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PubMed1992

Diurnal variation of amylase secretion is coupled with alterations of beta-adrenoceptors in the rat parotid gland.

Diurnal changes in the neurotransmitter receptors are important for studying the receptor function in neurophysiology. The purpose of this study is to gain an insight into the regulatory mechanisms of the diurnal variation of amylase secretion. Rat salivary amylase levels showed a diurnal variation with two peaks, a marked peak at 13 h and a lesser peak at 21 h. This increase in salivary amylase levels was completely inhibited by pretreatment of rats with the beta-adrenergic antagonist propranolol, but not by the alpha-adrenergic antagonist phentolamine. Amylase level in parotid tissue homogenate also showed a diurnal change, but there was only one peak, at 13 h. The number of maximal binding sites (Bmax) for [3H]dihydroalprenolol (DHA) in parotid membranes showed a diurnal variation with two marked peaks at 13 and 21 h, but the affinity of parotid beta-adrenoceptors for agonists or antagonists did not show any diurnal changes. Phosphorylation of nuclear non-histone proteins in the rat parotid gland showed diurnal variation with two marked peaks at 13 and 21 h. These results indicate that a diurnal variation in the number of rat parotid beta-adrenoceptors, which is presumably regulated by gene expression, is coupled with a change in salivary amylase secretion.

Amylases↗
PubMed1984

In vitro and in vivo effect of proglumide on cholecystokinin-stimulated amylase release in mouse pancreatic acini.

The effect of proglumide on cholecystokinin (CCK)-stimulated amylase release was studied in vitro and in vivo in dispersed acini from mouse pancreas. In an in vitro study, proglumide at concentrations between 0.3-10 mM inhibited CCK-stimulated amylase release dose-dependently, while proglumide did not influence the basal amylase release at concentrations between 0-3 mM. Dose-response curves to CCK for amylase release shifted to the right with increase in proglumide concentration. This inhibition by proglumide was reversible. In addition, the effect of proglumide was selective for CCK and its related peptide, and this drug did not inhibit other secretagogues such as carbachol or gastrin releasing peptide in mouse acini. In contrast to its inhibitory effect in vitro, in vivo administration of proglumide (500 mg/kg/day, i.p., for 5 days) to mice did not cause the rightward shift of the dose-response curve to CCK for amylase release from dispersed acini.

Amylases↗
PubMedDate not supplied

Separation of alpha-amylase by reversed micellar extraction. Effect of solvent type and cosolvent concentration on the transfer process.

The recovery of alpha-amylase from the crude enzyme preparation by the reversed micellar liquid-liquid extraction was investigated. The reversed micellar solution was formed by dissolving a cationic surfactant Aliquat 336 in six different alkanes (cyclohexane, n-hexane, isooctane, n-octane, n-decane, and n-dodecane) respectively with addition of a cosolvent n-octanol. It was found that a minimal quantity of n-octanol was needed for Aliquat 336 to dissolve in apolar solvent and form reversed micelles. Furthermore, this minimal amount of n-octanol needed was found to be different when Aliquat 336 was dissolved in different alkanes. It tended to increase with the number of carbon atoms in alkane and also depended on the solvent structure. During the forward extraction process, it was revealed that a high value of solubilization of protein in Aliquat 336 reversed micelles could be achieved when four out of the six alkanes (cyclohexane, n-hexane, isooctane, n-octane) were used as the solvent for Aliquat 336. After a full forward and backward extraction cycle, however, a high recovery of both the protein mass and alpha-amylase activity in the stripping solution could be obtained only when two out of the six alkanes (n-hexane and isooctane) were used as the solvent for Aliquat 336. When n-hexane and isooctane were used as the solvent for Aliquat 336, up to 80% of the total alpha-amylase activity in the crude enzyme preparation could be recovered at the end of extraction cycle, meanwhile alpha-amylase could be concentrated about 1.4-fold. In the cases of other four alkanes (cyclohexane, n-octane, n-decane, and n-dodecane) as solvent, most of the alpha-amylase activity in the crude enzyme preparation would be denatured after an extraction cycle.

1-Octanol↗
PubMed1990

The anticoordinate changes of pancreatic lipase and colipase activity to amylase activity induced by adrenalectomy in normal and diabetic rats.

