Search PubMed⌕ Search

Biomedical subjects

R W Alexander

Publications and source records attributed to R W Alexander.

At least 37 records · Page 2Linked to original sources
PubMed1998

Activation of microhelix charging by localized helix destabilization.

We report that aminoacylation of minimal RNA helical substrates is enhanced by mismatched or unpaired nucleotides at the first position in the helix. Previously, we demonstrated that the class I methionyl-tRNA synthetase aminoacylates RNA microhelices based on the acceptor stem of initiator and elongator tRNAs with greatly reduced efficiency relative to full-length tRNA substrates. The cocrystal structure of the class I glutaminyl-tRNA synthetase with tRNAGln revealed an uncoupling of the first (1.72) base pair of tRNAGln, and tRNAMet was proposed by others to have a similar base-pair uncoupling when bound to methionyl-tRNA synthetase. Because the anticodon is important for efficient charging of methionine tRNA, we thought that 1.72 distortion is probably effected by the synthetase-anticodon interaction. Small RNA substrates (minihelices, microhelices, and duplexes) are devoid of the anticodon triplet and may, therefore, be inefficiently aminoacylated because of a lack of anticodon-triggered acceptor stem distortion. To test this hypothesis, we constructed microhelices that vary in their ability to form a 1.72 base pair. The results of kinetic assays show that microhelix aminoacylation is activated by destabilization of this terminal base pair. The largest effect is seen when one of the two nucleotides of the pair is completely deleted. Activation of aminoacylation is also seen with the analogous deletion in a minihelix substrate for the closely related isoleucine enzyme. Thus, for at least the methionine and isoleucine systems, a built-in helix destabilization compensates in part for the lack of presumptive anticodon-induced acceptor stem distortion.

Amino Acyl-tRNA Synthetases↗
PubMed1998

Temporal dispersion of activation of phospholipase C-beta1 and -gamma isoforms by angiotensin II in vascular smooth muscle cells. Role of alphaq/11, alpha12, and beta gamma G protein subunits.

Activation of phospholipase C (PLC) is one of the earliest events in angiotensin II (Ang II) type 1 (AT1) receptor (R)-mediated signal transduction in vascular smooth muscle cells (VSMCs). The coupling mechanisms of AT1 Rs to PLC, however, are controversial, because both tyrosine phosphorylation of PLC-gamma and G protein-dependent PLC-beta activation pathways have been reported. The expression of PLC-beta1, furthermore, has not been consistently demonstrated in VSMCs. Here we identified the PLC subtypes and subunits of heterotrimeric G proteins involved in AT1 R-PLC coupling using cultured rat VSMCs. Western analysis revealed the expression of PLC-beta1, -gamma1, and -delta1 in VSMCs. Ang II-stimulated inositol trisphosphate (IP3) formation measured at 15 s, which corresponds to the peak response, was significantly inhibited by electroporation of antibodies against PLC-beta1, but not by anti-PLC-gamma and -delta antibodies. Electroporation of anti-Galphaq/11 and -Galpha12 antibodies also showed significant inhibition of the Ang II-induced IP3 generation at 15 s, while anti-Galphai and Galpha13 antibodies were ineffective. Furthermore, in VSMCs electroporated with anti-Gbeta antibody and cells stably transfected with the plasmid encoding the Gbetagamma-binding region of the carboxyl terminus of beta-adrenergic receptor kinase1, the peak Ang II-stimulated PLC activity (at 15 s) was significantly inhibited. The tyrosine kinase inhibitor, genistein, had no effect on the peak response to Ang II stimulation, but significantly inhibited IP3 production after 30 s, a time period which temporally correlated with PLC-gamma tyrosine phosphorylation in response to Ang II. Moreover, electropor-ation of anti-PLC-gamma antibody markedly inhibited the IP3 production measured at 30 s, indicating that tyrosine phosphorylation of PLC-gamma contributes mainly to the later phase of PLC activation. Thus, these results suggest that: 1) AT1 receptors sequentially couple to PLC-beta1 via a heterotrimeric G protein and to PLC-gamma via a downstream tyrosine kinase; 2) the initial AT1 receptor-PLC-beta1 coupling is mediated by Galphaq/11beta gamma and Galpha12 beta gamma; 3) Gbeta gamma acts as a signal transducer for activation of PLC in VSMCs. The sequential coupling of AT1 receptors to PLC-beta1 and PLC-gamma, as well as dual coupling of AT1 receptors to distinct Galpha proteins, suggests a novel mechanism for a temporally controlled, highly organized and convergent Ang II-signaling network in VSMCs.

