Comparison of spontaneously released endothelium-derived relaxing factor in cerebral and extracerebral arteries in rabbits

To investigate regional differences in spontaneously released endothelium-derived relaxing factor (EDRF), a bioassay of spontaneously released EDRF was performed on rabbit basilar, ear, common carotid and thoracic arteries using an isometric tension measurement technique and a measurement of cyclic guanosine monophosphate (cGMP) content in the vascular smooth muscle. The amount of spontaneously released EDRF was higher in the basilar artery than in any other arteries examined (p < 0.01). The levels of cGMP were 57.3 +/- 4.4 (n = 7) in basilar, 26.5 +/- 4.3 (n = 6) in ear, 24.5 +/- 2.3 (n = 11) in common carotid, and 30.3 +/- 3.8 pmol/g tissue (n = 8) in thoracic artery with endothelium, while endothelium-denuded arteries showed 24.2 +/- 6.6 (n = 5), 17.5 +/- 5.1 (n = 6), 20.1 +/- 2.9 (n = 7) and 14.4 +/- 2.3 pmol/g tissue (n = 8) in the same order. Haemoglobin (10(-5) M, incubated with the artery for 5 min, significantly reduced the level of cGMP in all vessels with endothelium: 35.3 +/- 4.4 (basilar), 16.0 +/- 2.1 (ear), 14.0 +/- 1.9 (common carotid) and 8.7 +/- 1.2 pmol/g tissue (thoracic artery). Since endothelium-dependent relaxation is associated with a rise in the cGMP content of the smooth muscle cell, the data of cGMP measurement in addition to the bioassay of spontaneously released EDRF in tension measurement suggests that the spontaneous release of EDRF is much greater in the basilar artery than in extracerebral arteries. It is concluded that the intensity of the spontaneously released EDRF is relatively higher in the intracerebral artery than in the extracerebral artery.     

Detailed examination of vascular lesions triggered by an inhibitor of endothelium-derived relaxing factor

BACKGROUND: Inhibition of an endothelium-derived relaxing factor (EDRF) may contribute to the pathogenesis of thrombotic arterial occlusions. EXPERIMENTAL DESIGN: We measured the blood pressure and urinary excretion of protein, sodium, and potassium and histologically examined the brains, hearts, and kidneys in normotensive Wistar Kyoto rats (WKY) and stroke-prone spontaneously hypertensive rats (SHRSP) fed on a diet containing: (a) EDRF inhibitor (L-N-nitroarginine:L-NNA); (b) L-arginine, which reverses the effect of L-NNA; or (c) both L-NNA and L-arginine for 1 to 8 weeks. In addition, we examined L-NNA-treated SHRSP, the blood pressures of which were lowered using hydralazine. Furthermore, we produced and examined Goldblatt's renal hypertensive rats, which are of a different type from those resulting from the L-NNA treatment. RESULTS: Both WKY and SHRSP rats fed on a diet containing L-NNA suffered from hypertension and cerebral infarctions in a dose-dependent manner. Cerebral infarctions occurred whether or not SHRSP rats were treated with an antihypertensive agent when they were fed a high dosage of L-NNA. In contrast, SHRSP rats, treated simultaneously with both L-NNA and L-arginine, suffered few cerebral infarctions, although they were severely hypertensive. In addition, there were no cerebral infarctions in Goldblatt's renal hypertensive rats, although they suffered from advanced hypertension. CONCLUSIONS: The data indicate that the inhibition of EDRF injures the vessel walls and encourages platelet adhesion to the damaged areas. The adhering platelets narrow the lumen with resultant thrombotic arterial occlusions. Pathophysiologic conditions that decrease EDRF synthesis appear to play an important role in cerebral, renal, and myocardial infarctions.     

