Glass transition of nanometric polymer films probed by picosecond ultrasonics.

The possibility to measure the glass transition temperature in poly(methyl methacrylate) (PMMA) films by picosecond ultrasonics with thicknesses ranging from 458 nm to 32 nm is demonstrated. A shift of the longitudinal acoustic eigenmodes towards lower frequencies with temperature is observed accompanied by a change in the temperature-frequency slopes at the glass transition temperature. The contributions to the frequency shift from changes in film thickness and sound velocity are discussed and the latter is extracted below the glass transition temperature. Finally, the advantages and disadvantages of the current approach in a comparison to other methods based on acoustic measurements in the GHz regime are reviewed.

Regulation of aspartate-derived amino acid homeostasis in potato plants (Solanum tuberosum L.) by expression of E. coli homoserine kinase.

The availability of the carbon backbone O-phosphohomoserine (OPHS) is critical to methionine (met) and threonine (thr) synthesis. OPHS derives from homoserine and is formed by homoserine kinase (HSK). To clarify the function of HSK in cellular metabolism, the E. coli HSK ortholog thrB was expressed in potato plants targeting the EcHSK protein to chloroplasts and to the cytosol. Both approaches resulted in up to 11 times increased total HSK enzyme activity. Transgenic plants exhibited reduced homoserine levels while met and thr did not accumulate significantly. However, the precursor cysteine and upstream intermediates of met such as cystathionine and homocysteine did indicating an accelerated carbon flow towards the end products. Coincidently, plants with elevated cytosolic levels of EcHSK exhibited a reduction in transcript levels of the endogenous HSK, as well as of threonine synthase (TS), cystathionine beta-lyase (CbL), and met synthase (MS). In all plants, cystathionine gamma-synthase (CgS) expression remained relatively unchanged from wild type levels, while S-adenosylmethionine synthetase (SAMS) expression increased. Feeding studies with externally supplied homoserine fostered the synthesis of met and thr but the regulation of synthesis of both amino acids retained the wild type regulation pattern. The results indicate that excess of plastidial localised HSK activity does not influence the de novo synthesis of met and thr. However, expression of HSK in the cytosol resulted in the down-regulation of gene expression of pathway genes probably mediated via OPHS. We integrated these data in a novel working model describing the regulatory mechanism of met and thr homeostasis.

Effect of sulfur availability on the integrity of amino acid biosynthesis in plants.

Amino acid levels in plants are regulated by a complex interplay of regulatory circuits at the level of enzyme activities and gene expression. Despite the diversity of precursors involved in amino acid biosynthesis as providing the carbon backbones, the amino groups and, for the amino acids methionine and cysteine, the sulfhydryl group and despite the involvement of amino acids as substrates in various downstream metabolic processes, the plant usually manages to provide relatively constant levels of all amino acids. Here we collate data on how amino acid homeostasis is shifted upon depletion of one of the major biosynthetic constituents, i.e., sulfur. Arabidopsis thaliana seedlings exposed to sulfate starvation respond with a set of adaptation processes to achieve a new balance of amino acid metabolism. First, metabolites containing reduced sulfur (cysteine, glutathione, S-adenosylmethionine) are reduced leading to a number of downstream effects. Second, the relative excess accumulation of N over S triggers processes to dump nitrogen in asparagine, glutamine and further N-rich compounds like ureides. Third, the depletion of glutathione affects the redox and stress response system of the glutathione-ascorbate cycle. Thus, biosynthesis of aromatic compounds is triggered to compensate for this loss, leading to an increased flux and accumulation of aromatic amino acids, especially tryptophan. Despite sulfate starvation, the homeostasis is kept, though shifted to a new state. This adaptation process keeps the plant viable even under an adverse nutritional status.

Capsaicin desensitization of the nasal mucosa reduces symptoms upon allergen challenge in patients with allergic rhinitis.

