Allergological characterisation of the storage mite Acarus gracilis(Acari: Acaridae)

BACKGROUND: Storage mites of the genus Acarus can be responsible for allergic sensitisation in domestic environments.Acarus gracilis is a frequent species in some geographical regions of the Iberian Peninsula. Since the allergenicity of this mite has not been described before, the objectives of this study were to characterise it immunologically, and to compare it with the closely related and more extensively studied speciesAcarus siro. METHODS: Extracts from A. gracilis and A. siro cultures were characterised by Lowry, 1D and 2D-SDS and IEF. Zymogram, and determination of different enzymatic activities were performed. Skin prick solution of A. gracilis was tested in consecutive patients attending the Hospital of Mérida (Extremadura, Spain). Serum samples from eight individuals with positive skin prick test were collected. IgE determination, immunoblot and immunoblot-inhibition studies were performed. RESULTS: Extracts of both species showed a very similar protein and allergenic profile. Allergens at 14 and 17 kDa were clearly recognised in both extracts by serum samples. Immunoblot-inhibition studies demonstrated that both extracts were totally inhibited by the opposite one. Enzymatic activity was similar in both cases with the most important differences being in kallikrein, serine protease and collagenase activities. CONCLUSION: The storage mite A. gracilis has a similar protein and allergen profile to A. siro and can induce allergic sensitisation. Due to the higher prevalence of this species respect to A. siro in some regions, more studies are needed to determine the clinical significance of sensitisation to this storage mite species

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A calix[4]arene ureidopeptide dimer self-assembled through two superposed hydrogen bond arrays.

Dimerization of calix[4]arene ureidopeptides is demonstrated for the first time. Two calix[4]arenes tetrasubstituted in the upper rim with -NHCONH(L)LeuNHC(8)H(17) (1) and -NHCONH(L)Leu(D)Leu-OMe (2) were prepared and studied by NMR, circular dicroism, and gel permeation chromatography. Compound 2 self-assembles through urea-urea hydrogen bonds, as well as by an additional set of hydrogen bonds provided by the peptide side chains, with participation of the ester carbonyls. The absence of such group in 1 causes the monomer structure to be favored in this case.

Toward an artifical acetylcholinesterase.

The methanolysis of choline p-nitrophenylcarbonate in chloroform containing 1% methanol is catalyzed with turnover by ditopic receptors 1 and 2, consisting of a calix[6]arene connected to a bicyclic guanidinium by means of a short spacer. The calix[6]arene subunit strongly binds to the trimethylammonium head group through cation-pi interactions, whereas the guanidinium moiety is deputed to stabilize through hydrogen bonding reinforced by electrostatic attraction the anionic tetrahedral intermediate resulting from methoxide addition to the ester carbonyl. The observed cholinesterase activity had been anticipated on the basis of the ability of the ditopic receptors 1 and 2 to bind strongly to the choline phosphate DOPC, which is a transition state analogue for the BAc2-type cleavage of choline esters.

Dimeric capsules by the self-assembly of triureidocalix

A number of calix[6]arenes bearing ureas at the upper rim positions of alternate rings 1, 3 and 5 were prepared and studied in detail by NMR spectroscopy and gel permeation chromatography. N-Unsubstituted ureas were shown to dimerize through a cyclic array of hydrogen bonds to give cylindrical cavities capable of encapsulating small molecules such as dichloromethane, benzene and fluorobenzene. Slow equilibria between dimer and monomer were observed in [D6]DMSO-CDCl3 mixtures. By contrast, N-substituted ureas are monomeric. All urea monomers with bulky O-substituents display a solvent-dependent, slow equilibrium between C3v and Cs cone conformations.

Changes of plasma L-arginine levels in spontaneously hypertensive rats under induced hypotension.