Dopamine releases endothelium-derived relaxing factor via alpha 2-adrenoceptors in canine vessels: comparisons between femoral arteries and veins

1. We investigated the role of vascular smooth muscle alpha-adrenoceptor subtypes in the vasoconstrictor response of femoral arteries and veins to dopamine and whether the vasoconstriction is modified by endothelium-dependent relaxation mediated via the activation of alpha 2-adrenoceptors in ring preparations of femoral arteries and veins from mongrel dogs. 2. Dopamine contracted both arteries and veins in a dose-dependent manner and this contraction was inhibited by pretreatment with phentolamine or prazosin. Pretreatment with yohimbine shifted the dose-response curve for dopamine to the right in femoral veins, but not in arteries. 3. Phenylephrine contracted femoral arteries and veins in a dose-dependent manner and this contraction was inhibited by pretreatment with prazosin. 4. Clonidine produced a bell-shaped dose-response curve in femoral veins and this curve was shifted upwards by pretreatment with NG-nitro-L-arginine (L-NNA). In contrast, femoral arteries were not affected by clonidine. NG-Nitro-L-arginine potentiated contractile responses to dopamine in both veins and arteries. This potentiation was inhibited by yohimbine or by the removal of the endothelium in both arteries and veins. 5. These results suggest that dopamine contracts femoral arteries via stimulation of alpha 1-adrenoceptors and contracts femoral veins via stimulation of both alpha 1- and alpha 2-adrenoceptors and that these contractions are attenuated by the vasodilator action of dopamine via alpha 2-adrenoceptor-mediated release of endothelium-derived relaxing factor.     

Atorvastatin does not alter the anticoagulant activity of warfarin

Twelve patients chronically maintained on warfarin were administered 80 mg atorvastatin for 2 weeks. Mean prothrombin times decreased slightly, but only for the first few days of the two-week treatment period. Thus atorvastatin had no consistent effect on the anticoagulant activity of warfarin and adjustment in warfarin dosing should not be necessary.     

Maternal diabetes status does not influence energy expenditure or physical activity in 5-year-old Pima Indian children

A novel series of matrix metalloproteinase (MMP) inhibitors is described. Incorporation of a terminal alpha-mercaptoketone or alpha-mercaptoalcohol in the zinc binding domain of a series of inhibitors led to compounds exhibiting low nanomolar activity against collagenase-1 (MMP-1), stromelysin (MMP-3), and gelatinase-B (MMP-9).     

Granulocyte colony-stimulating factor does not enhance phagocytosis or microbicidal activity of human mature polymorphonuclear neutrophils in vitro

The direct effects of human granulocyte colony-stimulating factor (hG-CSF) on mature polymorphonuclear neutrophils (PMNs) in vitro were studied with regard to chemotaxis, superoxide production, and phagocytosis and microbicidal activity against the following viable microorganisms: Staphylococcus aureus, serum-resistant Pseudomonas aeruginosa, and Candida albicans. Recombinant hG-CSF (rhG-CSF) acted as a chemoattractant for human PMNs in a dose-dependent manner. The chemotactic response of PMNs to N-formyl-methionyl-leucyl-phenylalanine (FMLP) was not enhanced by rhG-CSF at any of the concentrations used. rhG-CSF did not induce the generation of superoxide by itself. However, rhG-CSF was able to prime human PMNs and to enhance O2- release stimulated by FMLP in a dose-dependent manner. rhg-CSF did not enhance phagocytosis or killing of the three species of microorganisms by normal PMNs. With PMNs obtained from patients who had hematological disorders or solid tumors, no enhancement of the microbicidal activity was observed in most cases. Microbial killing mediated by PMNs depended on the ratio of PMNs to target organisms. We concluded from these facts that the most important effect of rhG-CSF was to increase the number of the peripheral PMNs and not to enhance the functions of mature PMNs.     

Diltiazem stimulates parathyroid hormone secretion in vivo whereas felodipine does not