Patients with birch pollen allergic rhinitis were treated locally, out of season, in the nasal cavity with capsaicin (30 microM) or saline. The capsaicin treatment resulted in a statistically significant reduction of symptoms upon allergen challenge, which lasted for 2 months. Saline had no effect on the symptom score upon allergen challenge. Neither capsaicin nor saline treatment had any effect on allergen challenge-induced nasal mucosal swelling monitored by acoustic rhinometry. Allergen challenge-induced eosinophil migration to the nasal mucosa was affected by neither capsaicin nor the saline treatment. The finding that capsaicin treatment reduces allergic symptoms indicates that selective, non-peptide neurokinin receptor antagonists may be an alternative in the future in the treatment of nasal allergy. However, owing to the pain involved in local capsaicin treatment this treatment is unlikely to be of clinical use.

Heavy physical exercise decreases nitric oxide levels in the nasal airways in humans.

A continuous production of nitric oxide (NO) takes place in human nasal airways. NO in the nasal airways is mainly derived from the paranasal sinuses. The factors that regulate NO synthesis in the upper airways are presently not known. We have investigated the effects of physical exercise on NO levels in the nasal airways. Nasal cavity NO levels were measured by chemiluminescence technique in five healthy non-smoking male subjects before, during and after 5 min of maximal exercise (245 W) on an ergometer cycle. In addition, in one subject NO levels were measured directly in the maxillary sinus during exercise. Nasal cavity NO levels were decreased by 47% after only 1 min of exercise compared with the control situation. A maxima 76% reduction was found at the end of the exercise period and thereafter NO levels slowly increased, reaching basal levels again in about 15-20 min. NO levels in the sinus decreased in a similar manner during exercise. The decrease in nasal cavity NO levels cannot be explained merely by dilution of nasal air due to changes in nasal cavity volume or increased ventilation. We conclude that the excretion of NO in the nasal airways is decreased acutely during heavy short term physical exercise.

Calcium-independent and steroid-resistant nitric oxide synthase activity in human paranasal sinus mucosa.

Nitric oxide (NO) is present in the human nasal airways and originates primarily from the paranasal sinuses. Immunohistochemical studies and messenger ribonucleic acid (mRNA) in situ hybridization indicate that a type-2 NO synthase (NOS) is constitutively expressed in healthy sinus epithelium. We have further characterized sinus NOS activity by studying the enzymatic conversion of L-arginine to L-citrulline in biopsies from sinus mucosa. Maxillary sinus biopsies were obtained from nine healthy subjects during reconstructive facial surgery. In addition, nasal NO concentrations in nine controls were compared with those found in five patients treated with high systemic doses of glucocorticosteroids. Finally, the effects of i.v. L-arginine infusion on nasal cavity NO concentrations were studied in six healthy subjects. Ca(2+)-independent NOS activity was found in all biopsies and was five times higher than Ca(2+)-dependent activity (179 +/- 64 and 36 +/- 17 pmol.g-1.min, respectively). There was no difference in nasal NO levels between controls (344 +/- 21 parts per billion (ppb)) and steroid-treated patients (342 +/- 36 ppb). Nasal NO levels increased up to 35% following i.v. infusion of L-arginine. We conclude that NOS activity in healthy sinus mucosa is predominantly Ca(2+)-independent and this NOS is not downregulated by systemic steroids. Furthermore, L-arginine infusion increases nasal airway NO excretion in vivo, indicating that the substrate concentration is a rate-limiting factor under basal conditions. These findings further support the notion that sinus NOS is identical or very closely related to the type-2 NOS; however, the regulation of expression seems to be fundamentally different from that described previously for this NOS isoform.

Nasal vasoconstriction and decongestant effects of nitric oxide synthase inhibition in the pig.

