Showing papers on "Amylase published in 1989"

Acute pancreatitis and normoamylasemia. Not an uncommon combination.

Abstract: A consecutive series of 352 attacks of acute pancreatitis (AP) was studied prospectively in 318 patients. AP was ascertained by contrast-enhanced CT scan in all but four cases in which diagnosis was made at operation or autopsy. Sixty-seven of these cases (19%) had normal serum amylase levels on admission (i.e., less than 160 IU/L, a limit that includes 99% of control values), a figure considerably higher than generally admitted. When compared to AP with elevated serum amylase, normoamylasemic pancreatitis was characterized by the following: (1) the prevalence of alcoholic etiology (58% vs. 33%, respectively, p less than 0.01), (2) a greater number of previous attacks in alcoholic pancreatitis (0.7 vs. 0.4, p less than 0.01); and (3) a longer duration of symptoms before admission (2.4 vs. 1.5 days, p less than 0.005). In contrast AP did not appear to differ significantly in terms of CT findings, Ranson's score, and clinical course, when comparing normo- and hyperamylasemic patients, although there was a tendency for normoamylasemic patients to follow milder courses. Serum lipase was measured in 65 of these normoamylasemic cases and was found to be elevated in 44 (68%), thus increasing diagnostic sensitivity from 81% when amylase alone is used to 94% for both enzymes. A peritoneal tab was obtained in 44 cases: amylase concentration in the first liter of dialysate was greater than 160 IU/L in 24 cases (55%), and lipase was greater than 250 U/L in 31 cases (70%). Twelve of these 44 cases had low peritoneal fluid and plasma concentrations for both enzymes. Thus little gain in diagnostic sensitivity was obtained when adding peritoneal values (96%) to serum determinations. AP is not invariably associated with elevated serum amylase. Multiple factors may contribute to the absence of hyperamylasemia on admission, including a return to normal enzyme levels before hospitalization or the inability of inflamed pancreases to produce amylase. Approximately two thirds of cases with normal amylasemia were properly identified by serum lipase determinations. AP does not appear to behave differently when serum amylase is normal or elevated, and should therefore be submitted to similar therapeutic regimens in both conditions.

Biochemical evidence that starch breakdown by Bacteroides thetaiotaomicron involves outer membrane starch-binding sites and periplasmic starch-degrading enzymes.

Abstract: Bacteroides thetaiotaomicron can utilize amylose, amylopectin, and pullulan as sole sources of carbon and energy. The enzymes that degrade these polysaccharides were found to be primarily cell associated rather than extracellular. Although some activity was detected in extracellular fluid, this appeared to be the result of cell lysis. The cell-associated amylase, amylopectinase, and pullulanase activities partitioned similarly to the periplasmic marker, acid phosphatase, when cells were exposed to a cold-shock treatment. Two other enzymes associated with starch breakdown, alpha-glucosidase and maltase, appeared to be located in the cytoplasm. Intact cells of B. thetaiotaomicron were found to bind 14C-starch. Binding was probably mediated by a protein because it was saturable and was decreased by treatment of cells with proteinase K. Results of competition experiments showed that the starch-binding proteins had a preference for maltodextrins larger than maltohexaose and a low affinity for maltose and maltotriose. Both the degradative enzymes and starch binding were induced by maltose. These findings indicate that starch utilization by B. thetaiotaomicron apparently does not involve secretion of extracellular enzymes. Rather, binding of the starch molecule to the cell surface appears to be a first step to passing the molecule through the outer membrane and into the periplasmic space.

Pancreatic duct obstruction in rabbits causes digestive zymogen and lysosomal enzyme colocalization.

Abstract: The pancreatic duct of anesthetized rabbits was cannulated and, in some animals, flow of pancreatic exocrine secretions was blocked by raising the cannula to a vertical position. Blockage for 3-7 h caused a rapid and significant rise in serum amylase activity and an increase in amylase activity within the pancreas. The concentration of lysosomal enzymes in the pancreas was not altered but they became redistributed among subcellular fractions and, as a result, an increased amount was recovered in the 1,000-g, 15-min pellet, which was enriched in zymogen granules. Immunofluorescence studies indicated that lysosomal enzymes become localized within organelles which, in size and distribution, resemble zymogen granules. They also contain digestive enzyme zymogens. Blockage of pancreatic secretions also caused lysosomal enzyme-containing organelles to become more fragile and subject to in vitro rupture. These changes noted after short-term pancreatic duct obstruction are remarkably similar to those previously noted to occur during the early stages of diet and secretagogue-induced experimental pancreatitis, observations that have suggested that colocalization of digestive enzyme zymogens and lysosomal hydrolases might result in intracellular digestive enzyme activation and be an important early event in the evolution of those forms of experimental acute pancreatitis.