Angiotensin II↗
PubMed1998

G-Protein binding domains of the angiotensin II AT1A receptors mapped with synthetic peptides selected from the receptor sequence.

The vascular angiotensin II type 1 receptor (AT1AR) is a member of the G-protein-coupled receptor superfamily. We mapped the G-protein binding domains of the AT1AR using synthetic peptides selected from the receptor sequence, which interfere with AT1AR-G-protein coupling. Membrane GTPase activity was used as a measure of the functional coupling in rat vascular smooth muscle cells. Peptides corresponding to the N-terminal region of the second intracellular loop (residues 125-137), the N-terminal region of the third intracellular loop (217-227) and the juxtamembranous region of the C-terminal tail (304-316) inhibited angiotensin II-induced GTPase activation by 30%, 30%, and 70%, respectively. The latter two domains (217-227 and 304-316) are predicted to form amphiphilic alpha-helices. Only the peptide representing residues 217-227 stimulated basal activity (45%). No synthetic peptide had a significant effect on either the number or the affinity of the AT1AR binding. These observations indicate that domains of the second and third regions and the cytoplasmic tail of the AT1AR interact with G-proteins, and that multiple contacts with these receptor domains may be important for binding and activation of the G-proteins.

Amino Acid Sequence↗
PubMed1998

p38 Mitogen-activated protein kinase is a critical component of the redox-sensitive signaling pathways activated by angiotensin II. Role in vascular smooth muscle cell hypertrophy.

Angiotensin II induces an oxidant stress-dependent hypertrophy in cultured vascular smooth muscle cells. To investigate the growth-related molecular targets of H2O2, we examined the redox sensitivity of agonist-stimulated activation of the mitogen-activated protein kinase (MAPK) family. We show here that angiotensin II elicits a rapid increase in intracellular H2O2 and a rapid and robust phosphorylation of both p42/44MAPK (16-fold) and p38MAPK (15-fold). However, exogenous H2O2 activates only p38MAPK (14-fold), and diphenylene iodonium, an NADH/NADPH oxidase inhibitor, attenuates angiotensin II-stimulated phosphorylation of p38MAPK, but not p42/44MAPK. Furthermore, in cells stably transfected with human catalase, angiotensin II-induced intracellular H2O2 generation is almost completely blocked, resulting in inhibition of phosphorylation of p38MAPK, but not p42/44MAPK, and a subsequent partial decrease in angiotensin II-induced hypertrophy. Specific inhibition of either the p38MAPK pathway with SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H- imidaz ole) or the p42/44MAPK pathway with PD98059 (2-(2'-amino-3'-methoxyphenyl)oxanaphthalen-4-one) also partially, but significantly, attenuates angiotensin II-induced hypertrophy; however, simultaneous blockade of both pathways has an additive inhibitory effect, indicating that the hypertrophic response to angiotensin II requires parallel, independent activation of both MAPK pathways. These results provide the first evidence that p38MAPK is a critical component of the oxidant stress (H2O2)-sensitive signaling pathways activated by angiotensin II in vascular smooth muscle cells and indicate that it plays a crucial role in vascular hypertrophy.

Angiotensin II↗
PubMed1998

Oscillatory and steady laminar shear stress differentially affect human endothelial redox state: role of a superoxide-producing NADH oxidase.