The effects of systemic administration of the nitric oxide synthase inhibitor N omega-nitro-L-arginine on the vasculature of the pig nasal mucosa were compared with the effects of alpha-adrenoceptor agonists and of sympathetic nerve stimulation. A novel in vivo pig model was used in which standard measurements of nasal mucosal blood flow were complemented with measurements of nasal cavity volume using an acoustic rhinometer. The vasodilatory effects of capsaicin, substance P. acetylcholine, vasoactive intestinal polypeptide and nitroprusside, before and after nitric oxide synthase inhibition, were also investigated. N omega-nitro-L-arginine evoked a marked, slowly developing, long-lasting reduction in nasal vascular conductance with a parallel increase in nasal cavity volume (by approximately 30%) comparable in magnitude with the response upon alpha-adrenoceptor stimulation. L-Arginine partly reversed the N omega-nitro-L-arginine-evoked vasoconstriction, although nasal cavity volume was unaffected. Nasal vasodilation and decrease in nasal cavity volume resulting from capsaicin-evoked activation of sensory nerves were unaltered after administration of N omega-nitro-L-arginine. Nitric oxide synthase inhibition did not attenuate the vasodilator responses evoked by either substance P. acetylcholine, vasoactive intestinal polypeptide or nitroprusside. The present data suggest that nitric oxide synthase activity is of importance both for basal nasal vascular conductance and nasal cavity volume in the pig in vivo. In contrast, nitric oxide synthase inhibition does not reduce the vascular effects of a variety of vasodilating agents, including capsaicin, acetylcholine and substance P.

Capsaicin-, resiniferatoxin-, and lactic acid-evoked vascular effects in the pig nasal mucosa in vivo with reference to characterization of the vanilloid receptor.

Nasal cavity volume, mucosal and superficial skin blood flow as well as renal splenic vascular effects of capsaicin, resiniferatoxin and lactic acid were investigated, using a novel in vivo pig model. The present results show that locally intraarterially injected capsaicin, resiniferatoxin and lactic acid evoke similar vasodilatory responses, although with different duration, in the nasal mucosa and superficial skin as well as an increase in heart rate and mean arterial blood pressure. Nasal vascular responses evoked by capsaicin, resiniferatoxin and lactic acid were unaffected by the cyclooxygenase inhibitor diclofenac. Moreover, chlorisondamine did not alter the nasal vasodilatory responses evoked by capsaicin and lactic acid. However, chlorisondamine abolished sympathetic reflex-mediated vasoconstrictor effects of capsaicin in the spleen and kidney. Lactic acid-evoked vasodilation in the nasal mucosa and skin was inhibited by the 8-37 fragment of calcitonin gene-related peptide, a calcitonin gene-related peptide-receptor antagonist. Lactic acid-evoked vasoconstriction in the spleen and kidney was reduced but not abolished by chlorisondamine, suggesting that the effects of lactic acid are not exclusively reflex-mediated. Capsazepine did not inhibit the vasodilatation in the nasal mucosa evoked by capsaicin and lactic acid. [3H]Resiniferatoxin bound to pig nasal mucosa membranes with an affinity of 134 pM in a non-cooperative fashion; this binding behaviour contrasted to the apparent positive cooperativity (a Hill coefficient of 2.2) of specific resiniferatoxin binding to pig spinal cord preparations. Specific [3H]resiniferatoxin binding to nasal mucosa membranes was fully inhibited by capsaicin (Ki = 5 microM) and lactic acid (IC50 at pH 5.0) but not by capsazepine (up to 10 microM), in accord with the physiological findings. Capsazepine, by contrast, displaced [3H]resiniferatoxin from spinal vanilloid receptors with an affinity of 3 microM. These findings show the presence of vanilloid receptors in the pig nasal mucosa and suggest heterogeneity in the properties of vanilloid receptors in the pig. Furthermore, lactic acid evokes vascular effects similar to those of capsaicin and resiniferatoxin, possibly via interaction of protons and/or proton-generated substances at vanilloid receptors with a subsequent release of calcitonin gene-related peptide.

Effects of hCGRP 8-37 and the NK1-receptor antagonist SR 140.333 on capsaicin-evoked vasodilation in the pig nasal mucosa in vivo.