Characterization of salivary alpha-amylase binding to Streptococcus sanguis.

Abstract: The purpose of this study was to identify the major salivary components which interact with oral bacteria and to determine the mechanism(s) responsible for their binding to the bacterial surface. Strains of Streptococcus sanguis, Streptococcus mitis, Streptococcus mutans, and Actinomyces viscosus were incubated for 2 h in freshly collected human submandibular-sublingual saliva (HSMSL) or parotid saliva (HPS), and bound salivary components were eluted with 2% sodium dodecyl sulfate. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western transfer, alpha-amylase (EC 3.2.1.1) was the prominent salivary component eluted from S. sanguis. Studies with 125I-labeled HSMSL or 125I-labeled HPS also demonstrated a component with an electrophoretic mobility identical to that of alpha-amylase which bound to S. sanguis. Purified alpha-amylase from human parotid saliva was radiolabeled and found to bind to strains of S. sanguis genotypes 1 and 3 and S. mitis genotype 2, but not to strains of other species of oral bacteria. Binding of [125I]alpha-amylase to streptococci was saturable, calcium independent, and inhibitable by excess unlabeled alpha-amylases from a variety of sources, but not by secretory immunoglobulin A and the proline-rich glycoprotein from HPS. Reduced and alkylated alpha-amylase lost enzymatic and bacterial binding activities. Binding was inhibited by incubation with maltotriose, maltooligosaccharides, limit dextrins, and starch.

Very stable enzymes from extremely thermophilic archaebacteria and eubacteria

Abstract: Thirty-six thermophilic archaebacteria and nine extremely thermophilic eubacteria have been screened on solid media for extracellular amylase, protease, hemicellulase (xylanase), cellulase, pectinase and lipase activities. Extracellular enzymes were detected in 14 archaebacteria belonging to three different orders. Twelve of these were able to degrade starch and casein and the two Thermofilum strains were able to degrade starch, xylan and carboxymethylcellulose. Three of the eubacteria could degrade only starch. The other six (including four Thermotoga strains) all had activity against starch, xylan and carboxymethylcellulose, and one also had activity against casein. Some of the amylolytic archaebacteria released α-glucosidase, β-glucosidase, amylase and transglucosylase activities into liquid media containing starch or maltose. Thermotoga strain FjSS3B.1 released amylase, xylanase, cellulase and β-glucosidase activities into the medium when grown in the presence of substrates. When the partially purified enzymes from Thermotoga and some of the archaebacteria were compared with known thermostable enzymes the majority were found to be the most thermostable of their type. The β-glucosidase, xylanase and cellulase from Thermotoga and two α-glucosidases, a β-glucosidase, an amylase and a pullulanase from archaebacteria all have half-lives of at least 15 min at 105°C.

Influence of retention time on degradation of pancreatic enzymes by human colonic bacteria grown in a 3-stage continuous culture system.

Abstract: Hydrolytic enzymes were measured in gut contents from four sudden death victims. Pancreatic amylase and total protease activities decreased distally from the small bowel to the sigmoid/rectum region of the large intestine, showing that considerable breakdown or inactivation of the enzymes occurred during gut transit. To determine whether pancreatic enzymes were substrates for the gut microflora, mixed populations of bacteria were grown in a 3-stage continuous culture system on a medium that contained pancreatic extract as the sole nitrogen source. The multichamber system (MCS) was designed to reproduce in vitro, the low pH, high nutrient, fast growth conditions of the caecum and right colon and the neutral pH, low nutrient, slow growth conditions of the left colon. Results showed that pancreatic amylase was resistant to breakdown by intestinal bacteria compared with the peptide hydrolases in pancreatic secretions. Leucine aminopeptidase, trypsin and to a lesser degree, chymotrypsin, were easily degraded by gut bacteria, but pancreatic elastase was comparatively resistant to breakdown. Protein degradation in the MCS, as determined by enzyme activities, protein concentration and ammonia and phenol production, increased concomitantly with system retention time over the range 24-69 h. These results suggest that intestinal bacteria play an important role in the breakdown of hydrolytic enzymes secreted by the pancreas and that this process and protein fermentation in general, is likely to occur maximally in individuals with extended colonic retention times.

