Longer Pretreatment Research Articles

Rapid characterization of Maillard reaction products in heat-treated honey by nanoelectrospray ionization mass spectrometry.

Amadori rearrangement products (ARPs) and α-dicarbonyl compounds (α-DCs) are critical intermediates in the Maillard chemistry. The screening of artificially heated honey (AH) is currently based on chromatography-mass spectrometry, which is commonly accompanied with the longer pretreatment and detection time. Here, low-abundance ARPs were detected directly in high-sugar environment by nanoelectrospray ionization mass spectrometry (nanoESI-MS) coupled with borosilicate glass capillaries (O-tips). When O-tips were replaced by borosilicate theta capillaries (θ-tips), the microdroplets allowed the derivatization of α-DCs to be accomplished on the millisecond timescale, rather than hours in conventional protocols. The results indicated that two ARPs and α-DCs of m/z 235 were significantly up-regulated in AH. Meanwhile, the straightforward differentiation between naturally matured honey (NH) and AH was achieved by nanoESI-MS fingerprints combined with multivariate analysis. The method may provide a rapid characterization of Maillard reaction products (MRPs), which exhibits the great application potential in other complex food matrix.

Do β-adrenoceptor agonists induce homologous or heterologous desensitization in rat urinary bladder?

β3-Adrenoceptor agonists have recently been introduced for the symptomatic treatment of the overactive bladder syndrome. As such treatment is not curative, long-term treatment is anticipated to be required. As the susceptibility of β3-adrenoceptors to undergo agonist-induced desensitization is cell type- and tissue-dependent, we have explored whether pre-treatment with a β-adrenoceptor agonist will attenuate subsequent relaxation responses to freshly added agonist using rat urinary bladder as a model. We have used the prototypical β-adrenoceptor agonist isoprenaline, the β2-selective fenoterol and the β3-selective CL 316,243 and mirabegron as well as the receptor-independent bladder relaxant forskolin. We show that a 6-h pre-treatment with agonist can significantly reduce subsequent relaxation against KCl-induced smooth muscle tone, but agonist-induced desensitization was also observed with longer pre-treatments or against passive tension. The agonist-induced desensitization was prominent for the β2 component of rat bladder relaxation but much weaker or even absent for the β3 component. Moreover, β-adrenoceptor agonist pre-treatment reduced contractile responses to the muscarinic agonist carbachol and the receptor-independent stimulus KCl. Taken together these data do not support the hypothesis that the long-term clinical efficacy of β3-adrenoceptor agonists in the treatment of the overactive bladder syndrome will be limited by receptor desensitization. Rather they raise the possibility that such treatment may not only cause smooth muscle relaxation but also may attenuate hyper-contractility of the bladder.

Immuno-laser capture microdissection of frozen prolactioma sections to prepare proteomic samples

Laser capture microdissection (LCM) technology combined with immunohistochemistry (immuno-LCM) is a valuable tool to obtain specific target cell populations and therefore this technique enables more accurate proteomic profile. In this study, we optimized the regular immuno-LCM technique to isolate and stain pure prolactin cells from either normal human pituitary ( n = 6) or prolactioma ( n = 11). Compared with the routine procedure, more intense and specific staining could be obtained when sections were pretreated with 0.2% Triton X-100 for 4 min. Interestingly, longer pretreatment (0.2% Triton X-100 for 10 min) or higher concentration (2% Triton X-100 for 4 and 10 min) greatly impaired labeling intensity and cell shape. Further scanning electron microscope study revealed that the component extracted from the cell surface by Triton X-100 was lipid. Using the optimized immuno-LCM technique, more pure prolactin cells could be isolated and prepared for further proteomic analysis. Taken together, we reported an optimized immuno-LCM technique that could effectively dissect pure target cells in different type pituitary adenomas for further proteomics analysis.

The influence of electrochemical pre-treatment of B-doped diamond films on the electrodeposition of Pt

The influence of the substrate electrochemical pre-treatment in 0.5 mol L-1 H2SO4 on the Pt electrodeposition on boron-doped diamond, BDD, film electrodes was investigated. Platinum cannot be electrodeposited on a freshly prepared BDD electrode; however, potentiodynamic cycling or anodic potential steps at short times does activate the electrode. Anodic pre-treatment plays a dual role in the behavior of Pt deposition on BDD surfaces: Pt deposition is increased at short-term anodic pre-treatments, whereas at longer pre-treatment times Pt deposition was inhibited. These facts are explained in terms of wettability changes and passivation of the surface. Conversely, the oxide layer formed in these treatments increases the dispersion level of the catalyst.