The effect of adrenalectomy (Adx) on pancreatic lipase and colipase activities in normal, streptozotocin-induced diabetic, and obese Zucker rats was studied. It was found that Adx induced a rapid decrease in amylase activity, a rapid increase in lipase activity, and a slow increase in colipase activity in normal rats. Twenty days after Adx, the levels of amylase activity were 70% lower than control, whereas the activities of lipase and colipase were 55 and 25% higher than control, respectively. The replacement of hydrocortisone normalized the changes in the three enzyme activities. Injection of insulin partially corrected the amylase activity, but had no significant effect on the activities of lipase and colipase. In streptozotocin-induced diabetic rats and obese Zucker rats, amylase activity was decreased, and lipase and colipase activities increased, compared to normal rats. These changes of enzyme activity were further enhanced by Adx. It is concluded that Adx results in an anticoordinate change in pancreatic lipase, colipase, and amylase activities, suggesting that glucocorticoid also influences the synthesis of the pancreatic lipolytic enzymes.

Adrenal Glands↗
PubMed2000

Caffeine inhibits a low affinity but not a high affinity mechanism for cholecystokinin-evoked Ca2+ signalling and amylase release from guinea pig pancreatic acini.

Caffeine has been found to inhibit the formation and action of Ca2+-mobilizing inositol 1,4,5-trisphosphate (IP3) in pancreatic acinar cells. The aim of the present study was to investigate the effects of caffeine on cytoplasmic Ca2+ concentrations ([Ca2+]i) and amylase release in response to different agonists. [Ca2+]i was determined by cytofluorometry using fura-2 as indicator and amylase release with a substrate reagent. Stimulation with low concentrations of carbachol or cholecystokinin octapeptide (CCK-8) induces [Ca2+]i oscillations whereas higher concentrations cause sustained elevation of [Ca2+]i. The less efficacious agonists pilocarpine and CCK-JMV-180 evoke oscillations only. Caffeine inhibited carbachol-induced elevation of [Ca2+]i and amylase responses in a competitive manner, abolishing the responses to low and incompletely inhibiting the responses to high concentrations of the agonist. Also, the [Ca2+]i elevations by pilocarpine were abolished by caffeine. The effects on CCK-8-induced elevation of [Ca2+]i and amylase secretion were paradoxical, the caffeine inhibition being more pronounced at high than at low concentrations of CCK-8. This enigma was further emphasized by moderate effects of caffeine on the responses to CCK-JMV-180. The results indicate that carbachol, pilocarpine and high concentrations of CCK-8 elicit IP3-mediated responses and that CCK-JMV-180 and low concentrations of CCK-8 elevate [Ca2+]i and stimulate amylase release by another signal transduction mechanism.

Amylases↗
PubMed2001

alpha-Amylase production in high cell density submerged cultivation of Aspergillus oryzae and A. nidulans.

The effect of biomass concentration on the formation of Aspergillus oryzae alpha-amylase during submerged cultivation with A. oryzae and recombinant A. nidulans strains has been investigated. It was found that the specific rate of alpha-amylase formation in chemostats decreased significantly with increasing biomass concentration in the range of approx. 2-12 g dry weight kg(-1). When using a recombinant A. nidulans strain in which the gene responsible for carbon catabolite repression of the A. oryzae alpha-amylase gene (creA) was deleted, no significant decrease in the specific rate of alpha-amylase formation was observed. On the basis of the experimental results, it is suggested that the low value of the specific alpha-amylase productivity observed at high biomass concentration is caused by slow mixing of the concentrated feed solution in the viscous fermentation medium.

Aspergillus nidulans↗
PubMed1997

Purification of alpha-amylase by specific elution from anti-peptide antibodies.

Chimeric alpha-amylase, produced by recombinant yeast cells, was purified by immunoaffinity chromatography by use of an anti-peptide antibody and an eluent containing an antigen peptide. Chimeric alpha-amylase was adsorbed by the antibody against the peptide corresponding to the C-terminal region of target alpha-amylase, and specifically eluted by the eluent containing the antigen peptide used for immunization. A low concentration of the peptide could competitively elute adsorbed alpha-amylase, and the rate-limiting step of the elution was mass transfer of desorbed alpha-amylase. With this specific method, target proteins can be effectively eluted, and highly purified under mild conditions, from the antibody ligand showing a high-affinity for the adsorption step

Adsorption↗
PubMed1998

Secretion, purification, and characterisation of barley alpha-amylase produced by heterologous gene expression in Aspergillus niger.