The impact of two calcium channel blockers of different structure, diltiazem and felodipine, on PTH secretion was studied under hyper- and hypocalcemic conditions. Six healthy volunteers were investigated before and after treatment with felodipine, then after treatment with diltiazem. Under each of these three conditions, they received first a calcium infusion (0.109 mmol/kg over 130 min). Blood was drawn every 5-10 min for measurements of Ca2+ and intact PTH concentrations, and urine was collected over the infusion periods for measurements of calcium and creatinine. Basal levels of Ca2+ and intact PTH concentrations were similar under the three conditions. During calcium infusion, Ca2+ increased linearly from 1.27 to 1.51 mmol/L during the control period. Based on the whole response curve, Ca2+/time, this rise was less marked (P < 0.002) during each of the calcium channel blocker periods than under control conditions, although the three values of urinary calcium excretion were similar. In addition, PTH secretion was less suppressed on diltiazem than on felodipine therapy or during the control period (P < 0.04). During EDTA infusion, Ca2+ decreased in a linear way from 1.27 to 1.07 mmol/L during the control period. Based on the whole response curve, Ca2+/time, this decrease was more marked during felodipine than during diltiazem treatment or in the control period (P < 0.001). Although Ca2+ concentrations did not differ between the control and diltiazem periods, PTH levels were 1.3-fold higher (P < 0.0001) during diltiazem, but similar in the control and felodipine periods. These data demonstrate that diltiazem, but not felodipine, stimulates PTH secretion in vivo in man, with a maximal effect observed under hypocalcemic conditions.     

Phonological typicality does not influence fixation durations in normal reading.

Using a word-by-word self-paced reading paradigm, T. A. Farmer, M. H. Christiansen, and P. Monaghan (2006) reported faster reading times for words that are phonologically typical for their syntactic category (i.e., noun or verb) than for words that are phonologically atypical. This result has been taken to suggest that language users are sensitive to subtle relationships between sound and syntactic function and that they make rapid use of this information in comprehension. The present article reports attempts to replicate this result using both eyetracking during normal reading (Experiment 1) and word-by-word self-paced reading (Experiment 2). No hint of a phonological typicality effect emerged on any reading-time measure in Experiment 1, nor did Experiment 2 replicate Farmer et al.’s finding from self-paced reading. Indeed, the differences between condition means were not consistently in the predicted direction, as phonologically atypical verbs were read more quickly than phonologically typical verbs, on most measures. Implications for research on visual word recognition are discussed. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

The -514 polymorphism in the hepatic lipase gene (LIPC) does not influence androgen-mediated stimulation of hepatic lipase activity

The -514T allele of hepatic lipase is associated with increased high density lipoprotein-cholesterol levels in men, but not in women. This observation suggests that the -514C to T polymorphism may diminish the response of hepatic lipase to androgens. To test this hypothesis, five -514T and five -514C homozygous men were treated with the anabolic steroid stanozolol for 6 days. The mean increase in hepatic lipase activity was similar in the two groups (45+/-10 vs. 51+/-10 mmol x hr(-1) x l(-1), P = 0.5). To evaluate the association between the -514 polymorphism and hepatic lipase activity at different physiological androgen concentrations, hepatic lipase genotypes and activities were measured in 44 men and 40 premenopausal women. The effect of the -514T allele on hepatic lipase activity was significant and quantitatively similar in both sexes. These data indicate that the -514 polymorphism does not influence the response of hepatic lipase activity to androgens, and that the effects of this polymorphism on hepatic lipase activity are independent of androgen action.     

Biopsy forceps disinfection technique does not influence Helicobacter pylori culture

OBJECTIVES: Culturing Helicobacter pylori (Hp) has a low sensitivity rate, and is affected by factors such as the number of biopsies, transport, and culture conditions. Hp detection is also influenced by omeprazole, antibiotics, bismuth salts, or benzocaine use. Disinfection procedures based on glutaraldehyde are highly effective in eliminating any Hp contamination of endoscopic equipment. However, the possibility that some residual glutaraldehyde present in biopsy forceps after decontamination could affect Hp viability has not yet been investigated. METHODS: Antral specimens from 25 patients with active gastric or duodenal ulcer obtained with three forceps (sterilized with ethylene oxide, glutaraldehyde, or glutaraldehyde-phenolate) were streaked on appropriate media, and results of culture evaluated. RESULTS: Helicobacter pylori was isolated in 17 patients. Positivity of culture was independent of the way the forceps were sterilized, and the number of colonies (mean +/- SD) was similar for the three types of forceps (475 +/- 312, 533 +/- 242, and 550 +/- 225 colony-forming units [CFUs] for ethylene oxide, glutaraldehyde, and glutaraldehyde-phenolate, respectively). Moreover, the incubation time since isolation was also similar (6.0 +/- 1.3, 5.8 +/- 1.2, and 5.7 +/- 1.2 days for ethylene oxide, glutaraldehyde, and glutaraldehyde-phenolate disinfected forceps, respectively). CONCLUSION: The use of glutaraldehyde to sterilize biopsy forceps is not responsible for the false-negative results of Hp culture.     