A novel pig in vivo model was used to study vascular effects of capsaicin, substance P and calcitonin gene-related peptide (CGRP) in the nasal mucosa and skin. An acoustic rhinometer was used to measure changes in nasal cavity volume, mainly representing changes in capacitance vessels in the vascular beds. The non-peptide NK1-receptor antagonist SR 140.333 and the CGRP-receptor antagonist hCGRP 8-37 were used to investigate the role of substance P and CGRP, respectively, in capsaicin-evoked vasodilation mediated through activation of sensory C-fibre afferents. In this study we show that SR 140.333 is a potent inhibitor of substance P-induced vasodilation in the nasal mucosa whereas it has no effect on the capsaicin-evoked responses. Substance P only elicited a minor and shortlasting increase in superficial skin blood flow; this response, however, was completely blocked after administration of SR 140.333. Capsaicin-evoked vasodilation in the skin was slightly reduced by SR 140.333. CGRP-induced vasodilation in the nasal mucosa and skin was of much longer duration than the substance P-induced response, and was thus similar to the vascular effects mediated by capsaicin. hCGRP 8-37 significantly reduced both the CGRP- and capsaicin-mediated vasodilation in the nasal mucosa and the decrease of nasal cavity volume. Although the peak vasodilation in the skin in response to capsaicin, was unaltered by blockade of CGRP-receptors, the integrated response was significantly reduced by hCGRP 8-37. The present results show that vasodilatory responses to activation of afferent nerves in the pig nasal mucosa and superficial skin are mainly dependent on CGRP, while NK1-receptor mechanisms seem to be of no or minor importance.

Sensory neuropeptides and nitric oxide in nasal vascular regulation.

1. RR may act as a preferential capsaicin antagonist in the pig nasal mucosa in vivo. However, the present data reveal a narrow concentration range for the selective actions of RR. Moreover, RR has systemic cardiovascular side effects despite local i.a. infusion in the IMA. 2. Acoustic rhinometry is a useful method for investigations of changes in nasal cavity volume in the pig in vivo. 3. The NK1-receptor antagonist RP-67,580 lacks NK1-receptor blocking properties in the pig in vivo. In contrast, CP-96,345 and SR 140.333 significantly blocked SP-mediated vascular effects in the pig nasal mucosa and superficial skin, indicating species dependent NK1-receptor selectivity. Capsaicin-induced vasodilatation in the IMA was not attenuated after administration of CP-96,345 and SR 140.333 whereas the superficial blood flow in the nasal mucosa and skin was slightly reduced. The CGRP-receptor antagonist hCGRP 8-37 markedly reduced the capsaicin-evoked vascular effects in the pig nasal mucosa and superficial skin. 4. Vanilloid receptors, as revealed by 3H-RTX binding, are present in the pig nasal mucosa although with different characteristics compared to vanilloid receptors in the pig dorsal horn. Capsaicin, RTX and LA evoked vasodilatation in the pig nasal mucosa in a similar fashion, indicating activation of sensory nerves. The LA (proton)-evoked vasodilatation was significantly attenuated after local i.a. infusion of hCGRP 8-37, closely resembling the results obtained from the capsaicin challenge before and after CGRP-receptor blockade. Capsazepine did not reduce the capsaicin-and LA-evoked vasodilation in the pig nasal mucosa. This agrees well with the observation that capsazepine did not inhibit RTX binding to vanilloid receptors in pig nasal mucosal membranes. 5. Capsaicin desensitisation of the human nasal mucosa attenuated the subjective pain response as well as the reduction of the cross-sectional area in the nasal cavity evoked by LA and hypertonic saline. This finding gives further support to the hypothesis that protons may act as endogenous ligands to the vanilloid receptor also in man. 6. Systemic administration of the NOS inhibitor L-NNA significantly reduced basal nasal V Con and increased C Vol in the pig. The effects evoked by L-NNA were similar in magnitude to those of phenylephrine and UK 14304, although of much longer duration. Administration of L-NNA did not reduce the vasodilator responses to SP and ACh, suggesting that these substances may mediate their vascular effects via one or several other mechanisms beside the NO/cGMP pathway. Moreover, capsaicin-, VIP-, and nitroprusside-evoked vasodilatation was not reduced after NOS inhibition. 7. Heavy physical exercise and alpha-adrenoceptor agonists reduce nasal cavity NO levels acutely in man. This may be due to a reduced supply of substrates for NO synthesis in the paranasal sinus epithelium, the primary NO production site in the upper airways. However, prolonged use of the alpha 2-adrenoceptor agonist oxymetazoline for 10 days, did not reduce basal nasal cavity NO levels. Nasal cavity NO levels and C Vol were not altered after topical administration of the NOS inhibitor L-NAME. Nor did we see any change in C Vol after local challenge with NO gas in the nasal cavity. The present results indicate that the human nasal mucosa is largely insensitive to NO gas in contrast to the bronchial mucosa and lung. 9. In conclusion, the present results suggest that vanilloid receptors are present on sensory nerves in the pig nasal mucosa and that LA (protons) may act as an endogenous ligand to this receptor. Sensory neuropeptides, especially CGRP, may be of importance for nasal congestion upon sensory nerve activation. Hence, selective, non-peptide CGRP-receptor antagonists may be of potential use in nasal disorders characterised by nasal congestion. NO is of importance for basal nasal vascular regulation. However, whether NOS inhibitors have potential as useful nasal de