Two human myeloma cell lines, amylase-producing KMS-12-PE and amylase-non-producing KMS-12-BM, were established from a patient, having the same chromosome marker, t(11;14)(q13;q32).

Abstract: Two human myeloma cell lines, KMS-12-PE and KMS-12-BM, were established from a 64-year-old woman with a non-producing type of multiple myeloma. The KMS-12-PE line originated from the pleural effusion and the KMS-12-BM from the bone marrow. These two lines showed the same chromosome marker, t(11:14)(q13:q32). However, their phenotypes of surface markers differed from each other. KMS-12-BM cells were positive to CD20, CD38 and PCA-1. showing the plasmacytoid (immature plasma cell) stage of B-cell differentiation, while KMS-12-PE cells were positive to CD38 and PCA-1, but not to CD20, indicating the terminal differentiated stage of B-cells. As seen in the pleural effusion of the patient. KMS-12-PE cells ectopically produced a salivary type of amylase, but KMS-12-BM cells did not. Interestingly, the chromosome abnormality of del(1)(p22----pter) near the region of 1p21, where the amylase gene was assigned, was noticed in as many as 76% of KMS-12-PE cells.

Characterization of resistant starch from autoclaved wheat starch

Abstract: The nature of a particular type of in vitro undigestible starch, i.e. resistant starch (RS), was studied. This type of undigestible starch is found in products processed at relatively high moisture contents, such as baking and autoclaving. The RS was concentrated by enzymic digestion of available starch and protein and recovered by centrifugation. RS from autoclaved wheat starch was evaluated by various methods. The crystallinity was studied by X-ray diffraction and DSC. The degree of polymerization (DP) was evaluated with gel permeation chromatography and iodine binding and the starch content and linearity by enzymic methods. The results indicate that RS consist of crystallized, linear α-glucans of relatively low molecular weight (DP approx. 65). The recovery of RS after various drying procedures, and the introduction of a thermostable amylase (Termamyl®) in the RS assay was also studied. Oven-drying of the fibre residues used in the analysis had no significant effect on RS recovery, whereas freeze-drying was accompanied by a slight increase in RS content. The use of Termamyl®, in the assay of RS in the fibre residues, decreased the yield of RS by 10–15%.

Receptor occupation, calcium mobilization, and amylase release in pancreatic acini: effect of CCK-JMV-180.

Abstract: We examined the relationships between receptor occupation, calcium mobilization, and stimulated amylase release for cholecystokinin octapeptide (CCK-8) and for CCK-JMV-180, an analogue of the COOH-terminal heptapeptide of CCK having the structure Boc-Tyr(SO3)-Nle-Gly-Trp-Nle-Asp-2-phenylethyl ester using dispersed acini from rat pancreas. CCK-8 and CCK-JMV-180 each bind to two classes of CCK receptors: one class has a high affinity for CCK-8 and CCK-JMV-180 and the other class has a low affinity for CCK-8 and CCK-JMV-180. Mobilization of cellular calcium was assessed by measuring cytosolic calcium with a fluorescent indicator and by measuring outflux of radioactive calcium from preloaded cells. In terms of causing an increase in cytosolic calcium or an increase in calcium outflux, CCK-JMV-180 was 50-60% as efficacious as CCK-8. Analysis of the relationship between receptor occupation and calcium mobilization caused by CCK-8 and CCK-JMV-180 in combination indicates that calcium mobilization is caused by occupation of low-affinity CCK receptors. Comparison of the dose-response curve for calcium mobilization and amylase release stimulated by CCK-8 or CCK-JMV-180 indicates that very low concentrations of each peptide stimulate amylase release without causing detectable calcium mobilization. At these very low concentrations, CCK-8 or CCK-JMV-180 do not cause potentiation of amylase release when combined with vasoactive intestinal peptide.