Efficient production of recombinant barley alpha-amylase has been achieved in Aspergillus niger. The cDNA encoding alpha-amylase isozyme 1 (AMY1) and its signal peptide was placed under the control of the Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter and the A. nidulans trpC gene terminator. Secretion yields up to 60 mg/l were obtained in media optimised for alpha-amylase activity and low protease activity. The recombinant AMY1 (reAMY1) was purified to homogeneity and found to be identical to native barley AMY1 with respect to size, pI, and immunoreactivity. N-terminal sequence analysis of the recombinant protein indicated that the endogenous plant signal peptide is correctly processed in A. niger. Electrospray ionisation/mass spectrometry gave a molecular mass for the dominant form of 44,960 Da, in accordance with the loss of the LQRS C-terminal residues; glycosylation apparently did not occur. The activities of recombinant and native barley alpha-amylases are very similar towards insoluble and soluble starch as well as 2-chloro-4-nitrophenol beta-D-maltoheptaoside and amylose (degree of polymerisation = 17). Barley alpha-amylase is the first plant protein efficiently secreted and correctly processed by A. niger using its own signal sequence.

Aspergillus niger↗
PubMed2000

Amylase mRNA expression in Crassostrea gigas during feeding cycles.

A Crassostrea gigas digestive gland copy DNA (cDNA) library constructed in the lambda phage ZapII (Stratagene, La Jola, USA) was screened with an amylase heterologous proble. To get access to the complete cDNA, a polymerase chain reaction extension was conducted using DNA extracted from the phages. The complete cDNA sequence is 1688 base pairs (EMBL = Y08370). The deduced protein sequence is 519 aminoacids long with a 19 aminoacid signal peptide. Similarity with Pecten maximus amylase is 72%. A 3-day nutrition experiment with a cyclic algal food supply was carried out. Amylase enzyme activities and mRNAs were individually measured on five animals, nine times a day. Messenger RNAs were quantified by dot hybridization using the previously characterized cDNA as probe. Variation of amylase mRNA was observed, in relation with the level of activity of the enzyme. Coordinated changes in RNA and enzyme levels suggested a possible transcriptional regulation of amylase in C. gigas as in vertebrates.

Amino Acid Sequence↗
PubMed2005

A primary culture of parotid acinar cells retaining capacity for agonists-induced amylase secretion and generation of new secretory granules.

Exocrine acinar cells, like parotid cells, have difficulty in maintaining their functions in cell lines or in primary cultures. For this reason, molecular studies on exocrine cell functions are unsatisfactory. To examine the mechanisms whereby the functions of parotid acinar cells are maintained, we attempted to establish a system for primary culture and transfection of exogenous genes. Acinar cells were dispersed from rat parotid glands by digestion with enzymes and were cultured in a medium containing rat serum. Most of the cultured cells had secretory granules that contained amylase, suggesting that they were derived from acinar cells, although they spread on the dish surface and formed filopodia. The cultured cells retained both granules and the ability to release amylase in response to beta-adrenergic and cholinergic agonists, even 48 h after dispersion. However, the total amount of amylase in the cells decreased rapidly from 24 to 48 h after dispersion. These results suggested that amylase synthesis was more damaged than the machinery for exocytosis during culture in vitro. VAMP2 gene fused with enhanced green fluorescence protein was transfected into the dispersed acinar cells, and VAMP2 protein was expressed and localized to amylase-containing granules, as normally seen for endogenous VAMP2 protein. This indicated that new granules were generated, and that protein sorting was functional. The cells cultured by this method maintained their functions for at least 48 h. They can be used for examining the effects of exogenous genes on parotid acinar cell functions, such as regulated exocytosis and the maturation of secretory granules.

Adrenergic beta-Agonists↗
PubMed1998

Duodenal secretion of phospholipase A2, amylase, and bicarbonate in chronic pancreatitis.