Distinctive roles for prolactin and growth hormone in the activation of signal transducer and activator of transcription 5 in pancreatic islets of langerhans.

Although the beta-cells of the pancreatic islets of Langerhans express both prolactin (PRL) and GH receptors, we have observed that PRL is considerably more effective than GH in the up-regulation of islet function in vitro. This study examined whether differences in the activation of the Janus kinase 2/signal transducer and activator of transcription (STAT) 5 signaling pathway by these closely related receptors may be involved in this disparity. The activation of STAT5B by PRL was biphasic, with an initial peak within 30 min, a nadir between 1 and 3 h, and prolonged activation after 4 h. In contrast, the response to GH was transient for 1 h. The importance of the long-term activation of STAT5B by PRL was supported by the similar dose response curves for STAT5B activation and the PRL-induced increases in insulin secretion and islet cell proliferation. Because the pulsatile secretion of GH affects its actions in other target tissues, the ability of pretreatment with either hormone to affect subsequent stimulation was also examined. Surprisingly, the response to PRL was inhibited by prior exposure for less than 3 h to either PRL or GH and disappeared with a longer pretreatment with either hormone. Similar to other tissues, the response to GH was inhibited by any length of prior exposure to GH. However, pretreatment with PRL had no effect. These experiments are the first demonstration of the transient desensitization of the PRL receptor by either PRL or GH pretreatment in any tissue and the desensitization of GH stimulation in islet cells. These observations provide insight into the mechanisms that regulate the desensitization of these receptors and, more importantly, allow the long-term activation of STAT5B by the PRL receptor. These results may apply to other members of the cytokine superfamily of receptors. We also demonstrate that the increase in islet cell proliferation required continuous stimulation with PRL, whereas the smaller effect with GH occurred with either continuous or pulsatile stimulation. In summary, this study demonstrates that islets are sensitive to the temporal pattern of stimulation by these hormones and provides a new basis for understanding their physiological roles in the regulation of islet function.

Cardiovascular effects of nicotine, chlorisondamine, and mecamylamine in the pigeon.

Chlorisondamine and mecamylamine are nicotinic antagonists that produce both ganglionic and central blockade. Chlorisondamine, when administered as a large systemic dose, produces a persistent central block, despite being charged. The present study evaluated the cardiovascular effects of chlorisondamine. Shortly after administration, chlorisondamine (0.10, 1, and 10 mg/kg i.m.) lowered blood pressure significantly and decreased heart rate at the low dose (0.1 mg/kg i.m.) and increased heart rate at the high dose (10 mg/kg i.m.). Mecamylamine (1 and 10 mg/kg i.m.) also lowered blood pressure and heart rate. After both antagonists, heart rate returned to baseline values within 90 min and blood pressure within 24 h. Low doses of nicotine (0.01-0.03 mg/kg i.m.) lowered blood pressure but did not affect heart rate. Higher doses (0.10-3.2 mg/kg i.m.) transiently increased blood pressure and heart rate. Subsequent to antagonist administration, nicotine was administered to determine whether either drug blocked the cardiovascular effects of nicotine. Chlorisondamine (0.1, 1, and 10 mg/kg i.m.) administered 30 min before nicotine blocked the increases in blood pressure and heart rate. Only the high dose (10 mg/kg i.m.) of chlorisondamine administered 24 h before nicotine produced a blockade of nicotine's pressor effect. This block diminished within 3 days. Mecamylamine (1 mg/kg i.m.) antagonized only nicotine's tachycardic effect. Longer pretreatment with mecamylamine (10 mg/kg, 24 h before nicotine challenge) did not antagonize the cardiovascular effects of nicotine. Thus, chlorisondamine produces a longer lasting blockade of nicotine's cardiovascular effects than mecamylamine.

Post-antifungal effect and effects of sub-MIC concentrations on previously treated Candida spp.: influence of exposure time and concentration.