Effect of vanadate administration on blood glucose and insulin levels as well as on the exocrine pancreatic function in streptozotocin-diabetic rats.

Abstract: In the present study, streptozotocin-induced diabetic rats with their corresponding controls, were treated orally with sodium metavanadate A gradual increase of the vanadate concentration up to 08 mg/ml in the drinking water, lowered the blood glucose levels of the diabetic animals to normal values without changing the insulin levels On the other hand, vanadate did not affect the blood glucose levels of the non-diabetic animals; it did however induce lower levels of circulating insulin in these animals The lowering of the glycaemic values of the diabetic animals was closely related to the consumption of vanadate When the treatment was ceased, the blood glucose levels rose rapidly The diabetic animals responded to the vanadate treatment with two sensitivities; while the large majority of the diabetic animals displayed stable normoglycaemic values, others had fluctuating values Amylase content in the exocrine pancreas of these two subgroups of animals was studied separately and compared to that from the non-treated control and diabetic animals The presence of amylase in the pancreatic acinar cells was assessed by the protein A-gold immunocytochemical approach and biochemical determinations Amylase was found to be very low in the non-treated diabetic animals Lowering of the blood glucose levels induced by the vanadate treatment restored the amylase to levels similar to those of the controls However, vanadate-treated diabetic animals with fluctuating levels of blood glucose, demonstrated only a partial recovery of amylase Thus, vanadate treatment was found to have a normalizing effect on blood glucose levels in diabetic animals as well as restoring amylase content in the pancreas of diabetic animals This appeared to be closely related to the glycaemic values of the diabetic animals

Serum amylase, isoamylase, and lipase in the acute abdomen. Their diagnostic value for acute pancreatitis

Abstract: We evaluated the diagnostic value of serum amylase, isoamylase, and lipase for the diagnosis of acute pancreatitis from sera of patients with acute abdominal pain. Comparison was first made in condition A between 32 patients with image-proven pancreatitis and 414 patients with nonpancreatic acute abdomen (the control group), then in condition B, between 62 pancreatitis patients with or without image proof and the control group. We found (a) that patients with image-proven pancreatitis suffer a more severe clinical course than those without; (b) that the sensitivity, positive predictive value, and accuracy in condition B are higher than in condition A at any cutoff level; (c) that none of the enzyme assays is specific at the upper reference limit, but their diagnostic yields are much improved by raising cutoff levels to about three or four times the upper limit; and (d) that at these selected cutoff levels, amylase had a diagnostic value similar to p-isoamylase or lipase in both conditions (sensitivity 84% and 92% for amylase in conditions A and B, respectively; specificity 98% and 98%; positive predictive value 75% and 90%; negative predictive value 99% and 99%; accuracy 91% and 97%). In conclusion, at an appropriately selected cutoff level, amylase can be effectively used as the first-line test and isoamylase or lipase as adjunct tests for acute abdominal conditions.

Effects of n-alcohols on junctional coupling and amylase secretion of pancreatic acinar cells.

Abstract: We have tested the effects of alcohols differing by their alkyl chain length on the membrane channels and amylase secretion of rat pancreatic acinar cells. In intact acini, alcohols with a chain of seven, eight, or nine carbons (C-7, C-8, and C-9) induced dye uncoupling and increased basal amylase release. These effects were readily reversible after alcohol removal. By contrast, an alcohol with a chain of 15 carbons (C-15) and several alcohols with chains of fewer than six carbons (C-2, C-4, and C-6) did not uncouple acinar cells and had no effects of amylase secretion. Neither did alkanes and oxidized derivatives of C-7 and C-8 alcohols did not affect dye coupling. Double patch-clamp experiments on pairs of acinar cells, under conditions of strong cytosolic Ca2+ and pH buffering, showed that C-7, C-8, and C-9 alcohols blocked completely and reversibly the electrical conductance of junctional channels. Furthermore, studies of single voltage-clamped acinar cells revealed that the uncoupling alcohols did not affect the resting nonjunctional membrane conductances. Thus the alcohols that did not affect acinar cells coupling did not affect amylase secretion, whereas the alcohols that caused uncoupling increased secretion. The latter effect was not mediated by changes in the conductance of nonjunctional membrane, cytosolic Ca2+, and pH and, as revealed by an immunological hemolytic plaque assay for amylase, had a time course consistent with the rapid (within 1 min) inhibition of coupling. These data provide new support for the view that the regulation of cell-to-cell communications is correlated with that of digestive enzyme secretion.

