Biomedical subjects

M A Manso

Publications and source records attributed to M A Manso.

At least 19 recordsLinked to original sources

PubMed2006

Redox-sensitive modulation of CD45 expression in pancreatic acinar cells during acute pancreatitis.

CD45, a transmembrane protein tyrosine phosphatase required for signal transduction in leukocytes, has recently been found in pancreatic acinar cells. We have investigated the relationship between kinetic expression of CD45 on acinar cells during acute pancreatitis (AP) and the ability of these cells to produce tumour necrosis factor-alpha (TNF-alpha) through mechanisms sensitive to the cellular redox state. Flow cytometric analysis showed a significant decrease in the constitutive expression of CD45 in acinar cells from six hours onwards after inducing AP by bile-pancreatic duct obstruction (BPDO) in parallel with a significant increase in acinar TNF-alpha production. Changes in protein expression on the acinar cell surface preceded CD45 mRNA down-regulation, which was not found until 12 hours after BPDO. N-Acetylcysteine treatment delayed and reduced the down-regulation of CD45 expression induced by AP and prevented acinar cells from producing TNF-alpha. Our results show that CD45 expression is down-regulated in acinar cells during acute pancreatitis by redox-sensitive mechanisms, and they support the notion that CD45 negatively controls the production of cytokines in pancreatic acinar cells.

Acute Disease↗

PubMed2005

Isolation and characterisation of a Lactobacillus helveticus ITG LH1 peptidase-rich sub-proteome.

Lactobacillus helveticus strains, one of the most nutritionally fastidious lactic acid bacteria, have a potent proteolytic system that makes them very interesting for different uses in the dairy industry. Its applications concern from cheese ripening to the preparation of fermented milk products with biologically active peptides. The cell-free extract (CFE) of Lactobacillus helveticus strain ITG LH1 was analysed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), using IPG immobiline dry strips (pH 4-7). With the aim to study the proteolytic enzymes expressed by Lactobacillus helveticus ITG LH1 grown in milk medium, a two step-chromatography methodology, based on ion exchange and affinity chromatography, was developed for the preparation of a peptidase-rich sub-proteome from the CFE of stationary growing cells. Several affinity chromatography columns were tested and among them a HiTrap Chelating column was selected as it provided the best performance for the enrichment in peptidases. Peptidase activities were studied using different beta-Naphtylamide (beta-NA) derivatives and specific activities were increased 50- to 100-fold by this chromatographic procedure. Sub-proteome characterisation was performed by 2D-PAGE, pH 4-7, followed by protein digestion with trypsin, analysis by MALDI-TOF mass spectrometry and subsequent database searches using peptide mass fingerprints. Among the most abundant proteins seven peptidases were present, namely the two general aminopeptidases (PepN, PepC), three dipeptidases (PepDA, PepV, PepQ) and two endopeptidases (PepO, PepO3), all of them corresponding to the catalytic classes of metallo- or cysteine-peptidases. Several stress proteins (such as heat shock proteins DnaK and GroEL) and other enzymes implied in bacterial metabolism, namely in the carbohydrate pathways (such as LDH), were also identified in the peptidase-rich sub-proteome.

Aminopeptidases↗

PubMed2003

N-acetylcysteine prevents intra-acinar oxygen free radical production in pancreatic duct obstruction-induced acute pancreatitis.

Although oxygen free radicals (OFR) are considered to be one of the pathophysiological mechanisms involved in acute pancreatitis (AP), the contribution of acinar cells to their production is not well established. The aim of the present study was to determine the effect of N-acetylcysteine (NAC) in the course of AP induced by pancreatic duct obstruction (PDO) in rats, directly analysing by flow cytometry the quantity of OFR generated in acinar cells. NAC (50 mg/kg) was administered 1 h before and 1 h after PDO. Measurements by flow cytometry of OFR generated in acinar cells were taken at different PDO times over 24 h, using dihydrorhodamine-123 as fluorescent dye. Histological studies of pancreas and measurements of neutrophil infiltration in the pancreas, pancreatic glutathione (GSH), malondialdehyde (MDA) levels, plasma amylase activity and hemoconcentration were carried out in order to assess the severity of AP at different stages. NAC effectively blunted GSH depletion at early AP stages and prevented OFR generation found in acinar cells as a consequence of AP induced by PDO. This attenuation of the redox state impairment reduced cellular oxidative damage, as reflected by less severe pancreatic lesions, normal pancreatic MDA levels, as well as diminished neutrophil infiltration in pancreas. Hyperamylasemia and hemoconcentration following AP induction were ameliorated by NAC administration at early stages, when oxidative stress seems to be critical in the development of pancreatitis. In conclusion, NAC reinforces the antioxidant defences in acinar cells, preventing OFR generation therefore attenuating oxidative damage and subsequently reducing the severity of PDO-induced AP at early stages of the disease.