High nitric oxide production in human paranasal sinuses.

Nitric oxide (NO) is present in air derived from the nasal airways. However, the precise origin and physiological role of airway-derived NO are unknown. We report that NO in humans is produced by epithelial cells in the paranasal sinuses and is present in sinus air in very high concentrations, close to the highest permissible atmospheric pollution levels. In immunohistochemical and mRNA in situ hybridization studies we show that an NO synthase most closely resembling the inducible isoform is constitutively expressed apically in sinus epithelium. In contrast, only weak NO synthase activity was found in the epithelium of the nasal cavity. Our findings, together with the well-known bacteriostatic effects of NO, suggest a role for NO in the maintenance of sterility in the human paranasal sinuses.

Capsaicin de-sensitization of the human nasal mucosa reduces pain and vascular effects of lactic acid and hypertonic saline.

The present study was initiated to investigate the effects of hypertonic saline (15%) or low pH (1 M lactic acid, pH 2) applied to the human nasal mucosa. Patients suffering from birch-pollen allergy, which had been de-sensitized with capsaicin, were compared to non-treated, healthy controls. Five patients were pre-treated with an intranasal, unilateral application of 30 microM capsaicin for 15 min during three consecutive days. Six weeks later we applied 50 microliters of hypertonic saline (15%) to the inferior turbinate on the capsaicin-pre-treated side of the patients as well as to the controls. Symptom score, using a visual analogue scale (VAS), and the cross-sectional area of the nasal cavity were measured bilaterally using acoustic rhinometry at different intervals. The same procedure was repeated one week later with lactic acid. Provocation with lactic acid and hypertonic saline caused a significantly higher symptom score in controls as compared to capsaicin-pre-treated patients. Furthermore, application of lactic acid caused a significant reduction in cross-sectional area of the nasal cavity suggesting vasodilatation in controls compared to capsaicin-pre-treated patients. The reactions to hypertonic saline were generally lower but the differences in symptom score between capsaicin-pre-treated and non-treated persons remained. The results implies that capsaicin-sensitive afferents are involved in low pH- and hypertonicity-mediated reactions in the human nasal mucosa. Furthermore, local capsaicin de-sensitization causes a very long-lasting loss of sensory reactivity to these agents.

Ruthenium red selectively attenuates capsaicin induced vasodilation in pig nasal mucosa.