Nicardipine, a dihydropyridine type calcium channel blocker, was infused at two flow-rates into spontaneously hypertensive (SH) and control normotensive Wistar-Kyoto (WKY) rats (young, 6-week-old and adult, 23-week-old, n = 5) under pentobarbital anesthesia, to cause hypotension. Mean arterial blood pressure and the concentrations of plasma amino acids and norepinephrine (NE) were measured before infusion and at each step of the infusion. The reduction in blood pressure caused by nicardipine induced a decrease in plasma L-arginine concentration in both young and adult SH rats, this effect being larger in adult rats. There was no significant change in plasma levels of L-arginine in age-matched WKY rats. The concentration of other amino acids did not change in both rat strains. On the contrary, there was an increase in plasma NE concentration in both SH and WKY rats after infusion with nicardipine. Plasma L-arginine concentration showed a good inverse correlation with the logarithm of plasma NE concentration in SH and WKY rats and the correlation was expressed as Y = -alpha log(X) + m (Y, plasma L-arginine concentration (nmol/mL); X, plasma NE concentration (pmol/mL); alpha, a slope; and m, an intercept). alpha, 43.0 and 4.35 for 23-week-old SH and WKY rats, respectively, and 17.0 and 4.0 for 6-week-old SH and WKY rats, respectively. The present data together with previous data suggest a direct noradrenergic stimulation of the synthesis of nitric oxide (NO) from L-arginine. The findings also indicate an impairment of the L-arginine metabolism or pools in SH rats compared with WKY rats. The deficiency of L-arginine increases with the age of SH rats and could be related to the development and maintenance of hypertension due to inefficient production of NO.

Age-related weakening of baroreflex-mediated sympathetic activity in spontaneously hypertensive rats in response to blood pressure reduction.

Nicardipine, a dihydropyridine type calcium channel blocker, was infused into 4-, 6-, and 23-wk-old spontaneously hypertensive (SH) and age-matched normotensive Wistar-Kyoto (WKY) rats (under sodium thiobutabarbital anesthesia and ventilation, n = 4) through the left femoral vein, resulting in the reduction of blood pressure. In each rat, mean arterial blood pressure, heart rate, and the concentration of plasma catecholamines (CAs), norepinephrine (NE), and epinephrine (E) were concomitantly determined, and the correlations between these three variables were studied. During the infusion of nicardipine, the plasma concentration of CAs was measured with an automatic detection system in blood samples collected from the right femoral artery of each rat. The reduction in blood pressure induced by nicardipine brought about an increase in plasma CA levels. The blood pressure correlated well with the logarithm of plasma NE or E concentration according to the formula Y= -alpha log (X) + m (Y, blood pressure; X, concentration of plasma NE or E; a, slope; and m, intercept). The slopes (as) of 6-wk-old and 23-wk-old SH rats were significantly greater than those of aged-matched WKY rats, meaning that the increment in plasma CAs in response to a decrease in blood pressure was smaller in SH than in WKY rats of similar ages. However, no significant differences were found between the as of 4-wk-old SH and WKY rats. We conclude that the increment in the baroreflex-mediated sympathetic activity in response to a drop in blood pressure induced by nicardipine is similar or greater in prehypertensive SH than in normotensive WKY 4-wk-old rats, while the increment becomes smaller in SH rats with the onset of hypertension (6-wk-old rats), and is much less in fully hypertensive adult (23-wk-old) SH rats than in age-matched WKY rats. On the basis of these findings and previous data obtained by neurography, we conclude that plasma CAs can be used to evaluate baroreflex-mediated sympathetic activity countering the blood pressure reduction caused by calcium antagonists.

Comparison of the sympathetic nervous system activity between spontaneously hypertensive and Wistar-Kyoto rats to respond to blood pressure reduction.

Two types of calcium antagonists, diltiazem and nicardipine, were separately infused in 23-28 week-old spontaneously hypertensive (SHR) and age-matched normotensive Wistar-Kyoto (WKY) rats (under sodium thiobutabarbital anesthesia and ventilation, n = 4) through the left femoral vein, resulting in the reduction of blood pressure. In each rat, mean arterial blood pressure, heart rate and the concentration of plasma catecholamines (CAs), norepinephrine (NE) and epinephrine (E), were concomitantly determined and the correlations between these three values were studied for each calcium antagonist. Plasma concentration of CAs were measured in blood samples collected during the infusion from the right femoral artery of each rat by the automatic sensitive and selective detection system. The reduction of blood pressure induced by the calcium antagonists brought about an increase in plasma CAs levels. The blood pressure correlated well with the logarithm of plasma NE and E concentration and the relations were expressed as Y = -alpha log(X)+m (Y, blood pressure; X, concentration of plasma NE or E; alpha, slope; and m, intercept). The alpha s of SHR rats were greater than those of WKY rats for the calcium antagonists employed, meaning that the increment of plasma CAs responding to a decrease in blood pressure was smaller in SHR than in WKY rats. It was concluded that the contribution of the sympathetic nervous system to maintaining blood pressure reduced by diltiazem and nicardipine is less in SHR than in WKY rats.