Acetylcysteine↗

PubMed2003

Major pathological mechanisms of acute pancreatitis are prevented by N-acetylcysteine.

AIM: To analyze the capability of N-acetylcysteine (NAC) to prevent major intra-acinar pathogenic mechanisms involved in the development of acute pancreatitis (AP). METHODS: AP was induced by pancreatic duct obstruction (PDO) in rats. Some animals received NAC (50 mg/kg) 1 h before and 1 h after PDO. During a 24-hour period of PDO, plasma amylase activity and pancreatic glutathione and malondialdehyde levels were measured. Cytosolic Ca(2+) levels and enzyme (amylase and trypsinogen) load in acinar cells were also analyzed by flow cytometry, and histological analysis of the pancreas was performed by electron microscopy. RESULTS: NAC avoided glutathione depletion at early AP stages, thereby preventing pancreatic oxidative damage, as reflected by normal malondialdehyde levels. By limiting oxidative stress, NAC treatment effectively prevented the impairment of Ca(2+) homeostasis found in acinar cells from early AP onwards, thus protecting the pancreas from damage. In addition, lower quantities of digestive enzymes were accumulated within acinar cells. This finding, together with the significantly lower hyperamylasemia observed in these animals, suggests that NAC treatment palliates the exocytosis blockade induced by PDO. CONCLUSION: By preventing oxidative stress at early AP stages, NAC administration prevents other pathological mechanisms of AP from being developed inside acinar cells, thus palliating the severity of disease.

Acetylcysteine↗

PubMed2003

N-acetylcysteine induces beneficial changes in the acinar cell cycle progression in the course of acute pancreatitis.

Oxygen free radicals (OFR) are produced in the course of acute pancreatitis (AP). In addition to injurious oxidative effects, they are also involved in the regulation of cell growth. The aim of the present study was to examine the relationship between the effectiveness of N-acetyl-l-cysteine (NAC) to prevent the generation of OFR and the changes in the cell-cycle pattern of acinar cells in the course of AP induced in rats by pancreatic duct obstruction (PDO). NAC (50 mg/kg) was administered 1 h before and 1 h after PDO. Flow-cytometric measurement of OFR generation in acinar cells was carried out using dihydrorhodamine as fluorescent dye. Plasma amylase activity, pancreatic glutathione (GSH) content and TNF-alpha plasma levels were also measured. The distribution of acinar cells throughout the different cell-cycle phases was analysed at different AP stages by flow cytometry using propidium iodide staining. NAC administration reduced the depletion of pancreatic GSH content and prevented OFR generation in acinar cells of rats with PDO-induced acute pancreatitis. As a result, AP became less severe as reflected by the significant improvement of hyper-amylasaemia and maintenance of plasma TNF-alpha levels at values not significantly different from controls were found. NAC administration inhibited progression of cell-cycle phases, maintaining acinar cells in quiescent state at early PDO times. The protection from oxidative damage by NAC treatment during early AP, allows the pancreatic cell to enter S-phase actively at later stages, thereby allowing acinar cells to proliferate and preventing the pancreatic atrophy provoked by PDO-induced AP. The results provide evidence that OFR play a critical role in the progression of acinar cell-cycle phases. Prevention of OFR generation of acinar cells in rats with PDO-induced AP through NAC treatment, not only protects pancreas from oxidative damage but also promotes beneficial changes in the cell cycle progression which reduce the risk of pancreatic atrophy.

Acetylcysteine↗

PubMed2003

Angiotensin I converting enzyme-inhibitory activity of bovine, ovine, and caprine kappa-casein macropeptides and their tryptic hydrolysates.