The effect of ruthenium red, a blocker of transmembrane Ca2+ fluxes, on vasodilation in pig nasal mucosa induced by capsaicin, nicotine, bradykinin or histamine was evaluated in an in vivo preparation. To decrease toxic systemic effects pretreatment with ruthenium red was performed locally, intra-arterially in a nasal artery. Pretreatment with two doses of ruthenium red was evaluated (0.25 and 2.5 mg kg-1). Ruthenium red, in the low dose, resulted in a marked attenuation of the capsaicin-induced nasal vasodilation while the effect on the nicotine-induced vasodilation was not as prominent. Pretreatment with the high dose significantly blocked the vasodilatory effects of capsaicin, nicotine, bradykinin and histamine. Our series indicates that low concentration ruthenium red selectively modulates capsaicin induced vasodilation in pig nasal mucosa in vivo, probably via a direct blocking effect on cation channels opened by capsaicin. A high concentration of ruthenium red may exert a general inhibitory effect on transmembrane Ca2+ transport.

Effects of NK1 receptor antagonists on vasodilation induced by chemical and electrical activation of sensory C-fibre afferents in different organs.

The effects of the non-peptide NK1 receptor antagonists, CP 96,345 and RP-67,580, were investigated in a model using anaesthetized pigs. Both the blood flow in the internal maxillary and the bronchial artery (ultrasonic flowmetry) and the superficial blood flow in nasal mucosa and the skin (laser-Doppler flowmetry) were monitored simultaneously. Vasodilation induced by substance P administered i.v. systemically was blocked by pretreatment with CP-96,345, 3 mg kg-1 but not by RP-67,580. CP-96,345 had no effects on the vasodilation induced by calcitonin gene-related peptide or vasoactive intestinal polypeptide. The capsaicin-induced vasodilation in the superficial blood flow of the nasal mucosa and the skin, was reduced after the CP-96,345 pretreatment. The vasodilation induced by capsaicin infusion in the internal maxillary or the bronchial artery was not affected by the CP-96,345 pretreatment. Electrical stimulation of the vagal nerve induced a vasodilation in the bronchial circulation which was not attenuated by pretreatment with CP-96,345. In the nasal mucosa and the skin NK1 receptors seem to be involved in the vasodilation in the superficial small vessels, due to chemical activation of sensory C-fibre afferents. Furthermore, CP-96,345 is a useful tool in the evaluation of NK1 receptor-mediated responses. RP-67,580 which has been shown to have NK1 antagonistic properties in the rat has no such effects in the domestic pig.

Preliminary report: validity of symptom analysis and daytime polysomnography in diagnosis of sleep apnea.

The aim of this study was twofold: first, to see if the prevalence of the sleep apnea syndrome (SAS) in a given population could be fairly estimated by our patient questionnaire, mainly based upon the 1979 American Sleep Association definition of SAS; and second, to investigate whether the severity of SAS could be similarly accurately measured by daytime polysomnography (DPSG), as an alternative to the more demanding all-night polysomnography (NPSG). Of 42 patients consecutively examined due to rhonchopathy, 18 had the clinical diagnosis of SAS, which was based on the three symptoms--snoring, sleep disturbances and diurnal hypersomnia--if reported to occur habitually. In 11 patients the diagnosis was established by NPSG [apnea index (AI) greater than 10]. However, in only 10 of the 18 cases NPSG indicated the diagnosis giving a positive predictive value of 56%. When comparing DPSG versus NPSG in 36 patients, the AI ranged from -23 to +65, and the mean AI value was found to be twice as high in the former (mean difference 9.0 +/- 18.4; p less than 0.01). The positive predictive value of DPSG was 63% (10/16). Both the self-report and DPSG were burdened with some 25% false-positive results, and DPSG gave far too variable AI values to be reliable in staging the disease. On the other hand, the negative predictive values were high, 96% (23/24) and 100% (20/20), respectively, indicating their usefulness for screening purposes.