Release of secretory phospholipase A2 from rat neuronal cells and its possible function in the regulation of catecholamine secretion.

Here we show that secretory phospholipase A2 (sPLA2) that is immunochemically indistinguishable from type II sPLA2 is (i) stored in neuroendocrine cells, (ii) released in response to neurotransmitters or depolarization, and (iii) involved in the regulation of catecholamine secretion by these cells. Rat brain synaptic vesicle fractions contained PLA2 activity, which was neutralized completely by an antibody raised against rat type II sPLA2. sPLA2 immunoreactive with anti-(type II sPLA2) antibody was released from synaptosomes in response to depolarization evoked by a high concentration of potassium in the presence of Ca2+. Rat pheochromocytoma PC12 cells, which differentiated into adherent cells similar to sympathetic neurons in response to nerve growth factor, were used for the detailed analysis of the dynamics and function of sPLA2 in neuronal cells. Antibody against rat type II sPLA2 precipitated approximately 80% of the PLA2 activity in PC12 cell lysates. Transcript for type II sPLA2 was detected in PC12 cells by reverse transcriptase-PCR. When neuronally differentiated PC12 cells were stimulated with carbamylcholine or potassium, sPLA2 was released into the medium and reached a maximal approximately 40% release by 15 min. Inhibitors specific to type II sPLA2 suppressed catecholamine secretion by PC12 cells which had been activated by carbamylcholine. Furthermore, treatment of PC12 cells with exogenous type II sPLA2 alone elicited catecholamine secretion. These observations indicate that sPLA2 released from neuronal cells may regulate the degranulation process leading to release of neurotransmitters and are compatible with our earlier finding that this enzyme is involved in the degranulation of rat mast cells.

Desensitization and selective down-regulation of rat cardiac beta 1-adrenoceptors by prolonged in vivo infusion of T-0509, a beta 1-adrenoceptor full agonist.

We studied the effects of prolonged infusion of a selective beta 1-adrenoceptor (beta 1AR) full agonist, T-0509 [(-)-(R)-1-(3,4-dihydroxyphenyl)-2-[(3,4- dimethoxyphenethyl)amino]ethanol hydrochloride], with regard to its inotropic effect in vivo and cardiac beta AR density. The results were compared with those for isoproterenol. Continuous infusion of isoproterenol at doses of 2.5-40 micrograms/kg/hr, s.c. for 6 days shifted the dose-response curves of isoproterenol (i.v.) for LVdP/dtmax to the right and increased the ED50 values up to fourfold. Isoproterenol infusion at 40 micrograms/kg/hr reduced the density of both beta 1- and beta 2ARs by 36% and 43% respectively, in left ventricular membranes. Following 6-day infusion of T-0509 at doses sufficient to induce a positive inotropic effect (5-40 micrograms/kg/hr), the ED50 value of T-0509 (i.v.) for LVdP/dtmax was also increased up to fourfold. In contrast to isoproterenol, infusion of T-0509 caused selective down-regulation of beta 1ARs by 30% without changing the number of beta 2ARs. These results indicate that long-term application of a selective beta 1AR full agonist causes desensitization to its inotropy in vivo, with subtype-selective down-regulation of beta 1ARs in cardiac ventricles.

Relation between blood pressure and plasma catecholamine concentration after administration of calcium antagonists to rats.

Three types of calcium antagonists, diltiazem, verapamil and nicardipine, were separately infused into Sprague-Dawley (SD) rats (under pentobarbital anesthesia n = 5) through the left femoral vein at four different flow rates. Mean arterial blood pressure, heart rate and the concentration of plasma catecholamines (CAs), epinephrine (E), norepinephrine (NE) and dopamine (DA), were measured for each calcium antagonist, and the correlations between them were studied. Blood samples were collected within the infusion from common jugular vein. Plasma concentrations of CAs were determined by a HPLC-ethylenediamine condensation reaction-peroxyoxalate chemiluminescence detection system (HPLC-ED-PO-CL). The plasma concentration of CAs increased corresponding to the blood pressure reduction. The reduction induced by each calcium antagonist correlated with the logarithm of plasma NE concentration. The relation was expressed as Y = -alpha log X+m (Y, blood pressure; X, concentration of plasma NE; alpha, slope; and m, intercept). The correlation coefficients (rs) were -0.950 (diltiazem), -0.975 (verapamil) and -0.978 (nicardipine) (versus -0.734 for control). The alpha for nicardipine (108.4) was greater than those of diltiazem (85.4) and verapamil (80.8) (versus 31.0 for control), meaning that blood pressure reduction was greater in the case of nicardipine than diltiazem and verapamil, with an identical increment of plasma NE concentration. These data indicate that the contribution of the sympathetic nervous system to maintaining blood pressure reduced by nicardipine is less than that observed following the infusion of diltiazem and verapamil. Similar good inverse correlations between blood pressure and the logarithm of plasma concentration of E were observed with the three drugs infused (r = -0.928, -0.966, and -0.948 for diltiazem, verapmil and nicardipine, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