This work evaluated the angiotensin-converting enzyme (ACE)-inhibitory activities of bovine, ovine, and caprine kappa-casein macropeptides (CMPs) and their tryptic hydrolysates. The results obtained indicate that bovine, ovine, and caprine CMPs exhibited moderate in vitro ACE-inhibitory activities that increased considerably after digestion under simulated gastrointestinal conditions. Active peptides could also be produced from CMPs via proteolysis with trypsin, with tryptic hydrolysates exhibiting a more extensive ACE-inhibitory activity than intact CMPs during simulated gastrointestinal digestion. Two active fractions were chromatographically separated from the tryptic hydrolysate of the bovine CMP, but their complexity hampered the assignment of the ACE-inhibitory activity to specific peptide sequences. Evidence for the release of the strong ACE-inhibitory tripeptide IPP was found upon simulation of the gastrointestinal digestion of peptides released by trypsin from the CMP sequence. These findings might help to promote further exploitation of cheese whey in the preparation of nutraceuticals for inclusion in the composition of functional food products with high added values.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed2002

Time-course of oxygen free radical production in acinar cells during acute pancreatitis induced by pancreatic duct obstruction.

The time-course of oxygen free radicals (OFR) generation within acinar cells was studied at different stages of acute pancreatitis (AP) induced in rats by duct obstruction (PDO) for 48 h by flow cytometry, using dihydrorhodamine-123 (DHR) as fluorescent dye. Parallel measurements of the most common markers of oxidative stress such as glutathione (GSH) depletion and malondialdehyde (MDA) levels in pancreas were also performed. OFR production significantly increased within acinar cells at early stages of AP, concomitant with a marked depletion in pancreatic GSH. Lipid peroxidation was significantly enhanced 6 h after PDO, suggesting that the antioxidant defence system of the cell is overwhelmed by OFR production. Both MDA and OFR production in acinar cells decreased to normal values at late AP stages, thus allowing the recovery of pancreatic GSH levels 48 h after PDO. Among the two types of acinar cells differentiated by flow cytometry, R1 and R2, it was the R2 population that showed higher values of DHR dye. However, no differences between the two cell types were found regarding the amount of OFR generation. Our results demonstrate that individual acinar cells significantly contribute to produce large amounts of OFR at early stages of AP. The two existing populations of acinar cells displayed similar behaviour regarding oxidative stress over the course of the disease.

Acute Disease↗

PubMed2002

Platelet aggregation inhibitory activity of bovine, ovine, and caprine kappa-casein macropeptides and their tryptic hydrolysates.

Kappa-casein macropeptide (CMP) is one of the components of whey and is obtained as a by-product in cheesemaking. There has been increasing interest in research to find new uses of cheese industry by-products in order to improve their value and promote their use. Human platelet aggregation inhibitory activities of bovine, ovine, and caprine CMPs and their tryptic hydrolysates were studied. CMPs from the three species exhibited in vitro antithrombotic properties similar to the activity of the gamma-fibrinogen 400-411 peptide. Inhibitory activities increased following hydrolysis with trypsin. Active sequences were identified among the tryptic peptides by reversed-phase high-performance liquid chromatography with on-line mass spectrometry.

Animals↗

PubMed2001

Improved method for the simultaneous determination of whey proteins, caseins and para-kappa-casein in milk and dairy products by capillary electrophoresis.

A capillary electrophoresis method for the simultaneous determination of whey proteins, caseins and their degradation products, such as para-kappa-casein, was proposed. The effect of several parameters (pH, ionic strength and concentration of urea in the electrophoresis buffer and applied voltage) on the analysis time and on the separation efficiency of the major milk proteins was studied. Using a hydrophilically coated capillary, in combination with electrophoresis buffer 0.48 M citric acid-13.6 mM citrate-4.8 M urea at pH 2.3, and a separation voltage of 25 kV, a complete separation of beta-lactoglobulin and para-kappa-casein was achieved, permitting the quantification of both components.

Animals↗

PubMed2000

Heterogeneous distribution of plasma membrane glycoconjugates in pancreatic acinar cells.