Atherosclerotic lesions are found opposite vascular flow dividers at sites of low shear stress and oscillatory flow. Since endothelial proinflammatory genes prominent in lesions are regulated by oxidation-sensitive transcriptional control mechanisms, we examined the redox state of cultured human umbilical vein endothelial cells after either oscillatory or steady laminar fluid shear stress. Endothelial oxidative stress was assessed by measuring activity of the superoxide (O2.- )-producing NADH oxidase (a major source of reactive oxygen species in vascular cells), intracellular O2.- levels, induction of the redox-sensitive gene heme oxygenase-1 (HO-1), and abundance of Cu/Zn superoxide dismutase (Cu/Zn SOD), an antioxidant defense enzyme whose level of expression adapts to changes in oxidative stress. When cells were exposed to oscillatory shear (+/-5 dyne/cm2, 1 Hz) for 1, 5, and 24 hours, NADH oxidase activity and the amount of HO-1 progressively increased up to 174+/-16% (P<0.05) and 505+/-111% (P<0.05) versus static conditions, respectively, whereas levels of Cu/Zn SOD remained unchanged. This upregulation of HO-1 was completely blocked by the antioxidant N-acetylcysteine (NAC, 20 mmol/L). In contrast, steady laminar shear (5 dyne/cm2) induced NADH oxidase activity and NAC-sensitive HO-1 mRNA expression only at 1 and 5 hours, a transient response that returned toward baseline at 24 hours. Levels of Cu/Zn SOD mRNA and protein were increased after 24 hours of steady laminar shear. Furthermore, intracellular O2.-, as measured by dihydroethidium fluorescence, was higher in cells exposed to oscillatory than to laminar shear. These data are consistent with the hypothesis that continuous oscillatory shear causes a sustained activation of pro-oxidant processes resulting in redox-sensitive gene expression in human endothelial cells. Steady laminar shear stress initially activates these processes but appears to induce compensatory antioxidant defenses. We speculate that differences in endothelial redox state, orchestrated by different regimens of shear stress, may contribute to the focal nature of atherosclerosis.

Arteriosclerosis↗
PubMed1998

Oscillatory shear stress stimulates adhesion molecule expression in cultured human endothelium.

Low and oscillatory shear stresses are major features of the hemodynamic environment of sites opposite arterial flow dividers that are predisposed to atherosclerosis. Atherosclerosis is a focal inflammatory disease characterized initially by the recruitment of mononuclear cells into the arterial wall. The specific characteristics of the hemodynamic environment that facilitate the generation of arterial inflammatory responses in the presence of, for example, hyperlipidemia are unknown. We show here that prolonged oscillatory shear stress induces expression of endothelial cell leukocyte adhesion molecules, which are centrally important in mediating leukocyte localization into the arterial wall. Vascular cell adhesion molecule-1 was upregulated an average 9-fold relative to endothelial monolayers in static culture. Intercellular adhesion molecule-1 and E-selectin exhibited 11-fold and 7.5-fold increases, respectively. Upregulation of these adhesion molecules was associated with enhanced monocyte adherence. Cytokine stimulation of surface vascular cell adhesion molecule-1 was maximally induced after 6 and 8 hours of cytokine incubation. Oscillatory shear stress for these time periods elicited respective vascular cell adhesion molecule-1 levels of 16% and 30% relative to those observed for cytokine stimulation. Surface intercellular adhesion molecule-1 induction by cytokine stimulation for 24 hours was found to be approximately five times the level detected after 24 hours of oscillatory shear stress. Experiments performed in the presence of the antioxidant N-acetylcysteine demonstrated that the expression of vascular cell adhesion molecule-1 could be almost totally abolished, whereas that of intercellular adhesion molecule-1 was typically reduced by approximately 70%. These results imply that oscillatory shear stress per se is sufficient to stimulate mononuclear leukocyte adhesion and, presumptively, migration into the arterial wall. These results further indicate that atherosclerotic lesion initiation is likely related, at least in part, to unique signals generated by oscillatory shear stress and that the mechanism of upregulation is, to some extent, redox sensitive.

Acetylcysteine↗
PubMed1998

Ribosomal proteins neighboring 23 S rRNA nucleotides 803-811 within the 50 S subunit.