Phospholipase A2 has been suggested to be involved in the pathogenesis and pathophysiology of acute pancreatitis. We determined phospholipase A2 and amylase activities in duodenal juice collected during a secretin test from 30 consecutive patients who were suspected to have chronic pancreatitis or biliary disease. The patients underwent endoscopic retrograde cholangiopancreatography (ERCP) the following day. In the 8 patients with ERCP findings of advanced chronic pancreatitis, the mean outputs of phospholipase A2, amylase, and bicarbonate were reduced by 74%, 72%, and 60% compared to the respective values in the 13 (control) patients without a diagnosis of any pancreatic disorder or jaundice. In the 3 patients with recurrent pancreatitis but normal ERCP findings and in the 6 patients with jaundice the output values were not significantly reduced compared to those in the patients without any pancreatic disorder or jaundice. The outputs of amylase and phospholipase A2 were not significantly interrelated, whereas the outputs of phospholipase A2 and bicarbonate correlated well. Receiver characteristic (ROC) curves confirmed the high specificity and sensitivity of phospholipase A2 or bicarbonate output in patients with ERCP findings of advanced chronic pancreatitis compared to those with no changes in pancreatic ducts, with similar probability values of 0.880 +/- 0.111 (SEM), compared to the respective lower value of amylase, 0.676 +/- 0.118. Phospholipase A2 and bicarbonate output proved of equal value as markers of chronic pancreatitis and were superior to amylase output in the secretin test.

Acute Disease↗
PubMed1998

Extracellular alpha-amylase from Thermus filiformis Ork A2: purification and biochemical characterization.

An extracellular alpha-amylase produced by the thermophilic bacterium Thermus filiformis Ork A2 was purified from cell-free culture supernatant by ion exchange chromatography. The molecular mass was estimated to be 60,000 Da by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was rich in both basic and hydrophobic amino acids, presenting the following NH2-terminal amino acid sequence: Thr-Ala-Asp-Leu-Ile-Val-Lys-Ile-Asn-Phe. Amylolytic activity on soluble starch was optimal at pH 5.5-6.0 and 95 degrees C, and the enzyme was stable in the pH range of 4.0-8.0. Calcium enhanced thermostability at temperatures above 80 degrees C, increasing the half-life of activity to more than 8 h at 85 degrees C, 80 min at 90 degrees C, and 19 min at 95 degrees C. Ethylenediaminetetraacetic acid (EDTA) inhibited amylase activity, the inhibition being reversed by the addition of calcium or strontium ions. The alpha-amylase was also inhibited by copper and mercuric ions, and p-chloromercuribenzoic acid, the latter being reversed in the presence of dithiothreitol. Dithiothreitol and beta-mercaptoethanol activated the enzyme. The alpha-amylase exhibited Michaelis-Menten kinetics for starch, with a K(m) of 5.0 mg.ml-1 and kcat/K(m) of 5.2 x 10(5) ml.mg-1s-1. Similar values were obtained for amylose, amylopectin, and glycogen. The hydrolysis pattern was similar for maltooligosaccharides and polysaccharides, with maltose being the major hydrolysis product. Glucose and maltotriose were generated as secondary products, although glucose was produced in high levels after a 6-h digestion. To our knowledge this is the first report of the characterization of an alpha-amylase from a strain of the genus Thermus.

Amino Acid Sequence↗
PubMed2005

Antioxidant status, alpha-amylase production, growth, and survival of hemoglobin bearing Escherichia coli exposed to hypochlorous acid.

In the present work, two matched strains of E. coli that bear a recombinant R-amylase gene (MK57) or the R-amylase gene and vgb (MK79-hemoglobin expressing strain) were exposed to HOCl. In these cells, glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), alpha-amylase production, growth and lethality were assessed in the presence and absence of HOCl. It was observed that the hemoglobin makes cells highly susceptible to killing by HOCl. The maximum survival for both strains was with stationary phase cells at any concentration of HOCl. Both strains grown in the presence of 0.0125-0.075 mg/liter HOCl showed a substantial increase in SOD activity and GSH level, with MK79 being the most increased strain in this respect, while the level of CAT activity was decreased in a dose depended manner. Growth of MK57 and MK79 strains decreased as HOCl concentration increased. However, HOCl at concentration above zero enhanced alpha-amylase production (about 2-fold) in both MK79 and MK57. Furthermore, total amylase production (at all HOCl concentrations) by MK79 was always greater than that by MK57. The results indicate that except for survival, the hemoglobin helps cells to grow better and produces more recombinant products and activates general defense systems more in response to oxidative stress when compared with the non-hemoglobin-containing counterpart.

Antioxidants↗
PubMed1981

Clinical significance of elevated serum and urine amylase levels in patients with appendicitis.