A fully automated HPLC method for the determination of catecholamines in biological samples utilizing ethylenediamine condensation and peroxyoxalate chemiluminescence detection.

A fully automated in-line extraction reversed-phase high-performance liquid chromatography (HPLC) method with chemiluminescence detection was developed for the analysis of human and rat plasma catecholamines (CAs), norepinephrine (NE), epinephrine (E) and dopamine (DA). N-Methyldopamine (N-MeDA) was used as an internal standard. The method involves collection of plasma samples, which are first diluted with a sample dilution buffer containing N-MeDA, and in-line extraction of CAs using a carboxylic acid small resin precolumn (SERUMOUT-CEX). This pre-extraction process was coupled with an HPLC system including reversed-phase mode separation on an analytical column (TSK gel ODS-80Ts), fluorogenic derivatization with ethylenediamine (ED) and finally postcolumn peroxyoxalate chemiluminescence reaction detection using bis [4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl]oxalate (TDPO) and hydrogen peroxide. The optimized mobile phase compositions, flow rates, operation timing for the adsorption and desorption of CAs in the precolumn, the separation in the analytical column and the optimum fluorogenic and chemiluminogenic reaction conditions were investigated. The detection limit for all the CAs was about 1 fmol (signal-to-noise ratio is 2). Excellent linearity of the calibration curves for CAs was observed in the range from 5 to 500 fmol for each CA using the internal standard. The relative standard deviations of the method for determining NE (183 fmol), E (23.6 fmol) and DA (6.1 fmol) in 50 microL of human plasma (n = 3) were 2.8, 2.7 and 3.1%, respectively, for the within-day assay and 5.0, 3.8 and 4.0%, respectively, for the between-day assay. The method was applicable to the determination of CAs in 25-50 microL of human or rat plasma.

Relations between blood pressure and plasma norepinephrine concentrations after administration of diltiazem to rats: HPLC-peroxyoxalate chemiluminescence determination on an individual basis.

A calcium antagonist, diltiazem, was infused continuously into Sprague-Dawley rats through the left femoral vein at four different flow rates. The mean arterial blood pressure and concentrations of plasma norepinephrine (NE) were measured in each single rat (n = 5) and the correlations between them were studied. Blood (150 microL) was collected 13 times during the infusion. Plasma NE was determined by HPLC-ethylenediamine condensation reaction-peroxyoxalate chemiluminescence detection system (HPLC-ED-PO-CL). In four cases from 5 rats, the blood pressure reduction caused by diltiazem was inversely correlated to logarithm of plasma NE concentration. The relation was expressed as Y = -alogX+m. The coefficients of correlation were -0.9506, -0.9293, -0.9341 and -0.8675, respectively. The correlation for the last rat was worse (r = -0.0799). The good correlation would imply that the sympathetic nervous system released NE to maintain blood pressure up to the normal level, responding to the sympathetic nervous system released NE to maintain blood pressure up to the normal level, responding to the blood pressure reduction caused by diltiazem. The present experiment proved the feasibility of the determination method of NE utilizing HPLC-ED-PO-CL detection in applying to the individual rats.

Sensitive determination of (-)-isoproterenol and (-)-(R)-1-(3,4- dihydroxyphenyl)-2-[(3,4-dimethoxyphenethyl)amino] ethanol (T-0509), a cardiotonic agent, in rat plasma utilizing a fully automated catecholamine analyser.