Cloning and nucleotide sequence of the gene (amyP) for maltotetraose-forming amylase from Pseudomonas stutzeri MO-19.

Abstract: The gene (amyP) coding for maltotetraose-forming amylase (exo-maltotetraohydrolase) of Pseudomonas stutzeri MO-19 was cloned. Its nucleotide sequence contained an open reading frame coding for a precursor (547 amino acid residues) of secreted amylase. The precursor had a signal peptide of 21 amino acid residues at its amino terminus. An extract of Escherichia coli carrying the cloned amyP had amylolytic activity with the same mode of action as the extracellular exo-maltotetraohydrolase obtained from P. stutzeri MO-19. A region in the primary structure of this amylase showed homology with those of other amylases of both procaryotic and eucaryotic origins. The minimum 5' noncoding region necessary for the expression of amyP in E. coli was determined, and the sequence of this region was compared with those of Pseudomonas promoters.

Altered regulation of β-amylase activity in mutants of Arabidopsis with lesions in starch metabolism

Abstract: Three classes of mutants of Arabidopsis thaliana (L.) Heynhold with alterations in starch metabolism were found to have higher levels of leaf amylase activity than the wild type when grown in a 12-hr photoperiod. This effect was dependent upon the developmental stage of the plants and was largely suppressed during growth in continuous light. The various amylolytic activities in crude extracts were separated by electrophoresis in nondenaturing polyacrylamide gels and visualized by activity staining. The increased amylase activity in the mutants was due to an up to 40-fold increase in the activity of an extrachloroplast β-amylase (EC 3.2.1.2). These observations indicate the existence of a regulatory mechanism that controls the amount of β-amylase activity in response to fluctuations in photosynthetic carbohydrate metabolism. It is paradoxical that β-amylase appears to be a highly regulated enzyme, but as yet no physiologically relevant function can be assigned to this enzyme due to the absence of starch in the cytoplasmic compartment of leaf cells.

Starch in human nutrition.

Abstract: Structure et composition, proprietes physicochimiques de l'amidon. Degradation enzymatique de l'amidon, principales enzymes. Digestion des produits alimentaires amylaces. Intervention de l'amidon dans le metabolisme des lipides et le diabete. Article synthese

Effects of islet hormones on amylase secretion and localization of somatostatin binding sites.

Abstract: The interaction of insulin and somatostatin on amylase secretion was examined in the isolated perfused rat pancreas. Exogenous insulin (10 mU/ml) significantly potentiated cholecystokinin- (CCK; 0.5 mU/ml) stimulated amylase secretion (12.47 +/- 2.9 micrograms/ml, n = 7). Glucose (16.7 mM) stimulated endogenous insulin secretion (523 +/- 66 microU/ml) and also significantly enhanced CCK-stimulated amylase secretion (13.41 +/- 2.8 micrograms/ml, n = 11). When somatostatin was included in the perfusion media, containing insulin and CCK, amylase secretion was reduced to 3.17 +/- 0.83 micrograms/ml (n = 7), a level comparable to that of CCK-stimulated amylase secretion alone. Similarly, addition of exogenous somatostatin to perfusion media, containing 16.7 mM glucose and CCK, reduced amylase secretion to 4.29 +/- 1.09 micrograms/ml (n = 9). The effect of somatostatin and insulin on carbamylcholine-stimulated amylase secretion was also examined. Exogenous insulin (50 mU/ml) potentiated carbamylcholine- (10(-8) M) stimulated amylase secretion, and addition of exogenous somatostatin to the media containing both insulin and carbamylcholine suppressed the insulin potentiation. Uptake of 125I-[Tyr11]somatostatin in the perfused pancreas was saturable as it decreased significantly with the addition of excess unlabeled somatostatin. Autoradiograms revealed uptake of the ligand by both the endocrine islets and the exocrine pancreas with the highest density of grains observed over the acini. These results support the hypothesis that islet peptides modulate the exocrine pancreas, that somatostatin inhibits amylase secretion by inhibiting the action of insulin, and that somatostatin may act directly on the exocrine pancreas via specific receptors on acinar cells.