This study evaluates the influence of exposure time and concentration on the post-antifungal effect (PAFE) and the effect of sub-MIC concentrations (1/4 x MIC) on Candida albicans and C. glabrata in the PAFE stage (PAFSE). This stage was induced by pretreatment for 1.5, 3 or 12 h with 1 x, 4 x or 8 x MIC of 4 antifungal agents fundamental to modern candidiasis therapy. The length of the 2 effects studied was dependent on the concentration of the antifungal agent applied during pretreatment, as well as on the exposure time. An increase in the dose and/or longer pretreatment prolonged the duration of the PAFE and PAFSE in both species and with all the antifungal agents. Significant PAFEs were always observed for amphotericin B and 5-fluorocytosine (0.8-13 h and 0.6-10.8 h, respectively). These values were increased (by 2.3-8.7 h and 1.5-7.8 h, respectively) by posterior exposure to 1/4 x MIC of the respective antifungal agent. Neither ketoconazole nor fluconazole were able to induce significant PAFEs, even with exposures of up to 12 h duration and a dose of 8 x MIC. However, treatment with 1/4 x MIC of each of the 2 azoles led to significant PAFSEs in both yeast species, of up to 6.5 h duration with ketoconazole and 1.7 h with fluconazole, if the concentrations and/or exposure times were sufficiently high.

Interspecific and intraspecific variation in seed size and germination requirements of Sarracenia (Sarraceniaceae)

Seed size and germination requirements of eight (of nine) Sarracenia species, and 13 populations of S. purpurea were studied. All species except for S. purpurea are restricted to the southeastern United States, whereas S. purpurea ranges across Canada, southward along the eastern United States into Maryland and Virginia (S. purpurea ssp. purpurea), and from New Jersey southward into northern Florida and the coast of the Gulf of Mexico (S. purpurea ssp. venosa). I tested the hypotheses that dormancy-breaking requirements vary predictably among species across a latitudinal gradient. I also sought to determine whether seed size and germination requirements were useful characters for resolving systematic and phylogenetic questions within this genus. Seed size varied significantly among species, but variability in seed size within S. purpurea exceeded the variability in seed size observed across all eight species studied. Seeds of all species are morphophysiologically dormant upon dispersal. Length of required cool, moist pretreatment varied among species, and germination in higher latitude populations is enhanced with longer pretreatment. In contrast, variability in germination requirements of subspecies, varieties, and populations of the geographically wide-ranging S. purpurea was not related clearly to geographic location (latitude or elevation). Germination requirements do not map onto a proposed phylogeny of Sarracenia, but observed differences in germination requirements of S. purpurea ssp. venosa var. burkii relative to other populations of S. purpurea support the recent proposal to elevate this variety to species status.

Influence of the organic solvents on the activity in water and the conformation of Candida rugosa lipase: Description of a lipase-activating pretreatment

The influence on lipase activity in water of a pretreatment on Candida rugosa lipase using water miscible and immiscible solvents was studied. The lipase activity in the hydrolysis of esteric substrates in aqueous media increases when the lipase was previously treated with various nearly anhydrous organic media. This activation, which was irreversible, was higher for longer pretreatment times. It was dependent on the pretreatment medium (water activity and solvent used). A relation between variations in the emission intensity and the activities of treated and untreated lipases was found. Activating pretreatment did not shift the peak of fluorescence emission but gave rise to variations in the secondary protein structure by increasing the helical nature. A similar increment in the hydrolysis rate in water can be obtained with the addition of an appropriate amount of solvent (acetonitrile or n-heptane) to the aqueous reaction medium.

Contrasting responses of sulphate and phosphate transport in barley (Hordeum vulgare L.) roots to protein-modifying reagents and inhibition of protein synthesis