We report the synthesis of a radioactive, photolabile oligodeoxyribonucleotide probe and its exploitation in identifying 50 S ribosomal subunit components neighboring its target site, nucleotides 803-811 in 23 S rRNA. Photolysis of the complex formed between the probe and 50 S subunits leads to site-specific probe photoincorporation into proteins L15, L17, and L20, labeled to greater extents, and L13 and L21, labeled to lesser extents. Portions of each of these proteins thus lie within 23 A of nucleotide U803. These results lead us to conclude that nucleotides 803-811 fall on the side of the L13-L17-L20-L21 protein cluster [Walleczek et al. (1988) EMBO J. 7, 3571-3576] that points from the back of the 50 S particle toward the peptidyl transferase center within the 50 S subunit. Such placement is consistent with the observation that an oligoDNA probe directed to nucleotides 803-811 decreases P-site binding of tRNA [Hill et al. (1990) Biochim. Biophys. Acta 1050, 45-50].

Affinity Labels↗
PubMed1998

Tumour necrosis factor alpha activates a p22phox-based NADH oxidase in vascular smooth muscle.

Increasing experimental evidence suggests that non-phagocytic cells express a potent superoxide (O2-.)-producing NADH oxidase that might be related to the phagocytic NADPH oxidase. Here we show that the cytokine tumour necrosis factor alpha (TNF-alpha) activates, in a time- and dose-dependent manner, a O2-.-producing NADH oxidase in cultured rat aortic smooth-muscle cells. Dose-response experiments for NADH showed an upward shift of the curve for TNF-alpha-treated cells, suggesting that TNF-alpha increased the amount of available enzyme. Using the anti-sense transfection technique, we further demonstrate that the molecular identity of this oxidase includes p22(phox) (the alpha subunit of cytochrome b558 and part of the electron transfer component of the phagocytic NADPH oxidase), which we recently cloned from a rat vascular smooth-muscle cell cDNA library. In addition, prolonged treatment with TNF-alpha increased p22phox mRNA expression without affecting p22phox mRNA stability, and only when transcriptional activity was intact. These findings identify a p22phox-containing NADH oxidase as a source for cytokine-induced free radical production in vascular smooth-muscle cells and clarify some of the mechanisms involved in the regulation of vascular oxidase activity.

Animals↗
PubMed1998

Sexual and physical abuse in women with fibromyalgia: association with outpatient health care utilization and pain medication usage.

OBJECTIVE: To evaluate the relationship between sexual and/or physical abuse and health care usage in patients with fibromyalgia (FM) and identify variables that may influence this relationship. METHODS: We assessed history of sexual/physical abuse, health care utilization, and medication usage, as well as related variables in 75 women with FM using standardized questionnaires, structured interviews, and laboratory pain perception tasks. RESULTS: Fifty-seven percent of FM patients reported a history of sexual/physical abuse. Compared to non-abused patients, abused patients reported significantly greater utilization of outpatient health care services for problems other than FM and greater use of medications for pain (P < or = 0.025). Consistent with our expectations, abused patients also were characterized by significantly greater pain, fatigue, functional disability, and stress, as well as by a tendency to label dolorimeter stimuli as painful regardless of their intensities (P < or = 0.05). Additional analyses suggested that the high frequency of sexual/physical abuse in our patients was associated primarily with seeking health care for chronic pain rather than the FM syndrome itself or genetic factors. CONCLUSION: There is an association in FM patients between sexual/physical abuse and increased use of outpatient health care services and medications for pain. This association may be influenced by clinical symptoms, functional disability, psychiatric disorders, stress, and abnormal pain perception. The relationships among these variables should be further tested in prospective, population-based studies.

Adolescent↗
PubMed1998

Determinants of cognitive performance in systemic lupus erythematosus.