Flow-cytometric studies of lectin binding to individual acinar cells have been carried out in order to analyse the distribution of membrane glycoconjugates in cells from different areas of the pancreas: duodenal lobule (head) and splenic lobule (body and tail). The following fluoresceinated lectins were used: wheat germ agglutinin (WGA), Tetragonolobus purpureus agglutinin (TP) and concanavalin A (Con A), which specifically bind to N-acetyl D-glucosamine and sialic acid, L-fucose and D-mannose, respectively. In both pancreatic areas, two cell populations (R1 and R2) were identified according to the forward scatter (size). On the basis of their glycoconjugate pattern, R1 cells displayed higher density of WGA and TP receptors than R2 cells throughout the pancreas. Although no difference in size was found between the cells from duodenal and splenic lobules, N-acetyl D-glucosamine and/or sialic acid and L-fucose residues were more abundant in plasma membrane cell glycoconjugates from the duodenal lobule. The results provide evidence for biochemical heterogeneity among individual pancreatic cells according to the distribution of plasma membrane glycoconjugates.

Animals↗

PubMed2000

Cell-cycle distribution of pancreatic cells from rats with acute pancreatitis induced by bile-pancreatic obstruction.

The aim of this study was to analyze, using electron microscopy, the morphological alterations that progressively appear in the pancreas of rats with acute pancreatitis induced by bile-pancreatic obstruction over 48 h. In addition, in order to ascertain the capability of pancreas regeneration at different stages of pancreatitis, the distribution of pancreatic cells throughout the different phases of the cell cycle was also analyzed by flow cytometry using propidium iodide staining. Interstitial edema, macrophage infiltration, vacuolization, and dilatation of endoplasmic reticulum were observed from 1.5 h after obstruction onward. Interestingly, cell cycle studies showed an increased proportion of S-phase cells at early stages of pancreatitis (1.5 h after obstruction), which leads to a significant increase in cells in G2/M phase 12 h after pancreatic obstruction. Histological studies revealed severe alterations in pancreas of rats with obstruction maintained over 48 h which affects the nuclear structure. Intracellular disorganization, apoptosis, and focal necrosis were observed at this stage. Furthermore, flow-cytometric analysis of cell DNA contents showed a significant decrease in the proportion of S and G2/M cells and a significant increase in G0/G1 cells, suggesting an arrest of almost all cells in quiescent states. These results suggest that rat pancreas cells are able to recover during the first 12 h after pancreatic obstruction. However, the gland would lose its ability to regenerate if the obstruction was maintained for longer periods.

Acute Disease↗

PubMed2000

Alterations in the glycoconjugates of pancreatic cell membrane induced by acute pancreatitis.

The alterations that progressively appear in plasma membrane glycoconjugates of rat pancreatic cells at different stages of acute pancreatitis induced by duct obstruction have been analyzed on individual cells by flow cytometry using the fluoresceinated lectins, wheat germ agglutinin (WGA), Tetragonolobus purpureus agglutinin (TP) and Concanavalin A (Con A), which specifically bind to N-acetyl D-glucosamine, L-fucose and D-mannose, respectively. Two populations of pancreatic cells were differentiated according to the forward scatter (size), which showed different density of saccharidic terminals located at external positions in the glycoconjugates of the plasma membrane. A significant increase in WGA and TP binding was found 1.5 h after pancreatic obstruction, which could be due to the fusion of zymogen granules with the plasma membrane as suggested by the basolateral exocytosis observed by electron microscopy at this stage. The most external sugar residues of membrane glycoconjugates are removed 12 h after pancreatic duct obstruction as a consequence of an advanced state of pancreatitis. The hydrolytic process reaches greater depths in the membrane 48 h after obstruction. At this stage a significant decrease in WGA, TP and ConA binding was found in all pancreatic cells, indicating the loss of N-acetyl D-glucosamine and/or sialic acid, L-fucose and even D-mannose which is located in the core of the glycan. The results provide information about the progressive degradation induced by acute pancreatitis in pancreatic cell membrane glycoconjugates.

Acute Disease↗

PubMed2000

Enzyme load in pancreatic acinar cells is increased in the early stages of acute pancreatitis induced by duct obstruction in rats.