Endothelin-1 receptor antagonism does not influence myocardial function in hypertensive dogs

BACKGROUND: As endothelin-1 exerts positive inotropic effects, the present study evaluated whether the hypotensive effects of the endothelin-1 receptor antagonist bosentan were partially related to a decrease in myocardial performance. METHODS: In group I, eight anaesthetized open-chest dogs with perinephritic hypertension received four cumulative doses of bosentan (B1-B4). In group II, eight animals received the same doses of bosentan after autonomic blockade. Indices of heart function were derived from the pressure-length loops obtained during vena cava occlusion. RESULTS: In group I, bosentan decreased left ventricular systolic pressure (LVSP) and mean aortic pressure (MAP) dose dependently, reaching 21% and 23% respectively at B4 (LVSP from 190 +/- 8 to 150 +/- 5 mmHg, P < 0.001; MAP from 167 +/- 7 to 128 +/- 5 mmHg, P < 0.001). These effects were only related to peripheral vasodilatation, without depression of myocardial contractility, as systemic vascular resistance dropped (from 670 +/- 83 to 446 +/- 53 mmHg mL-1 min-1 x 10(4); P < 0.05), and the end-systolic pressure-length relationship (ESPLR) remained unchanged (4.0 +/- 0.4 vs. 4.3 +/- 0.7 mmHg mm-1 kg-1). Concomitantly with pressure decline, heart rate tended to increase in this group (from 150 +/- 4 to 156 +/- 6 beats min-1). When autonomic system was blocked (group II), administration of bosentan induced similar hypotensive effects as in group I (26% and 28% reduction in LVSP and MAP respectively, P < 0.001) whereas ESPLR did not change (3.0 +/- 0.9 vs. 3.1 +/- 0.5mmHg-1 mm kg-1 ). Under these sympathetically blocked conditions, heart rate significantly fell after bosentan infusion (from 120 +/- 4 to 110 +/- 6 beats min-1, P < 0.001). CONCLUSIONS: Without influencing heart function, bosentan is an efficient and safe therapy that opens up new therapeutic perspectives in human essential hypertension.     

Identification of the 11,14,15- and 11,12, 15-trihydroxyeicosatrienoic acids as endothelium-derived relaxing factors of rabbit aorta

A number of endothelium-derived relaxing factors have been identified including nitric oxide, prostacyclin, and the epoxyeicosatrienoic acids. Previous work showed that in rabbit aortic endothelial cells, arachidonic acid was metabolized by a lipoxygenase to vasodilatory eicosanoids. The identity was determined by the present study. Aortic homogenates were incubated in the presence of [U-14C]arachidonic acid, [U-14C]arachidonic acid plus 15-lipoxygenase (soybean lipoxidase), or [U-14C]15-hydroxyeicosatetraenoic acid (15-HPETE) and analyzed by reverse phase high pressure liquid chromatography (RP-HPLC). Under both experimental conditions, there was a radioactive metabolite that migrated at 17.5-18.5 min on RP-HPLC. When the metabolite was isolated from aortic homogenates, it relaxed precontracted aortas in a concentration-dependent manner. Gas chromatography/mass spectrometry (GC/MS) of the derivatized metabolite indicated the presence of two products; 11,12,15-trihydroxyeicosatrienoic acid (THETA) and 11,14,15-THETA. A variety of chemical modifications of the metabolite supported these structures and confirmed the presence of a carboxyl group, double bonds, and hydroxyl groups. With the combination of 15-lipoxygenase, arachidonic acid, and aortic homogenate, an additional major radioactive peak was observed. This fraction was analyzed by GC/MS. The mass spectrum was consistent with this peak, containing both the 11-hydroxy-14, 15-epoxyeicosatrienoic acid (11-H-14,15-EETA) and 15-H-11,12-EETA. The hydroxyepoxyeicosatrienoic acid (HEETA) fraction also relaxed precontracted rabbit aorta. Microsomes derived from rabbit aortas also synthesized 11,12,15- and 11,14,15-THETAs from 15-HPETE, and pretreatment with the cyctochrome P450 inhibitor, miconazole, blocked the formation of these products. The present studies suggest that arachidonic acid is metabolized by 15-lipoxygenase to 15-HPETE, which undergoes an enzymatic rearrangement to 11-H-14,15-EETA and 15-H-11,12-EETA. Hydrolysis of the epoxy group results in the formation of 11,14,15- and 11,12,15-THETA, which relaxed rabbit aorta. Thus, the 15-series THETAs join prostacyclin, nitric oxide, and epoxyeicosatrienoic acids as new members of the family of endothelium-derived relaxing factors.     