Patients with systemic lupus erythematosus (SLE) may have a variety of neuropsychiatric syndromes. Assessment of cognitive functioning for these patients is complicated by increased prevalence and disease severity among groups obtained lower scores on measures of cognitive functioning in normative national samples. Cognitive ability was quantified in a diverse cohort of patients with SLE and a demographically matched group of control participants. Hierarchical regression demonstrated a small increase (6%) in explained variation in cognitive functioning when presence of SLE was added to the equation derived from demographic variables. No significant interaction was found between race and disease. These results suggest that increased frequency of cognitive impairment in African Americans with SLE is due to the additive effects of psychosocial variables.

Adult↗
PubMed1998

The study of the influence of flow on vascular endothelial biology.

It is now recognized that the mechanical environment of a cell has an influence on its structure and function. For the vascular endothelial cell that resides at the interface of the flowing blood and the underlying vessel wall, there is mounting evidence of the importance of flow and the associated wall shear stress in the regulation of endothelial biology. Not only is it a sensitive regulator of endothelial structure and function, but different flow environments will influence endothelial cell biology differently. Furthermore, there may be an interaction between the chemical environment of a cell and its mechanical environment. This is illustrated by the inhibition by steady laminar shear stress of the cytokine induction of VCAM-1. Results also are presented in which flow studies have been conducted using a co-culture model of the vessel wall. These experiments provide evidence of a quiescent endothelium; however, much more needs to be done to engineer the cell culture environment to make it more physiologic.

Animals↗
PubMed1998

Angiotensin II type 1 receptor: relationship with caveolae and caveolin after initial agonist stimulation.

Caveolae are membrane domains that have been implicated in signal transduction, and caveolins are major structural components of these domains. We found that all reported caveolin isoforms (caveolin-1, -2, and -3) were expressed in vascular smooth muscle cells (VSMCs); however, only caveolin-1 mRNA was regulated by angiotensin II (Ang II). Ang II (100 nmol/L) increased caveolin-1 mRNA, with a peak at 2 hours (193+/-6% of control, P<0.01, n=4). In contrast, Ang II significantly decreased caveolin-1 protein, with a nadir at 4 hours (64+/-5% of control, P<0.01, n=6). [35S]Methionine labeling showed that Ang II increased caveolin biosynthesis (226+/-33% of control labeling at 4 hours), suggesting that the transient decrease in caveolin protein levels is due to increased degradation. When cells were fractionated with sucrose, on agonist stimulation, AT1 receptors appeared in fraction 5 where caveolin was fractionated. This migration was blocked by low temperature and treatment with phenylarsine oxide, interventions that interfere with agonist-induced Ang II type 1 (AT1) receptor sequestration and tonic phase signaling. In addition, caveolin-1 coimmunoprecipitates with AT1 receptor only on agonist stimulation. These data support the concept that the caveola is a specialized signaling domain in VSMCs that can be dynamically accessed by the AT1 receptor. Because of the signaling and coupling proteins that are localized in caveolae and because of evidence that these proteins may interact directly with caveolin, caveola-AT1 receptor interaction likely represents an important focus for dynamic control of receptor signaling in VSMCs.

Angiotensin II↗
PubMed1998

Atherosclerosis as disease of redox-sensitive genes.

Accumulating evidence provides a compelling case that one of the major pathophysiologic mechanisms involved in the pathogenesis of atherosclerosis is enhanced oxidative stress and that the most important manifestation of this altered redox state is the modulation of a set(s) of proinflammatory genes that are regulated directly or indirectly by reactive oxygen species. Viewed in this perspective the oxidation of LDL is but one important consequence of a generalized metabolic abnormality of the arterial wall in atherosclerosis rather than being the core pathophysiological feature. The fact that hypercholesterolemia, hypertension, and AGEs related to diabetes mellitus all activate similar redox-sensitive proinflammatory genes associated with the pathogenesis of atherosclerosis provides the potential for the development of unifying concepts concerning the etiology of the disease. These concepts also provide additional evidence that antioxidants may be potentially attractive therapeutic agents.

Animals↗
PubMed1997

G protein-coupled receptor kinase 5 in cultured vascular smooth muscle cells and rat aorta. Regulation by angiotensin II and hypertension.