Trypsinogen and amylase content has been analysed by flow cytometry in individual pancreatic cells from rats with acute pancreatitis induced by pancreatic duct obstruction, from the earliest stages to 48 h after obstruction. Parallel morphological studies of the pancreas by electron microscopy and analysis of various parameters for the diagnosis of pancreatitis will allow research into the possible relationship between intracellular enzyme load and the severity of pancreatitis. Progressive increases in amylase activity in ascites and plasma, the volume of ascites, haematocrit, vacuolization, oedema and macrophage infiltration were observed between 1.5 h and 12 h after duct obstruction. A progressive increase in enzyme content was also observed in individual acinar cells at this stage. Interestingly, the larger increase was for trypsinogen, so that the trypsinogen/amylase ratio was significantly increased in all acinar cells by 12 h after duct obstruction. This represents a risk factor for the development of pancreatitis. Sections of pancreas taken from rats that had duct obstruction for 48 h showed massive dilatation and disorganization of the endoplasmic reticulum, focal apoptosis and necrosis. These severe alterations would affect enzyme synthesis, as reflected by the significant decrease in the intracellular enzyme load observed at this stage. However, not all acinar cells were affected equally by the damage induced by pancreatitis: R(1) cells appeared to be more sensitive than R(2) cells. In conclusion, intracellular accumulation of digestive enzymes occurs at early stages of pancreatitis, and this effect is proportionally greater for trypsinogen, a finding that could explain the degree of severity achieved in the course of pancreatitis.

Acute Disease↗

PubMed1999

Detection of the presence of soya protein in milk powder by sodium dodecyl sulfate capillary electrophoresis.

Two different commercial kits for sodium dodecyl sulfate capillary electrophoresis (SDS-CE) were evaluated for the detection of the presence of soya protein in milk powder. The results obtained showed that SDS-CE allowed the separation of the basic subunits of glycinin and the alpha and alpha' subunits of beta-conglycinin from the main milk protein peaks. However, a detection limit lower than 10% (w/w) of soya protein in total protein could not be achieved. The use of a tetraborate-EDTA sample treatment minimized interferences from milk proteins, allowing the detection of at least 1% (w/w) of soya protein in total protein. The addition of soya protein hydrolysates could not be determined using SDS-CE.

Calibration↗

PubMed1999

The recovery of acute pancreatitis depends on the enzyme amount stored in zymogen granules at early stages.

Little is known about the changes in pancreatic enzyme storage in acute pancreatitis. We have performed flow cytometric studies of zymogen granules from rats with acute pancreatitis induced by hyperstimulation with caerulein. A comparison was made with rats treated with hydrocortisone (10 mg/kg/day) over 7 days before inducing pancreatitis in order to find out whether the amount of enzymes stored in the pancreas plays a key role in the development of pancreatitis. The potentially therapeutic effect of L-364,718 (0.1 mg/kg/day, for 7 days), a CCK receptor antagonist, was assayed in the rats with caerulein-induced pancreatitis which had previously received the hydrocortisone treatment. A significant increase in the intragranular enzyme content was observed 5 h after hyperstimulation with caerulein. The highest values were reached in the rats previously treated with hydrocortisone. The greatest pancreatic enzyme load was parallel to the highest values in plasma amylase, edema and haematocrit observed. Acute pancreatitis was reversed seven days later. At this stage smaller granules appeared in the pancreas whose enzyme content was similar to that of controls when no treatment was applied after pancreatitis. In contrast, L-364,718 administration prevented the favourable evolution of pancreatitis since the antagonism exerted on CCK receptors induced a blockade of secretion of the large amounts of enzymes stored in the pancreas. Moreover, the enzyme content in zymogen granules was below normal values since the stimulatory CCK action on enzyme synthesis can be inhibited by L-364,718. Our results suggest that the efficiency of CCK antagonists, as potential therapy, would also depend on the load of enzymes in the pancreas when acute pancreatitis is produced.

Acute Disease↗

PubMed1999

Zymogen granule alterations in caerulein-induced pancreatitis developed during continuous hydrocortisone administration.

