Structural basis of the substrate specificity and instability in solution of a glycosidase from Lactobacillus plantarum.

Statistics from structural genomics initiatives reveal that around 50-55% of the expressed, non-membrane proteins cannot be purified and therefore structurally characterized due to solubility problems, which emphasized protein solubility as one of the most serious concerns in structural biology projects. Lactobacillus plantarum CECT 748T produces an aggregation-prone glycosidase (LpBgl) that we crystallized previously. However, this result could not be reproduced due to protein instability and therefore further high-resolution structural analyses of LpBgl were impeded. The obtained crystals of LpBgl diffracted up to 2.48Å resolution and permitted to solve the structure of the enzyme. Analysis of the active site revealed a pocket for phosphate-binding with an uncommon architecture, where a phosphate molecule is tightly bound suggesting the recognition of 6-phosphoryl sugars. In agreement with this observation, we showed that LpBgl exhibited 6-phospho-ß-glucosidase activity. Combination of structural and mass spectrometry results revealed the formation of dimethyl arsenic adducts on the solvent exposed cysteine residues Cys211 and Cys292. Remarkably, the double mutant Cys211Ser/Cys292Ser resulted stable in solution at high concentrations indicating that the marginal solubility of LpBgl can be ascribed specifically to these two cysteine residues. The 2.30Å crystal structure of this double mutant showed no disorder around the newly incorporated serine residues and also loop rearrangements within the phosphate-binding site. Notably, LpBgl could be prepared at high yield by proteolytic digestion of the fusion protein LSLt-LpBgl, which raises important questions about potential hysteretic processes upon its initial production as an enzyme fused to a solubility enhancer.

Efficacy and safety of ketotifen in young children with asthma.

Thirty children (aged 1 to 3 years) with mild to moderate asthma were entered into a 28-week double-blind, crossover study comparing the prophylactic effect of ketotifen and placebo. A patient daily diary card was used to document symptoms and concomitant medications taken during a 2-week baseline and subsequent 12-week drug/placebo period. This was followed by a 2-week washout and 12 more weeks of drug/placebo in a crossover design. Physician assessment was performed at the beginning, at the end of each period, and at 4-week intervals throughout the drug study. No significant difference was observed between ketotifen treatment and placebo treatment in any of the study parameters that were tested. A decrease in concomitant medication usage during the first treatment period with ketotifen was observed. The principle side effect of ketotifen therapy observed in this age group was weight gain. In contrast to studies in adults, no sedation was noted.

Selectivity of quercetin inhibition on stimulated amylase release in rat pancreatic acini.

Quercetin (Q) has been shown to inhibit Ca2+-dependent processes. The present study evaluates the effect of Q on amylase release stimulated by various agonists in dispersed rat pancreatic acini. Q inhibited amylase release stimulated by an optimal concentration of carbachol. The inhibition was dependent on Q concentration. Preincubation with Q was not necessary. Maximal inhibition (up to 60% of control) was reached at 50 microM of Q and was completely reversible. Full responsiveness of the acini to agonist stimulation was reestablished as early as 5 min upon the removal of Q. At 50 microM, Q inhibited stimulated amylase release by optimal concentrations of tetradecanoylphorbol-13-acetate (TPA) (10(-6) M), A23187 (3 x 10(-6) M), cholecystokinin C-terminal octapeptide (CCK-OP) (10(-9) M) and carbachol (3 x 10(-6) M), but not by vasoactive intestinal polypeptide (VIP) (3 x 10(-7) M). Instead, Q promoted amylase release stimulated by VIP. The inhibition of amylase secretion by Q occurred only at near optimal, optimal, and supraoptimal concentrations of TPA, A23187, CCK-OP, and carbachol. The potentiation effect of Q on VIP-stimulated amylase secretion was, however, seen at all concentrations of VIP used (10(-8) to 10(-6) M). Quercetin also inhibited protein kinase C activity from rat pancreas in a dose-dependent manner. Maximal inhibition (approximately 85%) was seen at 100 microM of Q. These results provide further support that the intermediary steps for stimulated enzyme secretion in pancreatic acini by TPA, A23187, CCK-OP, and carbachol involve calmodulin and/or protein kinase C, whereas VIP does not.(ABSTRACT TRUNCATED AT 250 WORDS)

Phorbol ester attenuates cholecystokinin-stimulated amylase release in pancreatic acini of rats.

