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Grouping strategies for exposure to inhalable dust, wheat allergens and alpha-amylase allergens in bakeries.

This paper describes repeated measurements of inhalable flour dust, wheat allergens and alpha-amylase allergens in the bakery industry. A total of 571 full-shift personal dust samples was collected. Wheat allergens and alpha-amylase allergens were measured in 449 and 507 samples, respectively, by the use of recently developed immunoassays. For all three measures of exposure, the main components of exposure variability were determined. Different grouping strategies for studying exposure-response relationships were compared. The specific job of a bakery worker was identified as the most important source of variability in inhalable flour dust concentrations. For exposure to wheat allergens, the job performed was also the most important source of variation, but type of bakery also explained some of the variability. For alpha-amylase allergen exposure, information on type of bakery was more important then job information. For exposure to inhalable dust and wheat allergens, a classification by job title would lead to sufficient contrast in average exposure levels. By contrast, a grouping strategy based on a combination of job and type of bakery appeared to be essential to obtain a useful classification of exposure to alpha-amylase allergens. 1997 British Occupational Hygiene Society.

Allergens↗

Stimulatory effects of vanadate on amylase release from isolated rat pancreatic acini.

The effects of vanadate on exocrine pancreatic function were examined in isolated rat pancreatic acini. Vanadate caused a concentration-dependent stimulation of amylase release above a concentration of 1 mM. Co-incubation of vanadate with vasoactive intestinal polypeptide, 8-bromoadenosine 3':5'-cyclic monophosphate, and the Ca2+ ionophore A23187 produced a synergistic pattern of amylase release, whereas co-incubation with cholecystokinin octapeptide (CCK-8), carbamylcholine, and 12-O-tetradecanoylphorbol 13-acetate produced an additive effect. Vanadate alone had no influence on acinar cyclic AMP content, Ca2+ efflux, or intracellular Ca2+ concentration. However, preincubation with vanadate prevented the plateau phase of CCK-8-induced Ca2+ transient increase from returning to baseline. Moreover, depletion of the intracellular Ca2+ pool by pretreatment of acini with CCK-8 in Ca2+-free medium (plus ethyleneglycol bis[beta-aminoethylether]-N,N'-tetraacetic acid) had no effect on subsequent stimulation by vanadate, although it abolished the response to both CCK-8 and carbamylcholine stimulation. The protein kinase C (PKC) inhibitors staurosporine and calphostin C significantly inhibited vanadate-stimulated amylase release, whereas the protein tyrosine kinase inhibitor genistein had no inhibitory effect. Moreover, vanadate caused a significant translocation of PKC from cytosol to membrane fraction in pancreatic acinar cells. This translocation was inhibited significantly by staurosporine and calphostin C but not by genistein. These results suggest that vanadate acts directly on pancreatic acini and stimulates amylase release by activating PKC without an effect on Ca2+ mobilization, cyclic AMP, or protein tyrosine kinase.

8-Bromo Cyclic Adenosine Monophosphate↗

Suitability and limitations of methods for characterisation of activity of malto-oligosaccharide-forming amylases.

The suitability and limitations of essential methods and reference substrates used for characterisation of activity of amylolytic enzymes is investigated. Saccharogenic, chromogenic and chromatographic methods are included. The results are discussed in relation to the measurement of reaction rates, determination of action mode and product specificity and the impact on identification and nomenclature of malto-oligosaccharide-forming amylases. An accurate determination of reaction rates using the saccharogenic methods strongly depends on the degree of polymerisation (DP) of the standards used and the hydrolysis products formed by the amylase. Particularly the use of glucose as standard can lead to overestimates due to the differences in the reducing potential of glucose and malto-oligosaccharides. The reliability of the chromogenic methods for determination of action mode depends on the DP of the substrate and the specificity of the amylase. For a characterisation of the starch hydrolysis products and the variation in the DP during hydrolysis, high performance anion-exchange chromatography with pulsed amperometric detection provided a fast and reliable method. A literature survey revealed varying and inconsistent use of nomenclature of malto-oligosaccharide forming amylases. Therefore a systematic approach identifying three main classes of activity is suggested using not only the mode of action and the DP of the major product but also the stage of hydrolysis at which this product is formed.