The uptake of sulphate into roots of barley seedlings is highly sensitive to phenylglyoxal (PhG), an arginine-binding reagent. Uptake was inhibited by >80% by a 1-h pre-treatment of roots with 0.45 mol · m(-3) PhG. Inhibition was maximal in pre-treatment solutions buffered between pH 4.5 and 6.5. Phosphate uptake, measured simultaneously by double-labelling uptake solutions with (32)P and (35)S, was less susceptible to inhibition by PhG, particularly at pH <6.5, and was completely insensitive to the less permeant reagent p-hydroxyphenylglyoxal (OH-PhG) administered at 1 mol · m(-3) at pH at 5.0 or 8.2; sulphate uptake was inhibited in -S plants by 90% by OH-PhG-treatment. Root respiration in young root segments was unaffected by OH-PhG pre-treatment for 1 h and inhibited by only 17% after 90 min pre-treatment. The uptake of both ions was inhibited by the dithiol-specific reagent, phenylarsine oxide even after short exposures (0.5-5.0 min). Sulphate uptake was more severely inhibited than that of phosphate, but in both cases inhibition could be substantially reversed by 5 min washing of treated roots by 5 mol · m(-3) dithioerythritol. After longer pre-treatment (50 min) with phenylarsine oxide, inhibition of the ion fluxes was not relieved by washing with dithioerythritol. Inhibition of sulphate influx by PhG was completely reversed by washing the roots for 24 h with culture solution lacking the inhibitor. The reversal was dependent on protein synthesis; less than 20% recovery was seen in the presence of 50 mmol · m(-3) cycloheximide. Sulphate uptake declined rapidly when -S roots were treated with cycloheximide. In the same roots the phosphate influx was little affected, small significant inhibitions being seen only after 4 h of treatment. Respiration was depressed by only 20% in apical and by 31% in basal root segments by cycloheximide pre-treatment for 2 h. Similar rates of collapse of the sulphate uptake and insensitivity of phosphate uptake were seen when protein synthesis was inhibited by azetidine carboxylic acid, p-fluorophenylalanine and puromycin. Considering the effects of all of the protein-synthesis inhibitors together leads to the conclusion that the sulphate transporter itself, or some essential sub-component of the uptake system, turns over rapidly with a half-time of about 2.5 h. The turnover of the phosphate transporter is evidently much slower. The results are discussed in relation to strategies for identifying the transport proteins and to the regulation of transporter activity during nutrient stress.

Activin A increases the number of follicle-stimulating hormone cells in anterior pituitary cultures

The mode of action of activin A on the anterior pituitary gland (AP) was investigated using primary cultured cells. The AP cells were cultured with activin A (1 ng/ml) at various cell densities for 24–96 h, and further incubated for 3 h with activin A-free medium. When the cells were pretreated with activin A for 48 h, follicle-stimulating hormone (FSH) secretion during the following 3-h incubation was increased only at a density of 1 × 10 5 cells/200 mm 2, but not at 2 × 10 5 or 4 × 10 5 cells/200 mm 2. A longer pretreatment (96 h) was required in order to induce this response at a density of 2 × 10 5 cells/200 mm 2. Luteinizing hormone (LH) secretion was not affected by activin A. Thus, the FSH secretory activity of the primary culture of AP was stimulated by activin A in a cell density-dependent manner. Furthermore, it was found that treatment with activin A (10 ng/ml) for 72 h increased the number of immunoreactive FSH cells by 25–41%, and that these newly induced FSH cells were low responders to gonadotropin-releasing hormone stimulation. The proportions of immunoreactive LH, thyroid-stimulating hormone, prolactin or growth hormone cells were not affected. From these results, we conclude that activin A increases FSH secretion by changing the cell population of pituitary gonadotropes.

Photorespiratory ammonium release by the cyanobacterium Anabaena cylindrica in the presence of methionine sulfoximine

A release of ammonium by non-nitrogen-fixing Anabaena cylindrica (grown on NH4Cl) in the presence of MSX (methionine sulfoximine) and absence of any external nitrogen source was found. In the light the release was maximal at 0.2 mM MSX, a concentration which did not affect net CO2 fixation nor the glycollate excretion, but inhibited the glutamine synthetase activity and the reassimilation of ammonium. It is suggested that the major source of the ammonium released is the photorespiratory conversion of glycine to serine as (1) the release was stimulated by increase in light intensity, (2) high CO2 (3%) lowered the release, if not given as a longer pretreatment (as CO2 or HCO 3 - ) when a stimulation was observed, (3) glyoxylate and glutamate stimulated the release, the latter compound particularly under nitrogen-deficient conditions and (4) isonicotinic acid hydrazide caused a reduced release of ammonium. Furthermore, a substantial part of the ammonium released by N2-fixing A. cylindrica in presence of MSX may thus originate from the glycollate pathway. The data show that in the light the glycine to serine conversion is active in cyanobacteria with a concomitant production of ammonium which is assimilated by glutamine synthetase.

The contracture produced by sodium removal in the non-pregnant rat myometrium.