12-O-tetradecanoylphorbol 13-acetate (TPA) and cholecystokinin octapeptide stimulate amylase secretion in dispersed pancreatic acini, presumably acting via the activation of protein kinase C. In this study, we examined TPA pretreatment on the subsequent response of rat pancreatic acini to secretagogues. Acini exposed to TPA (3 X 10(-7) M) at 37 degrees C reduced the subsequent amylase secretion as stimulated by cholecystokinin octapeptide and carbachol, but not by A23187 or VIP. The optimal effect was obtained after 5 min of preincubation with TPA. Longer incubation did not result in greater attenuation. The degree of attenuation was dependent on the concentration of TPA used in the pretreatment. Maximal effect was seen at TPA concentrations of 10(-7) M and higher. Preincubation with TPA resulted in alterations of the dose response of pancreatic acini to cholecystokinin octapeptide. A decrease in amylase secretion was obtained at optimal and suboptimal but not at supraoptimal concentrations of cholecystokinin octapeptide. The peak response to cholecystokinin octapeptide, furthermore, was shifted almost 1 log unit to the right, suggesting a decrease in cholecystokinin binding of the acini following TPA treatment. Binding studies demonstrated a reduction in the specific binding of 125I-labelled cholecystokinin octapeptide to acini following TPA treatment. Analysis of binding data revealed a decrease in affinity and binding capacity of the high-affinity component. No significant change in the binding capacity was detected with the low-affinity component, but a great increase in its affinity was observed. This suggests that the attenuation effect by TPA on the cholecystokinin octapeptide response in rat pancreatic acini in vitro is at the receptor level.

Effects of hydralazine, nitroglycerin, and food on estimated hepatic blood flow.

Several recent studies have shown that hydralazine and nitroglycerin may increase the apparent oral bioavailability of high-clearance drugs. It has been postulated that the mechanism responsible may be a vasodilator-induced transient increase in hepatic blood flow with an associated reduction in first-pass metabolism. To test this hypothesis, we examined the effect of hydralazine (25 mg) and sublingual nitroglycerin (2 doses of 0.6 mg separated by 30 minutes) on indocyanine green (ICG) blood clearance (ClB). Forty minutes after the start of nitroglycerin therapy, ICG ClB fell from a baseline of 648 +/- 98 to 607 +/- 151 ml/min, and was further decreased to 578 +/- 98 ml/min 80 minutes after dosing. Hydralazine induced no consistent effect on ICG ClB. ICG ClB was 744 +/- 376, 721 +/- 218, and 763 +/- 195 ml/min at baseline, 40 minutes, and 80 minutes after dosing. As a positive control, ICG ClB was assessed after a high-protein meal. After this meal, ICG ClB increased from 656 +/- 107 to 811 +/- 141 and 801 +/- 132 ml/min at 40 and 80 minutes after dosing. These data suggest that one or more mechanism(s) other than changes in hepatic blood flow are involved in the vasodilator-induced increase in the apparent oral bioavailability of high-clearance drugs.

Characteristics of lymphocyte and macrophage reactivity in otitis media with effusion.

On the basis of our observations and previously published data, it is proposed that in at least certain forms of chronic otitis media with effusion there is a relative predominance of macrophage--monocyte cells which are actively immunosuppressive for the proliferative response to mitogens and specific antigens, as well as for the synthesis of specific immunoglobulins. Such immunological hypo-responsiveness may be related to the pathogenesis of chronic otitis media with such effusions.

Immunologic aspects of otitis media with effusion. II. Nature of cell-mediated immunosuppressive activity in middle ear fluid.

The effects of middle ear effusions from patients with serous, seromucoid, mucoid, and purulent forms of otitis media with effusion on lymphoproliferative responses and polyclonal immunoglobulin synthesis were determined in peripheral blood and adenoidal lymphocytes. The responses were studied after stimulation of lymphocytes by phytohemagglutinin, pokeweed mitogen, purified protein derivative, herpes simplex virus, sheep red blood cells, or ovalbumin. Serous and seromucoid middle ear effusions resulted in significant suppression of proliferative responses to phytohemagglutinin, purified protein derivative, and herpes simplex virus and of polyclonal immunoglobulin synthesis in response to pokeweed mitogen, sheep red blood cells, and ovalbumin. The suppressive activity appeared to be associated with low-molecular-weight soluble products liberated by esterase-positive adherent population of cells in middle ear fluid.

Immunologic aspects of otitis media with effusion: characteristics of lymphocyte and macrophage reactivity.

The characteristics of cellular elements in the middle ear effusions, peripheral blood, and adenoidal tissue and ther effects of middle ear macrophages on the functional activity of lymphocytes were examined in 50 subjects with chronic otitis media. Macrophages were the predominant cell type in middle ear effusions, and they constituted up to 63% of the total number of cells. T cells were 11%-66% of the total number of cells. The proliferative responses induced by phytohemagglutinin or pokeweed mitogen in middle ear lymphocytes were generally low compared with the responses observed in peripheral blood and adenoidal lymphocytes. Cocultures of middle ear macrophages with adenoidal lymphocytes resulted in a significant depression of proliferative response and immunoglobulin synthesis in the lymphocytes. These observations suggest that macrophages in the middle ear may have a profound influence on the regulation of the immune response in the middle ear in patients who have otitis media with effusion.