Amylases↗

Specificity of amylases and cyclodextrin-glucanotransferase in reactions with 2-deoxy-maltooligosaccharides.

2-Deoxy-maltooligosaccharides of different chain length were tested as substrates for exo- and endo-amylases. Cleavage occurred with beta-amylase, yielding 2,2'-dideoxy-maltose, and with amyloglucosidase. With the alpha-amylase from Thermomonospora curvata tris-(2-deoxy)-maltotriose and the corresponding tetra- and pentasaccharides were formed. Porcine pancreatic alpha-amylase did not tolerate the deoxygenated substrate, nor were cyclization experiments with cyclodextrin-glucanotransferase (CGT) successful. In a coupling reaction with CGT, however, a series of transfer products to the acceptor 2-deoxyglucose were obtained.

Amylases↗

Evaluation of a new assay for pancreatic amylase: performance characteristics and estimation of reference intervals.

We have evaluated a new assay for the specific determination of pancreatic (P) isoamylase, the principle being based on a synergistic inhibitory effect of two monoclonal antibodies directed towards salivary (S) amylase. After 3 min incubation, activities were determined with maltoheptaoside-PNP as substrate on a Hitachi 705 analyzer at 25, 30 and 37 degrees C, respectively. Coefficients of variation ranged from 0.6 to 5.4% for within-run and 2.1 to 9.9% for day-to-day precision. Linearity held up to 1200 U/L (25 degrees C) and 2300 U/L (37 degrees C). Comparison of the new method with the wheat germ inhibitor technique showed an excellent correlation, with coefficients ranging from 0.990 to 1.00. Using purified P- and S-amylase we observed no inhibiting cross-reactivity of the antibodies with the P-isoenzyme, but an incomplete blockage of S-amylase: residual activity was approximately 2% at 25 and 30 degrees C, and 2.5% at 37 degrees C. The distribution pattern of P-, S- and total amylase activity in serum of healthy subjects was only slightly skewed to the right. We found neither an influence of sex nor of age on the reference ranges. In random urine samples, distribution of activities was strongly skewed. However, if the activity was related to the urinary creatinine concentration, an approximately normal distribution was obtained, allowing, as in serum, the establishment of upper and lower reference values.

Adolescent↗

Effects of K+ channel blockers and K+ ionophore on isoprenaline-induced secretion of amylase from rat parotid acini.

The involvement of K+ channels in regulating secretion of amylase from isolated rat parotid acini was studied in conjunction with beta-adrenoceptor function. It was observed that increasing the concentration of extracellular K- or adding K+ channel blockers enhanced the secretion of amylase. Among several K+ channel blockers, tetraethylammonium, apamin and charybdotoxin were effective to enhance secretion by 48, 69 and 84%, respectively. Glibenclamide was without effect. A low concentration of isoprenaline (10(-7) M) enhanced secretion by 154% and its simultaneous application with tetraethylammonium gave a synergistic effect, producing 371% stimulation. Combination of tetraethylammonium and a low concentration of carbachol (10(-6) M) did not give the synergistic effect. Isoprenaline at the concentration of 10(-6) M enhanced secretion by 313% and this was reduced to 116% by 10(-5) M valinomycin, a K+ ionophore. Valinomycin was without effect on carbachol (10(-5) M)-induced secretion. Somatostatin (10(-5) M) and morphine (10(-4) M) also reduced isoprenaline-induced secretion of amylase. These results suggested the regulation of Ca(2+)-activated K+ channels by isoprenaline in amylase secretory processes in parotid acini.

Adrenergic beta-Agonists↗

Evidence that the glucoamylases and alpha-amylase secreted by Aspergillus niger are proteolytically processed products of a precursor enzyme.