The fusion promotion activity of the NDV HN protein does not correlate with neuraminidase activity

Three activities, attachment, neuraminidase, and fusion promotion, have been associated with the hemagglutinin-neuraminidase (HN) protein encoded by paramyxoviruses such as Newcastle disease virus. The fusion promotion activity of the HN protein can be separated from its attachment activity by mutation (Sergel et al., 1993, Virology 193, 717-726). To determine if neuraminidase activity of the HN protein has any role in fusion promotion, two sets of mutants were characterized. First, a change of amino acid 193 from a serine to a proline and a change of amino acid 175 from isoleucine to a methionine diminished neuraminidase activity as previously reported. However, these mutant proteins retained fusion promotion activity. In addition, mutation of amino acid 200 from a histidine to a proline resulted in nearly twice the neuraminidase activity of wild-type as previously reported. This mutant also had wild-type levels of fusion promotion activity. Second, substitution of three leucine residues at amino acids 94, 96, and 97 with three alanines resulted in a mutant protein with full neuraminidase as well as full attachment activity but no fusion promotion activity. Thus, two sets of HN protein mutants demonstrate that the fusion promotion activity does not correlate with the level of neuraminidase activity.     

Amantadine does not have antiviral activity against Borna disease virus

We have investigated the antiviral activity of amantadine (AD) against Borna disease virus (BDV) in several culture cell systems. We present evidence that AD, in the range 5 to 10 microM, does not have antiviral activity against BDV. Treatment of BDV infected cells with AD for six days caused neither a reduction in the number of infected cells, nor a decrease in steady state levels of BDV RNA or proteins. Moreover, treatment of cells with AD prior infection did not affect BDV multiplication, whereas influenza A virus yield was less than 1% with respect to that obtained in untreated control cells.     

Thrombin receptor activating peptide does not stimulate platelet procoagulant activity

Platelets after challenge with alpha-thrombin alone, collagen alone or thrombin/collagen mixture were observed to increase the rate of activation of prothrombin by factor Xa in the presence of factor Va and calcium ion (platelet procoagulant activity) by a maximum of 25, 45 and 110 fold, respectively. The increase in platelet procoagulant activity due to these agonists has been described previously and arises from increased expression of phosphatidylserine on the platelet surface. When platelets were treated with the thrombin receptor activating peptide (TRAP) (SFLLRNPNDKYEPK), alone or in the presence of collagen or thrombin, no change in platelet procoagulant activity was observed at concentrations of TRAP sufficient to cause increased intracellular calcium levels and protein phosphorylation in a manner similar to that of thrombin. In addition, no increase in platelet procoagulant activity was seen upon treatment with TRAP in the presence of inactivated thrombin (PPACK-thrombin). These results suggest that the thrombin-mediated increase in procoagulant activity may be due to activation of a thrombin receptor distinct from the recently cloned G-protein-coupled receptor, or to other proteolytic events on the platelet surface.     

Experimental muscle pain does not cause long-lasting increases in resting electromyographic activity

The mutual links between muscle pain and resting electromyographic (EMG) activity are still controversial. This study described effects of experimental muscle pain on resting EMG activity in a jaw-closing muscle and a leg muscle. Pain was induced by injections of hypertonic saline into the muscles in 10 subjects. Injections of isotonic saline served as a control. The pain intensity was scored on visual analog scales (VAS) and surface and intramuscular wire EMGs were obtained from the resting muscles before, during, and after saline injections. EMG activity was analyzed in 30-s intervals and demonstrated, in both muscles, significant increases 30-60 s after injection of hypertonic saline, but not after injection of isotonic saline. In contrast to the transient increase in EMG activity, the pain sensation lasted up to 600 s after injection of hypertonic saline. It was concluded that acute muscle pain is unable to maintain longer-lasting resting muscle hyperactivity.     