1. Mechanical responses to Na removal were investigated in the circular and longitudinal muscles of the non-pregnant rat myometrium at 35 degrees C. In both muscles, reduction of the external Na concentration to less than 20 mM produced an initial acceleration of phasic contractions and a sustained tonic contracture. No difference was found with different Na substitutes (Tris-hydroxymethyl aminomethane, choline, dimethyl diethanol ammonium). However, when Mg was substituted for Na, only the tonic contracture was produced without the phasic contractions. 2. Readmission of 5-10 mM-Na, after exposure to Na-free solution, relaxed the contracture produced by Na removal. The degree of relaxation was dependent on the Na concentration readmitted and on the period of pre-treatment with Na-free solution, being stronger with longer pre-treatment. 3. In the presence of Na, excess Ca failed to increase the muscle tone. In the absence of Na, the tension development was closely related to the external Ca concentration up to 20 mM. In the absence of both Ca and Na, some tension remained. Even after pre-treatment with Ca-free solution containing 0.1-0.5 mM EGTA, removal of Na caused some mechanical response. A similar small tension development was observed when Na removal was repeated during prolonged absence of external Ca for more than 3 h. 4. Verapamil (2 X 10(-4) M) markedly suppressed the response to Na removal, but it did not block it, either in the presence or in the absence of Ca. Ouabain (10(-3) M) in the presence of verapamil potentiated the early phasic component of the response to Na removal, but the tonic component was little affected or even slightly reduced. 5. The results indicate that there are three components in the mechanical response to Na removal: the phasic and tonic components, which are highly Ca-dependent, and the third small tonic component, which is independent of external Ca. Most of the phasic and tonic responses seem to be due to an increase in Ca permeability, but this may be secondary to membrane depolarization. A Na-Ca exchange mechanism is also considered to contribute to the transient phase of the response to Na removal and to Na readmission.

Acute protection against arachidonate toxicity by hydrocortisone and dexamethasone in mice

1. 1. Pharmacologic doses of hydrocortisone sodium succinate (100 mg/kg) has a rapid protective action against arachidonate-induced mortality in mice when administered intravenously 5 to 60 min before intravenous infusion of arachidonate. 2. 2. Intravenous dexamethasone sodium phosphate (4 mg/kg) has a similar protective effect. 3. 3. The time course of the development of the protective effect of intravenous glucocorticoids suggests that DNA transcription and protein synthesis are not involved in the mechanism of this action. 4. 4. Longer pretreatment with glucocorticoids are required to reduce mortality when subcutaneous of intraperitoneal routes of administration of the steroids used.

Adenovirus early gene products may control viral mRNA accumulation and translation in vivo

The mechanisms controlling early adenovirus gene expression in vivo have been studied using inhibitors of protein synthesis. When inhibitors were added shortly before or at the onset of infection, viral mRNA from all early regions was transcribed, spliced and accumulated over a 7 hr period. After longer pretreatment, accumulation of several early mRNAs were suppressed. Addition of inhibitors 1 hr after infection enhanced the accumulation of viral mRNA in the cytoplasm. Translation of early mRNA selected on adenovirus DNA in a cell-free system reflected the amount of viral mRNA present. A viral coded product may therefore control accumulation of viral mRNA. A different pattern emerged when inhibitors of protein synthesis were removed at 5 hr postinfection and cells were pulse-labeled in vivo. If inhibitors were introduced at or before infection, early viral proteins were synthesized only after a lag of 1–3 hr. However, if treatment was introduced 1 hr post-infection, reversion of the protein synthesis block was instantaneous. It appears that protein synthesis inhibitors reveal an in vivo translational block for viral mRNA. This block could be overcome by preinfection with a related virus. Furthermore, no block was observed in a virus-transformed human embryonic kidney cell line (293) which expresses early region 1 of the viral genome. Viral gene product(s) encoded in early region 1 may control translation of early adenovirus messenger RNA in vivo.

The effect of 2,4-dichlorophenoxyacetic acid and (2-chloroethyl)-trimethylammonium chloride on chlorophyll synthesis in barley leaves

2.4-dichlorophenoxyacetic acid (2.4-D) and (2-chloroethyl)-trimethylammonium chloride (CCC) inhibit chlorophyll synthesis and protochlorophyllide 652 regeneration in 6-8 day old barley leaves whilst having little effect on the rates of protochlorophyll 632 synthesis from exogenous δ-aminelevulinic acid (ALA) and ALA-dehydratase activity. Longer pretreatments with 2.4-D and CCC show it is only after 50 to 60 hr that the rates of P632 production from exogenous ALA and ALA-dehydratase activity are affected. Similar response times were obtained for chloramphenicol (CAP). The results indicate that 2.4-D and CCC may act by directly inhibiting specific plastid-protein synthesis similar to CAP. Hence it seems that it is only those proteins (enzymes) having a rapid turnover that are affected first i.e. the enzymes necessary for ALA synthesis in the plastid.