A 125-kDa starch hydrolysing enzyme of Aspergillus niger characterised by its ability to dextrinise and saccharify starch [Suresh et al. (1999) Appl. Microbiol. Biotechnol. 51, 673-675] was also found to possess activity towards raw starch. Segregation of these activities in the 71-kDa glucoamylase and a 53-kDa alpha-amylase-like enzyme supported by antibody cross-reactivity studies and the isolation of mutants based on assay screens for the secretion of particular enzyme forms revealed the 125-kDa starch hydrolysing enzyme as their precursor. N-terminal sequence analysis further revealed that the 71-kDa glucoamylase was the N-terminal product of the precursor enzyme. Immunological cross reactivity of the 53-kDa amylase with antibodies raised against the precursor enzyme but not with the 71- and 61-kDa glucoamylase antibodies suggested that this enzyme activity is represented by the C-terminal fragment of the precursor. The N-terminal sequence of the 53-kDa protein showed similarity to the reported Taka amylase of Aspergillus oryzae. Antibody cross-reactivity to a 10-kDa non-enzymic peptide and a 61-kDa glucoamylase described these proteins as products of the 71-kDa glucoamylase. Identification of only the precursor starch hydrolysing enzyme in the protein extracts of fungal protoplasts suggested proteolytic processing in the cellular periplasmic space as the cause for the secretion of multiple forms of amylases by A. niger.

Amino Acid Sequence↗

Evidence that cleavage of the precursor enzyme by autocatalysis caused secretion of multiple amylases by Aspergillus niger.

The observation that a mutant strain of Aspergillus niger isolated for protease overproduction accumulated Taka-amylase supported an earlier report that processing of the precursor amylase by protease resulted in the secretion of multiple amylases. Studies using a mutant strain revealed that such processing was not due to aspergillopepsin but to autocatalysis by an inherent protease activity of the precursor and glucoamylase. Alignment of protease sequences with glucoamylase showed regions of consensus with serine carboxypeptidase of A. niger. Thus point mutations in this region due to ultraviolet radiation apparently caused the mutant to evolve with enhanced protease activity that degraded the precursor and accumulated Taka-amylase.

Amylases↗

Affinity column purification of amylases on protein inhibitors from wheat kernel.

An affinity column was devised for the purification of a large number of amylases inhibited by the albumin from wheat kernel. The procedure involved linking the protein inhibitors from wheat to Sepharose and then specifically eluting the amylase adsorbed to the gel with a high concentration of maltose. By this procedure, the amylases from Tenebrio molitor L. (yellow mealworm) larvae and chicken pancreas were purified to homogeneity with good yields for the first time, as shown by both alkaline and acidic electrophoresis. Human saliva alpha-amylase, purified by the same procedure, showed specific activity and electrophoretic patterns similar to those obtained by other workers with different techniques.

Adsorption↗

Control of postprandial hyperglycaemia by galactosyl maltobionolactone and its novel anti-amylase effect in mice.

The ability to control carbohydrate digestion is useful in the treatment of diabetes mellitus and obesity. In the present study, we examined whether recently developed 4(2)-O-beta-D-galactosyl maltobionolactone (LG2O) having anti-amylase activity is able to control postprandial blood glucose concentration in mice. In addition, we tried to determine how LG2O regulates carbohydrate delivery in the gut lumen by conducting in vivo and in vitro studies. Male non-diabetic ddY mice and KK-A(y) mice, a spontaneously diabetic strain, had free access to a carbohydrate rich diet supplemented with LG2O (3 or 10 g/kg) for 0.5 hr, and blood glucose concentration was measured. LG2O suppressed any steep increase in postprandial blood glucose concentration in both ddY and KK-A(y) mice. Corresponding to the blood glucose response, LG2O also markedly suppressed any increase in postprandial plasma insulin concentration. After ingestion of the diet, LG2O produced a 1.5-3.5 fold increase in the gut contents and reducible sugar content in the small intestine but not in the stomach. Although alpha-amylase activity in the stomach was much lower compared with the activity in the small intestine, LG2O still strongly inhibited alpha-amylase activity in the stomach. In contrast, LG2O had little or no influence on alpha-amylase activity in the proximal intestine. From the in vitro carbohydrate digestion stimulation, LG2O at 7.5 mM decreased glucose production by 75% for dextrin, 25% for alpha-starch and 60% for raw starch. In conclusion, administration of LG2O inhibits carbohydrate digestion in the gut, and produces significant improvements in both blood glucose and insulin response following ingestion as part of the diet, and this evidence provides support for its therapeutic potential in treating diabetes mellitus and obesity.