Depletion of striatal dopamine transporter does not affect psychostimulant-induced locomotor activity

The effect of neurotoxin-induced depletion of striatal dopamine transporter (DAT) binding sites on animals' responses to psychostimulants was investigated. Multiple 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or methamphetamine (METH) injections but not a single METH injection to Swiss Webster mice resulted in > 60% depletion of striatal DAT. MPTP-induced depletion of DAT did not affect METH- and cocaine-stimulated locomotor activity compared with the response of control mice. Pre-exposure to either the neurotoxic or the single non-neurotoxic dose of METH resulted in a marked locomotor sensitization in response to METH or cocaine challenge injections. The present results indicate that > 60% loss in striatal DAT binding sites has no effect on animals' responses to psychostimulants, and suggest that neural systems other than striatal DAT may contribute to the induction of locomotor sensitization to METH and cocaine.     

Cross-priming of CTL responses in vivo does not require antigenic peptides in the endoplasmic reticulum of immunizing cells

It has been proposed that the cross-priming of CTL responses in vivo involves the transfer to host APCs of heat shock protein glycoprotein 96-chaperoned antigenic peptides released from the endoplasmic reticulum (ER) of dying or infected cells. We have tested this possibility directly using TAP-deficient cell lines lacking antigenic ER peptides derived from two model Ags, the human adenovirus type 5 early regions E1A and E1B. Although both proteins were well expressed, the cells were not recognized by E1A- or E1B-specific CTLs unless the relevant epitope was either provided exogenously as a synthetic peptide or targeted to the ER in a TAP-independent fashion. Despite the absence of these ER peptides, the TAP1-/- cells were able to efficiently cross-prime E1A- and E1B-specific CTLs following immunization of syngeneic mice. These results indicate that, although purified peptide/glycoprotein 96 complexes are potent immunogens, the mechanism of CTL cross-priming in vivo does not depend upon antigenic peptides in the ER of immunizing cells.     

Inhibition of nitric oxide synthesis does not improve interleukin-2-mediated antitumor effects in vivo

BACKGROUND: Protein stability appears to be governed by non-covalent interactions. These can be local (between residues close in sequence) or non-local (medium-range and long-range interactions). The specific role of local interactions is controversial. Statistical mechanics arguments point out that local interactions must be weak in stable folded proteins. However, site-directed mutagenesis has revealed that local interactions make a significant contribution to protein stability. Finally, computer simulations suggest that correctly folded proteins require a delicate balance between local and non-local contributions to protein stability. RESULT: To analyze experimentally the effect of local interactions on protein stability, each of the five Che Y alpha-helices was enhanced in its helical propensity. alpha-Helix-promoting mutations have been designed, using a helix/coil transition algorithm tuned for heteropolypeptides, that do not alter the overall hydrophobicity or protein packing. The increase in helical propensity has been evaluated by far-UV CD analysis of the corresponding peptides. Thermodynamic analysis of the five Che Y mutants reveals, in all cases, an increase in half urea ([urea]1/2) and in Tm, and a decrease in the sensitivity to chemical denaturants (m). ANS binding assays indicate that the changes in m are not due to the stabilization of an intermediate, and the kinetic analysis of the mutants shows that their equilibrium unfolding transition can be considered as following a two-state model, while the change in m is found in the refolding reaction (m(k)f). CONCLUSIONS: These results are explained by a variable two-state model in which the changes in half urea and Tm arise from the stabilization of the native state and the decrease in m from the compaction of the denatured state. Therefore, the net change in protein stability in aqueous solution produced by increasing the contribution of native-like local interactions in Che Y is the balance between these two conflicting effects. Our results support the idea that optimization of protein stability and cooperativity involve a specific ratio of local versus non-local interactions.