL cholesterol. These findings suggest that arachidonic acid contributes mechanistically to high LDL-perturbed EC H2O2 generation and heightened endocytosis. Such cellular functional changes add to our understanding of endothelial perturbation, which has been hypothesized to be a major contributing factor in the pathogenesis of atherosclerosis.

Structural features and stability of an RNA triple helix in solution.

A 30 nt RNA with a sequence designed to form an intramolecular triple helix was analyzed by one-and two-dimensional NMR spectroscopy and UV absorption measurements. NMR data show that the RNA contains seven pyrimidine-purine-pyrimidine base triples stabilized by Watson-Crick and Hoogsteen interactions. The temperature dependence of the imino proton resonances, as well as UV absorption data, indicate that the triple helix is highly stable at acidic pH, melting in a single sharp transition centered at 62 degrees C at pH 4.3. The Watson-Crick and Hoogsteen pairings are disrupted simultaneously upon melting. The NMR data are consistent with a structural model where the Watson-Crick paired strands form an A-helix. Results of model building, guided by NMR data, suggest a possible hydrogen bond between the 2' hydroxyl proton of the Hoogsteen strand and a phosphate oxygen of the purine strand. The structural model is discussed in terms of its ability to account for some of the differences in stability reported for RNA and DNA triple helices and provides insight into features that are likely to be important in the design of RNA binding compounds.

Atherogenic levels of low-density lipoprotein increase hydrogen peroxide generation in cultured human endothelial cells: possible mechanism of heightened endocytosis.

Cultured human umbilical vein endothelial cells(EC) exposed to atherogenic low-density lipoprotein (LDL) levels have augmented reactive oxygen species generation. Confluent EC were incubated with 30-330 mg/dl LDL cholesterol and cellular hydrogen peroxide (H2O2) generation measured. EC incubated with 30 and 90 mg/dl LDL cholesterol showed similar low level H2O2 production. In contrast, EC exposed to 180 and 330 mg/dl LDL cholesterol have a marked, dose-related elevation in H2O2 generation. Subsequent studies have explored if direct EC exposure to H2O2 promotes cellular functional changes similar to those induced by high LDL levels (> 160 mg/dl cholesterol). Confluent EC were incubated with 0.1-10 mM H2O2 for 30 minutes and endocytosis measured and cytoskeletal structure examined. H2O2 exposure (0.5 and 1 mM) promoted heightened EC endocytosis, which similarly occurs with high LDL exposure. Likewise, cytoskeletal examination of EC perturbed with 1 mM H2O2 reveals structural remodeling with a marked increase in stress fibers, which similarly happens with high LDL levels. The above observations that high LDL levels cause increased EC H2O2 production, and direct H2O2 exposure promotes cellular functional changes similar to those induced by high LDL concentrations, suggest a modulatory role for reactive oxygen species. Thus LDL-induced reactive oxygen species generation may contribute mechanistically to endothelial perturbation, which has been hypothesized to be a major contributing factor in the pathogenesis of atherosclerosis.

Nursing care of a child with meningococcemia.

This case presentation will discuss the pathophysiology of a child in septic shock due to Neisseria meningitidis. The most prevalent nursing care concerns of this case encountered during the pediatric intensive care unit (PICU) and during the general floor stay will be addressed. The nursing skill required for identifying problems and planning care that clearly fall under the nursing domain also will be covered. In addition, the complexities of this case demonstrate that collaboration between the PICU nurse and the general pediatric nurse is imperative for successful patient outcome. A.W. was a 5 1/2-month-old infant transported to our PICU from a referral hospital in the state. Diagnosis at time of admission was meningococcemia, disseminated intravascular coagulopathy, septic shock, respiratory failure, and purpura fulminans. There was a 2- to 3-day history of a runny nose, cough, and vomiting. On the day of admission, A.W. had three seizures and developed a fever and a purpuric rash.

Atherogenic levels of low-density lipoprotein increase endocytotic activity in cultured human endothelial cells.

Cultured human umbilical vein endothelial cells (EC) exposed to atherogenic low-density lipoprotein (LDL) levels for protracted periods demonstrated heightened endocytosis. Confluent EC were incubated with LDL 90 to 240 mg/dl cholesterol for 1 to 4 days and endocytosis was measured by 14C-sucrose uptake. Control EC and cells incubated with 90 mg/dl LDL cholesterol showed similar uptakes of 14C-sucrose during all measured time periods. In contrast, EC exposed to 240 mg/dl LDL cholesterol showed an increase in endocytosis beginning at 2 days, whereas 160 mg/dl LDL cholesterol promoted increased uptake by 4 days. The endocytotic activity of LDL-perturbed EC is reduced to levels seen in control cells by cytochalasin B, an actin polymerization inhibitor. This finding suggests a modulatory role for the cytoskeleton in endocytosis changes. Examination of LDL-perturbed EC cytoskeleton reveals structural remodeling resulting in a marked increase in stress fibers. Cytochalasin B exposure causes a loss of stress fibers with the formation of globular filamental aggregates. Such LDL-induced cellular functional changes may contribute mechanistically to endothelial dysfunction, which is widely held to be a major contributing factor in the pathogenesis of atherosclerosis.

Native low density lipoprotein. Endothelial cell recruitment of mononuclear cells.

The effect of native low density lipoprotein (LDL) on human umbilical vein endothelial cell (EC) recruitment of mononuclear cells (Monos) was investigated. ECs were exposed to LDL at atherogenic concentrations (240 mg cholesterol [Chol]/dl) for as long as 4 days (LDL-treated ECs). LDL-treated ECs bound substantially greater amounts of freshly isolated human monocytes and U937 cells than did control ECs. The enhanced Mono binding was time and LDL concentration dependent. LDL-induced binding was reduced to control levels when cycloheximide was added together with LDL, indicating that de novo protein synthesis was required. Furthermore, this LDL effect was not a general feature of apolipoproteins, as high density lipoprotein in physiologically relevant concentrations (45 mg Chol/dl, 4 days) had no effect on EC-Mono binding. Conditioned media from LDL-treated EC cultures did not increase EC binding of Monos. In contrast, minimally modified LDL increased EC-Mono binding more than eightfold. In conclusion, LDL in concentrations associated with the premature development of atherosclerosis increased EC affinity for Monos. Such LDL-induced alterations in EC physiology likely represent a proinflammatory response and an early step in atherogenesis.

Bradykinin induces superoxide anion release from human endothelial cells.

The time-dependent release of superoxide anion (O2-) from bradykinin (Bk)-stimulated human umbilical vein endothelial cells (EC) was measured as the superoxide dismutase-inhibitable reduction of ferricytochrome C employing a novel application of microspectrophotometry. In the absence of Bk, O2- release by EC was not detectable. EC exposure to Bk (10(-6) to 10(-5) M) resulted in a rapid release of O2-. The release of O2- occurred within 5 minutes of exposure. O2- release was partially inhibited by indomethacin (63 +/- 6%), thus suggesting that arachidonic acid metabolism, through cyclooxygenase, contributes to EC O2- production. EC O2- release may be an important component in the pathophysiologic actions of Bk on vascular function.