Amylases↗

Effect of a purified amylase inhibitor on carbohydrate tolerance in normal subjects and patients with diabetes mellitus.

Slowing starch digestion by inhibiting amylase activity in the intestinal lumen should improve postprandial carbohydrate tolerance in patients with diabetes mellitus. Crude bean-derived amylase inhibitor ("starch blocker") that contains only minimal antiamylase activity, however, does not modify carbohydrate assimilation. To test the validity of the "starch blockade" concept, we assessed the effect of a partially purified bean-derived amylase inhibitor with increased antiamylase activity on carbohydrate tolerance in normal subjects and in patients with non-insulin-dependent diabetes mellitus. In comparison with a placebo, ingestion of this inhibitor with 50 g of starch substantially reduced postprandial increases in plasma concentrations of glucose and insulin in both normal subjects and those with diabetes. We conclude that a purified amylase inhibitor is effective and potentially beneficial in the treatment of diabetes mellitus.

Adult↗

Gibbestatin B inhibits the GA-induced expression of alpha-amylase expression in cereal seeds.

The expression of alpha-amylase in aleurone layers of barley is known to be induced by gibberellin A3 (GA). In the present study, gibbestatin B (GNB) was isolated from Streptomyces sp. C-39 as an inhibitor of the GA-induced expression of alpha-amylase in barley and rice, with IC50 values of 125 and 70 microM, respectively. GNB suppressed accumulation of GA-induced barley high-pI type B and rice RAmylA alpha-amylase transcripts. However, GNB showed no inhibitory activity on GUS expression in transgenic tobacco harboring the auxin-inducible par B promoter:: GUS fusion gene. The transcription of an abscisic acid (ABA)-inducible gene, HVA1, was unaffected by GNB. In addition, GNB prevented aleurone cells from cell death induced by GA. In tobacco and Arabidopsis plants, GNB suppressed the germination and retarded the growth of seedlings without toxicity. The growth of gai, spy and abi mutants was also retarded by GNB. Normal plants treated with GA-biosynthesis inhibitors and GA-defective and GA-signaling mutants normally have dwarf dark green leaves. However, dwarfed healthy green leaves were observed in normal plants treated with GNB. GA-induced stem elongation of plants was also detected in the presence of GNB. These analyses indicate that GNB inhibits the GA-induced expression of alpha-amylase by regulating one of the steps involved in ABA signaling, but not by acting as a weak ABA analog.

Abscisic Acid↗

Amylase inhibitors of pigeonpea (Cajanus cajan) seeds.

Pigeonpea (Cajanus cajan L) seeds were analysed quantitatively for amylase inhibitor (AI) activity and qualitatively, by an in-gel-detection method on polyacrylamide gels. At least four AI isoforms were identified in pigeonpea seeds. The AIs inhibit human salivary and bovine pancreatic amylase but fail to inhibit bacterial, fungal and endogenous amylase. Pigeonpea AIs were found to be active over a pH range of 4.5 to 9.5 and were heat labile. The isoelectric point of a major inhibitor is 6.2 AIs were tolerant to proteolysis by trypsin, chymotrypsin, bromelain and endogenous pigeonpea proteases. Pigeonpea AIs were synthesized during late seed development and also degraded during late germination. Addition of AIs or protease inhibitors (PIs) alone to a diet of Helicoverpa armigera larvae did not increase mortality. However, the larvae reared on a diet containing AIs and PIs in combination, showed increased mortality and adverse effects on larval growth and development. In vitro inhibition of Helicoverpa gut amylase revealed that only 22% activity is sensitive to inhibitors. Further investigations on interactions of pigeonpea AIs and PIs with Helicoverpa gut enzymes is necessary to develop strategies to strengthen defense mechanisms in pigeonpea against H. armigera.