BACKGROUND: The amount of enzymes stored in individual zymogen granules and the glycosylation of their membrane have been analysed in rats with acute pancreatitis induced by caerulein after hydrocortisone treatment. The consequences of prolonging hydrocortisone administration after pancreatitis and the use of the cholecystokinin (CCK) receptor antagonist, L-364,718, have also been evaluated. MATERIALS AND METHODS: Analysis was performed using flow cytometry. RESULTS: Caerulein-induced pancreatitis in rats previously treated for 7 days with hydrocortisone (10 mg kg-1 per day) revealed alterations in enzyme storage in the pancreas. Significant increases in amylase and trypsinogen contents in zymogen granules were observed, an effect associated with a reduction in L-fucose glycoconjugates. Pancreatitis persists 7 days later if hydrocortisone treatment is prolonged. At this stage, a reduced granule fucosylation was still observed, and a significant decrease in the amount of trypsinogen stored in the granules was found. However, hydrocortisone administration led to an increase in intragranular amylase quantities up to normal values, even when L-364,718 was simultaneously administered, but it reverted to plasma as a consequence of pancreatitis. The amount of N-acetyl D-glucosamine in the zymogen granule membrane was not altered by caerulein acute pancreatitis induced under continuous hydrocortisone treatment, but it was decreased by the administration of L-364,718 over 7 days after pancreatitis induction. CONCLUSIONS: The administration of hydrocortisone after the development of pancreatitis prevented recurrence of the disease. L-364,718 proved to be detrimental, not only failing to reduce the symptoms of pancreatitis but also altering the glycoproteins of zymogen granule membrane.

Acetylglucosamine↗

PubMed1999

Selective exocytosis of zymogen granules induces non-parallel secretion in short-term cholecystokinin-stimulated rats.

Parallel studies on pancreatic enzyme secretion and zymogen granule enzyme composition have been carried out in rats subjected to infusion of cholecystokinin (CCK) (1.25 microgram/kg per h) over 30 min. Flow cytometric analysis showed a significant decrease in the mean value of granule size after CCK stimulation. The amount of trypsinogen stored in each individual zymogen granule was significantly lower at 30 min of CCK infusion, but no variation in intragranular amylase content was observed. As a result, the amylase/trypsinogen ratio was significantly increased in the zymogen granules that remained in the pancreas of rats stimulated with CCK for 30 min. A significantly greater proportion of trypsin than amylase was secreted after 30 min CCK infusion. Our results support the existence of different types of granules loaded with different proportions of enzymes. We conclude that short-term CCK stimulation induces the selective release of large granules containing a high proportion of trypsinogen, which leads to a non-parallelism of enzyme secretion.

Amylases↗

PubMed1998

Effect of cholecystokinin blockade on the recovery of alterations induced by acute pancreatitis in glycoconjugates of rat zymogen granules.

Lectin-binding studies have been performed on rat zymogen granules to investigate alterations in the carbohydrate membrane composition that occur in acute pancreatitis induced by caerulein. The influence of treatment with hydrocortisone for seven days before inducing pancreatitis was also studied. Lectin labeling on zymogen granules was also analyzed seven days after inducing pancreatitis in rats that had previously received a hydrocortisone treatment. During this period L 364,718 (0.1 mg/kg)--specific cholecystokinin (CCK) receptor antagonist--was administered daily to some of the rats, and no treatment was applied to others. Using fluorescein-labelled T. purpureus (TP)lectin, a significant decrease in the amount of L-fucose in the granule membrane was observed in rats with caerulein-induced pancreatitis. This effect was directly caused by the pancreatitis and was not influenced by previous hydrocortisone treatment. Seven days later, the density of TP receptors in the granule membrane was similar to the controls both in L-364,718-treated and untreated rats. Therefore, we suggest that endogenous CCK is not an essential factor in the recovery of L-fucose containing glycoconjugates the granule membrane after pancreatitis. Acute pancreatitis did not alter the expression of wheat germ agglutinin (WGA) receptors in the zymogen granule membrane. WGA specifically binds N-acetyl glucosamine and sialic acids. L 364,718 administered for seven days after inducing pancreatitis significantly reduced WGA binding, untreated rats showed a normal zymogen granule membrane. Therefore, the blockade of CCK-induced alterations in membrane glycoconjugates enriched in N-acetyl glucosamine and sialic acid of newly formed granules after pancreatitis, a finding that could explain the delay in the regression of the disease.

Amylases↗