GRK5, a recently cloned member of the G protein-coupled receptor kinase family, has been shown to phosphorylate and participate in the desensitization of angiotensin II (Ang II) type 1A (AT1A) receptors. In this study, the effect of angiotensin II on GRK5 expression was examined in cultured vascular smooth muscle cells and aortas of Ang II-infused hypertensive rats. In vascular smooth muscle cells, Ang II (100 nM) up-regulated GRK5 mRNA as early as 1 h, with a peak at 16 h. This up-regulation was dose- and calcium-dependent. The increase in GRK5 mRNA was reflected in a smaller increase in protein expression, which nonetheless had functional significance since AT1 receptor phosphorylation was increased and phospholipase C activation was decreased following prolonged incubation with Ang II. In aortas of Ang II-infused hypertensive rats, both GRK5 mRNA and protein levels increased approximately 3-fold compared with sham-operated rats at 5 and 7 days, respectively. This up-regulation was blocked either by losartan or by the nonspecific vasodilator hydralazine. Since a subpressor dose of Ang II did not increase GRK5 mRNA levels and norepinephrine infusion also increased GRK5 mRNA expression, we conclude that Ang II-induced GRK5 up-regulation in rat aortas may be due to hypertension per se. Hormone- and hemodynamic stress-induced GRK5 regulation may provide a novel molecular basis for long-term regulation of agonist sensitivity of vascular cells.

Angiotensin II↗
PubMed1997

Placement of the alpha-sarcin loop within the 50S subunit: evidence derived using a photolabile oligodeoxynucleotide probe.

We report the synthesis of a radioactive, photolabile oligodeoxyribonucleotide probe and its exploitation in identifying 50S ribosomal subunit components neighboring the alpha-sarcin loop. The probe is complementary to 23S rRNA nt 2653-2674. Photolysis of the complex formed between the probe and 50S subunits leads to site-specific probe photoincorporation into proteins L2, the most highly labeled protein, L1, L15, L16 and L27, labeled to intermediate extents, and L5, L9, L17 and L24, each labeled to a minor extent. Portions of each of these proteins thus lie within 23 A of nt U2653. These results lead us to conclude that the alpha-sarcin loop is located at the base of the L1 projection within the 50S subunit. Such placement, near the peptidyl transferase center, provides a rationale for the extreme sensitivity of ribosomal function to cleavage of the alpha-sarcin loop.

Affinity Labels↗
PubMed1997

Angiotensin II-induced hypertension increases heme oxygenase-1 expression in rat aorta.

BACKGROUND: We investigated the in vivo effects of angiotensin (Ang) II-induced hypertension on heme oxygenase (HO) mRNA and protein expression, activity, and localization in rat aortas. METHODS AND RESULTS: Infusion of Ang II (0.7 mg x kg(-1) x d(-1)) increased HO-1 mRNA levels to 169+/-31%, 251+/-47%, 339+/-26%, and 370+/-74% of the control level at 1, 3, 5, and 7 days after operation, respectively. The HO-1 protein level at 7 days was markedly upregulated, as was HO activity. Treatment with either losartan (25 mg x kg(-1) x d(-1)) or hydralazine (15 mg x kg(-1) x d(-1)), both of which prevented the Ang II-induced hypertension, blocked HO-1 mRNA upregulation. Norepinephrine infusion (2.8 mg x kg(-1) x d(-1)) produced a degree of hypertension and degree of HO-1 mRNA upregulation similar to those of Ang II infusion, which was again blocked by treatment with hydralazine (382+/-18% and 150+/-30% of the control level, respectively). Immunohistochemical analysis demonstrated that HO-1 is expressed in medial smooth muscle and adventitial cells in normotensive rat aortas, and this is markedly increased in adventitial and endothelial cells in Ang II-induced hypertensive rat aortas. In contrast, HO-2 protein expression was not changed in hypertensive rat aortas. CONCLUSIONS: These findings indicate that HO-1 is upregulated in hypertensive rat aortas, apparently by mechanisms unique to Ang II and by hemodynamic stress.

Angiotensin II↗