Amylases↗

Amylase levels in preservation solutions as a marker of exocrine tissue injury and as a prognostic factor for pancreatic islet isolation.

Occurrence of primary graft nonfunction of pancreatic islets demands research for new methods of organ preservation during cold ischemia conditions. Digestive enzymes released during preservation injure the islets for subsequent rewarming and islet isolation processes. The aim of our study was to assess the amylase level in preservation solution as a marker of exocrine tissue injury, allowing the prognosis of islet yield and viability. The experiments undertaken on rats used three commercially available preservation solutions: ViaSpan (UW); Custodiol (HTK); and Euro-Collins (EC). After 180 minutes of cold ischemia, the highest islet recovery was observed among pancreata stored in UW solution (508 +/- 139 vs HTK 344 +/- 103; P <.05 vs EC 322 +/- 113; P <.05). These islets also revealed the highest insulin stimulation index in glucose static tests (1.19 +/- 0.30 vs HTK, 0.87 +/- 0.43; P <.01, vs EC.25 +/-.06; P <.001). The highest amylase level in the preservation solution was associated with a decreased yield of islets during the isolation process and lowest insulin stimulation index (increasing 139 +/- 18% for EC, 108 +/- 12% for HTK; P <.05 vs 87 +/- 10% for UW; P <.05). Our data strongly suggest, that the dynamic of amylase release during pancreas preservation at 4 degrees C correlates with a reduced number and viability of isolated islets. These results suggest that measurement of amylase levels after pancreas preservation may have potential clinical application as a marker to evaluate pancreatic tissue injury.

Amylases↗

Studies on the formation of alpha-amylase by Thermomonospora vulgaris.

Conditions affecting the formation of alpha-amylase by static cultures of the thermophilic actinomycete Thermomonospora vulgaris were studied. The organism failed to grow under submerged culture conditions or when the culture medium was devoid of CaCO3-alpha-Amylase was produced during the logarithmic phase of growth and maximum yield was obtained after 3 to 9 days of incubation. Growth and amylase formation took place only in a range from 45 degrees to 55 degrees C; optimum temperature was 55 degrees C. Of the tested carbon sources only starch induced enzyme formation. Maximum enzyme yield was obtained when starch concentration of the medium was 2% and when ammonium citrate served as a nitrogen source. Crushed clay pots could substitute for CaCO3 of the medium, but growth and amylase yield were less.

Amylases↗

The human pancreatic alpha-amylase isoforms: isolation, structural studies and kinetics of inhibition by acarbose.

A rapid method is proposed for isolating the two main components of human pancreatic alpha-amylase (HPA I and HPA II). The isoelectric point of HPA I (7.2), the main component, was determined using an isoelectrofocusing method and found to differ from that of HPA II (6. 6). The molecular mass of HPA I (55862+/-5 Da) and that of HPA II (55786+/-5 Da) were determined by performing mass spectrometry and found to be quite similar to that of the protein moiety calculated from the amino acid sequence (55788 Da), which indicates that the human amylase is not glycosylated. The structure of both HPA I and HPA II was further investigated by performing limited proteolysis. Two fragments with an apparent molecular mass of 41 kDa and 14 kDa were obtained by digesting the isoforms with proteinase K and subtilisin, whereas digestion with papain yielded two cleaved fragments with molecular masses of 38 kDa and 17 kDa. Proteinase K and subtilisin susceptible bonds are located in the L8 loop (A domain), while the papain cut which occurs in the presence of the calcium chelator EDTA is in the L3 loop (B domain). The kinetics of the inhibition of HPA I and HPA II by acarbose, a drug used to treat diabetes and obesity, were studied using an amylose substrate. The Lineweaver-Burk primary plots of HPA I and HPA II, which did not differ significantly, indicated that the inhibition was of the mixed non-competitive type. The secondary plots gave parabolic curves. All in all, these data provide evidence that two acarbose molecules bind to HPA. In conclusion, apart from the pI, no significant differences were observed between HPA I and HPA II as regards either their molecular mass and limited proteolysis or their kinetic behavior. As was to be expected in view of the high degree of structural identity previously found to exist between human and porcine pancreatic amylases, the present data show that the inhibitory effects of acarbose on the kinetic behavior of these two amylases are quite comparable. In particular, the process of amylose hydrolysis catalyzed by HPA as well as by PPA in both cases requires two carbohydrate binding sites in addition to the catalytic site.