During the 45 month period beginning January 1977, 251 patients with a pathologically confirmed diagnosis of acute appendicitis underwent celiotomy at the Medical College of Virginia Hospital. A preoperative serum or urine amylase determination was recorded in 155 of the patients (62 percent). Of this group, 15 patients (10 percent) had elevation of serum amylase or 2 hour urine amylase. Hyperamylasemia or hyperamylasuria directly led to misdiagnosis or treatment delay in 5 of the 15 patients. Appendiceal rupture occurred in three patients, two of whom had prolonged (greater than 1 month) hospitalizations directly attributable to the misdiagnosis. As a result of this study, we conclude that (1) acute appendicitis and elevated amylase levels may occur concurrently, (2) hyperamylasemia or hyperamylasuria should not dissuade the surgeon from early operation if other clinical features suggest appendicitis, and (3) abdominal pain and elevation of amylase level define significant intraabdominal disease, not specifically pancreatic disease.

Adolescent↗
PubMed1989

Effects of secretory nerve stimulation on the movement of rat parotid amylase into the circulation.

Stimulation of the nerves to the rat parotid gland demonstrated that increases in serum amylase levels can originate exclusively from this gland. However, whilst parasympathetic nerve stimulation caused a significant movement of parotid amylase into the blood, sympathetic nerve stimulation did not, despite the higher concentration of amylase in the saliva evoked. Experiments in which conscious animals were fed for a short period showed that parotid amylase reaches the blood during normal activity. The mechanisms underlying the movement of parotid amylase into the circulation are still unknown but there appears to be some dependence on the volume of saliva being produced.

Amylases↗
PubMed1989

Effects of liquid diet on amylase activity and mRNA levels in rat parotid gland.

Rats maintained on this diet demonstrated a dramatic reduction in amylase enzyme activity. This reduced level was evident through quantitation based on activity per mg gland protein, as well as on activity per gland wet weight. This was confirmed by the densitometric scanning of Western blots of sodium dodecyl sulphate-polyacrylamide gels of total parotid gland protein. Immune precipitation of enzyme activity from cell lysates showed that liquid-diet animals had lower levels of amylase than chow-fed controls. Isolation of poly (A+)-containing RNA followed by in vitro translation indicated that the steady-state level of amylase-specific mRNA had increased relative to the total mRNA pool. This outcome was also observed by dot-blot analysis of the cellular RNA using an amylase cDNA probe. Thus the regulation of amylase synthesis in rats maintained on liquid diets may take place at the level of protein translation, as well as of differential mRNA stability or gene transcription.

Amylases↗
PubMed1990

The effect of the chewing side on parotid amylase secretion in conscious rabbits.

This study was conducted to investigate whether changes in amylase output accompany changes in salivary flow which occur during feeding. New Zealand White rabbits were trained to eat food offered to them in the laboratory. After chronic parotid duct cannulation, saliva was collected and flow rates recorded bilaterally while the animals fed on fresh carrots or pelleted food. The amylase concentration (U/ml) obtained with carrots was greater than with pellets, but because of the differences in flow rate, the overall amylase output (U/min) for both foods did not differ. There was no difference in the amylase concentration in saliva collected either ipsilateral to or contralateral to the chewing side. However, amylase output was significantly greater ipsilateral than contralateral to chewing with both pellets (p less than 0.01) and carrots (p less than 0.05).

Amylases↗
PubMed1986

Mechanism of the inhibitory effect of trifluoperazine on isoprenaline-evoked amylase secretion from isolated rat parotid glands.

We have investigated the effects of the calmodulin antagonist trifluoperazine (TFP) on amylase secretion and adenosine 3':5'-monophosphate (cyclic AMP) metabolism using incubated parotid glands of young rats. Exposing unstimulated glands to 100 microM TFP doubled the basal rate of amylase and lactate dehydrogenase (LDH) release, but had no effect on either the parotid cyclic AMP or ATP contents. Isoprenaline (1 microM) stimulated amylase secretion and increased the tissue cyclic AMP content. 100 microM TFP inhibited these responses by 46% and 33%, respectively. N6,O2-dibutyryl adenosine 3',5'-monophosphate (dibutyryl cyclic AMP) mimicked the effect of isoprenaline on amylase release but 100 microM TFP had no effect on this response. 10 microM TFP inhibited F- -stimulated adenylate cyclase activity in a subcellular fraction isolated from the parotid by 32%. We conclude that TFP may inhibit isoprenaline-evoked amylase secretion from the rat parotid by an effect on either the catalytic or regulatory subunits of adenylate cyclase.

Adenylyl Cyclases↗