(-)-Isoproterenol (ISO), used as bronchodilator, and (-)-(R)-1-(3,4-dihydroxyphenyl)-2-[(3,4-dimethoxyphenethyl)amino]ethanol (T-0509), a new cardiotonic agent, were determined in rat plasma (only 25 microL) using a fully automated catecholamine (CA) analyser that includes in-line pre-extraction of CAs, coupled with an HPLC-ethylenediamine condensation reaction and a peroxyoxalate chemiluminescence detection system (HPLC-ED-PO-CL). The chromatographic conditions were: precolumn, SERUMOUT-CEX; buffer for delivering CAs in the precolumn, 10 mM potassium phosphate buffer (pH 7.5)/ethanol (92 + 8, v/v) (1 mL/min); precolumn clean-up solution, 4% phosphoric acid/acetonitrile (50 + 50, v/v) (1 mL/min); analytical column, TSKgel ODS-80Ts, 250 x 4.6 mm i.d.; eluent, 75 mM potassium acetate buffer (pH 3.2)/50 mM potassium phosphate buffer (pH 3.2)/acetonitrile (83.6 + 4.4 + 12, v/v/v for ISO and 76 + 4 + 20 v/v/v for T-0509) (1 mL/min); fluorogenic reagent solution, 105 mM ED and 175 mM imidazole in acetonitrile/ethanol (90 + 10, v/v) (0.25 mL/min); chemiluminogenic reaction solution, 0.25 mM TDPO, 150 mM hydrogen peroxide and 110 mM TFA in dioxane/ethyl acetate (50 + 50, v/v) (1.4 mL/min). Colterol, (+/-)-1-(3,4-dihydroxyphenyl)-2-[(1,1-dimethyl)ethylamino]ethanol, (+/-)-N-t-butylnorepinephrine was used as an internal standard (IS) for ISO, and (+/-)-1-(3,4-dihydroxyphenyl)-2-[(3,4,5- trimethoxyphenethyl)amino]ethanol, (+/-)-(3,4,5-trimethoxyphenethyl- aminomethyl)-3,4-dihydroxybenzylalcohol (T-1583) for T-0509. The detection limits for ISO and T-0509 were 1.3 and 0.9 fmol on injection, respectively.

13-Hydroxyacenaphato[1,2-b]quinolizinium bromide as a new flouorescence indicator.

13-Hydroxyacenaphtho[1,2-b]quinolizinium bromide (13-HQBr)was selected as a fluorescence indicator to determine basic compounds in non-aqueous media. This compound possesses an acidic phenolic hydroxyl group. It presents varying absorption (ROH, 408, 430 nm; RO(-) 456, 478 nm) and excitation spectra (ROH, 425 nm; RO, 471 nm) depending on the pH of the media, but the same emission fluorescence spectrum (ROH = RO(-), 526 nm) at different pH in buffered aqueous solutions. However, in acidic non-aqueous media (acetic, formic and trifluoroacetic acids), it can be observed that the fluorescence emission spectra differ for the ionized (lambda(em) = 530 nm) and non-ionized (lambda(em) = 440, 470 nm) forms. The fluorescence intensity at the characteristic peaks depends on the acid-base equilibria in the ground and excited states. Therefore, this property could be used to evaluate the concentration of basic compounds, showing a good linearity range between fluorescence intensity and basic sample concentration.

Hyperparathyroidism in metastases of prostatic carcinoma: a biochemical, hormonal and histomorphometric study.

Secondary hyperparathyroidism can develop as a result of bone metastases from prostatic cancer, but this has not been studied from the multiple aspects of biochemistry, hormonal status and histomorphometry. In 20 patients with stage-D prostatic cancer, a transiliac bone biopsy was performed for histomorphometric study. In all of them, molecular parathormone (PTH-M) and osteocalcin were determined by radioimmunoassay together with other parameters considered to be biological markers of bone remodelling. Of these 20 patients, only 2 (10%) had elevated PTH-M (240 +/- 20.6 pmol/l), differing significantly from the other 18 (58.6 +/- 11.7 pmol/l) and from controls (60.4 +/- 7.2 pmol/l). In the high PTH-M patients, corrected calcium was low (7.8 +/- 0.4 mg/dl) as compared to normal PTH-M patients (9.2 +/- 0.5 mg/dl, p less than 0.001), and this was also the case for serum phosphorus (2.2 +/- 0.6 vs. 3.2 +/- 0.3 and 3.4 +/- 0.4 mg/dl, respectively p less than 0.001). Alkaline phosphatase was raised in the patient groups as compared to controls (p less than 0.001) and was higher in the high PTH-M group (362 +/- 58 vs. 224 +/- 62 U/l, p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)