Acarbose↗

Cyclic AMP potentiates substance P-induced amylase secretion by augmenting the effect of calcium in the rat parotid acinar cells.

Potentiation of amylase secretion by the combination of isoproterenol and substance P was examined in perfused rat parotid acinar cells. Combined additions of substance P and isoproterenol evoked biphasic changes in amylase secretion, an initial large peak and the following sustained plateau: the magnitudes of the both responses were higher than the sum of the responses induced by each agonist alone. Isoproterenol also increased the maximum response and the apparent affinity (EC50) for substance P to evoke the initial peak response; the EC50 values were about 20 and 0.8 nM, respectively, in the absence and the presence of isoproterenol. On the other hand, 1 nM substance P was sufficient for evoking the maximum potentiation of the sustained plateau response. Substance P did not change the EC50 for isoproterenol. The effect of isoproterenol was mimicked with dibutyryl cyclic AMP and agonists that increase parotid cyclic AMP. Omission of Ca2+ or addition of 5 mM nickel chloride almost completely abolished the potentiation of the sustained plateau, but little decreased that of the initial peak. Depletion of Ca2+ in InsP3-sensitive intracellular stores with thapsigargin, on the other hand, decreased the initial peak response, but not the sustained plateau, to substance P. The potentiation was also observed between isoproterenol and Ca2+ ionophores. Switching to the solutions containing higher concentrations of Ca2+ during the continuous stimulation with isoproterenol or IBMX evoked a large, but transient, response of amylase secretion. Time course of changes in amylase secretion induced by isoproterenol and substance P in combination was very similar to that of substance P, but not of isoproterenol. Isoproterenol did not enhance the effect of substance P on [Ca2+]i. These results show that the potentiation is mainly, if not totally, caused by cyclic AMP-induced enhancement of the potency and the efficacy in the pathway regulated by Ca2+.

1-Methyl-3-isobutylxanthine↗

Study of starch fermentation at low pH by Lactobacillus fermentum Ogi E1 reveals uncoupling between growth and alpha-amylase production at pH 4.0.

Lactobacillus fermentum Ogi E1 is an amylolytic heterofermentative lactic acid bacterium previously isolated from ogi, a Benin maize sourdough. In the present study, the effect of different pH between 3.5 and 6.0 on starch fermentation products and alpha-amylase production was investigated. Whereas a pH of 5.0 was optimum for specific growth rate and lactic acid production, growth was only slightly affected at suboptimal pH of 4.0 and 6.0. Over a pH range of 6.0 to 3.5, yields of product formation from substrate and of biomass relative to ATP were constant. These results showed that L. fermentum Ogi E1 was particularly acid tolerant, and well adapted to the acid conditions that develop during natural fermentation of cereal doughs. This acid tolerance may partly explain the dominance of L. fermentum in various traditional African sourdoughs. Surprisingly, alpha-amylase production, unlike growth, dropped dramatically when the strain was cultivated at pH 4.0 with starch. With maltose as substrate, the yield of alpha-amylase relative to biomass remained unchanged at pH 4.0 and 5.0, unlike that observed with starch. Based on the distribution of enzyme activity between extra- and intracellular fractions and fermentation kinetics, it appears that starch was first hydrolyzed into dextrins by alpha-amylase activity, and maltose was produced from dextrins by extracellular enzyme activity, transferred into the cell and then hydrolyzed into glucose by intracellular alpha-glucosidase.

Bread↗