A novel collagenolytic protease from Mucor subtilissimus UCP 1262: Comparative analysis of production and extraction in submerged and stated-solid fermentation.

This work aimed to compare the production of collagenolytic proteases produced by M. subtilissimus UCP1262 in submerged fermentation (SF) and solid-state fermentation (SSF) as well as extracting in aqueous two-phase system (ATPS). Collagenolytic protease production was performed in using MS-2 culture medium (SF) and soybean bran as substrate (SSF). Subsequently, the fermented liquid from both fermentations were used for the extraction of enzyme by ATPS, it was verified the influence of different variables from a factorial design 23. In SSF the highest protease and collagenolytic activities were achieved with 362.66 U/mL and 179.81 U/mL, respectively. When compared with SF (26.33 and 18.70 U/mL) higher values were obtained in the activities. The protease partitioning from SF and SSF in ATPS showed a similar profile showing higher affinity for the polymer rich phase. The highest value for the response variable purification factor (3.49) was obtained in the system using SSF. Thus, SSF shows promise as a bioprocess for extracellular production of collagenolytic proteases, using of soybean bran as substrate had used sustainable raw material, aiming application this possible enzyme in the treatment of burns and postoperative scarring.

Erythrocyte acetylcholinesterase as biomarker of pesticide exposure: new and forgotten insights.

Acetylcholinesterase (AChE) acts on the hydrolysis of acetylcholine, rapidly removing this neurotransmitter at cholinergic synapses and neuromuscular junctions as well as in neuronal growth and differentiation, modulation of cell adhesion ("electrotactins") and aryl-acylamidase activity (AAA). This enzyme is also found in erythrocyte, as 160 kDa dimer that anchors to the plasma membrane via glycophosphatidylinositol. The function of this enzyme in erythrocytes has not yet been elucidated; however, it is suspected to participate in cell-to-cell interactions. Here, a review on erythrocyte AChE characteristics and use as biomarker for organophosphorus and carbamate insecticides is presented since it is the first specific target/barrier of the action of these pesticides, besides plasma butyrylcholinesterase (BChE). However, some past and current methods have disadvantages: (a) not discriminating the activities of AChE and BChE; (b) low accuracy due to interference of hemoglobin in whole blood samples. On the other hand, extraction methods of hemoglobin-free erythrocyte AChE allows: (a) the freezing and transporting of samples; (b) samples free of colorimetric interference; (c) data from only erythrocyte AChE activity; (d) erythrocyte AChE specific activity presents higher correlation with the central nervous system AChE than other peripheral ChEs; (e) slow spontaneous regeneration against anti-ChEs agents of AChE in comparison to BChE, thus increasing the chances of detecting such compounds following longer interval after exposure. As monitoring perspectives, hemoglobin-free methodologies may be promising alternatives to assess the degree of exposure since they are not influenced by this interfering agent.

Portable near infrared spectroscopy applied to fuel quality control.

Fuel quality control has gained interest in many countries owing to the potential damage of low-quality fuel to engines, the environment, and economy. Thus, the application of analytical techniques to verify quality control of fuels has become crucial. The portable micro-spectrometer in the near infrared region (microNIR) has gained credibility as a successful analytical technique in several quality control sectors. The possibility of real-time analysis using a nondestructive and reliable method is the main advantage of this methodology. In this work, chemometric models (PLS) were developed using microNIR data to determine the amount of biodiesel in diesel (LODBio = 0.5wt%; LOQBio = 1.8wt%; and RMSEPBio = 1.8wt%); sulfur in diesel (LODS = 2.4mgL-1; LOQS = 8.0mgL-1; and RMSEPS = 13.2mgL-1); gasoline, ethanol, and methanol in C-type gasoline (LODgas = 0.55wt%; LOQgas = 1.84wt%; and RMSEPgas = 0.81wt%; LODeth = 0.75wt%; LOQeth = 2.5wt%; and RMSEPeth = 3.81wt%; and LODmet = 0.85wt%; LOQmet = 2.84wt%; and RMSEPmet = 1.80wt%); and water, methanol, and ethanol in ethanol-hydrated fuel (EHF) (LODH2O = 0.04wt%; LOQH2O = 1.29wt%; and RMSEPH2O = 1.05wt%; LODmet = 0.52wt%; LOQmet = 1.73wt%; and RMSEPmet = 2.78wt%; and LODeth = 1.22wt%; LOQeth = 4.07wt%; and RMSEPeth = 4.41wt%). A total of 181 blends were prepared, with biodiesel and sulfur contents ranging from 0 to 100wt% and 10-500mgL-1, respectively. For gasoline blends, the gasoline, ethanol, and methanol contents ranged from 0.0 to 75.0wt%, 25.0-75.0wt%, and 0.0-50.0wt%, respectively. In the EHF control, the ethanol, water, and methanol contents ranged from 0.0 to 100.0wt%, 0.0-50.0wt%, and 0.0-50.0wt%, respectively. The proposed method presented high precision and accuracy in all cases, and the results showed that the microNIR technique had excellent performance in fuel quality control.

Sorbin in the porcine gastrointestinal tract and pancreas: an immunocytochemical analysis.

Sorbin is a 153-amino acid peptide that was initially discovered in the porcine duodenum. We have reported previously that this peptide regulates intestinal electrolyte transport and have described accumulation sites in the rat digestive tract. In the present study, we investigated the anatomical distribution and the site(s) of sorbin production in the porcine digestive tract using immunocytochemistry. The use of polyclonal antisera, which by cross-reaction studies were shown to be specific for different regions of the molecule, revealed a diversified distribution. Sorbin predominated in endocrine cells preferentially localized in the pyloric glands, duodenal crypts of Lieberkühn, and pancreatic islets; in the gastrointestinal tract, sorbin coexisted with Met-enkephalin or with substance P in a small fraction of serotonin-storing [enterochromaffin (ED)] cells, i.e. EC2 cells and EC1 cells, respectively; in the pancreas, sorbin coexisted with insulin in the beta-cells, also considered as serotonin-storing cells in the pig, and with EC cells in the exocrine pancreas. An enteric neuronal system containing sorbin was also reported. Our results demonstrate that sorbin is a component of the serotonin-storing cell type in the porcine gastrointestinal tract and pancreas, and suggest potential directions to investigate the functions of this new regulatory peptide.

Similarities in cellular expression and functions of melanin-concentrating hormone and atrial natriuretic factor in the rat digestive tract.

Melanin-concentrating hormone (MCH) is a cyclic peptide isolated first from salmon brain, then from rat and human hypothalamus. We have recently found expression of MCH messenger RNA and encoded peptides, e.g. MCH and neuropeptide-glutamic acid-isoleucine, within the rat gastrointestinal (GI) tract, but their cellular origin was unclear. Furthermore, similarities in the localization of rat atrial natriuretic factor (ANF) and rat MCH immunoreactivities within intestine suggested functional convergence. In the present study we determined first the presence and distribution of MCH messenger RNA and encoded peptides in the GI tract by combining in situ hybridization and immunohistochemical analysis. Our data revealed numerous MCH-containing cells located in the lamina propria and submucosa at both duodenal and colonic levels. Second, the localisation of MCH- and arginine vasopressin- or ANF-containing cells appears similar at the duodenal and colonic levels, respectively. Colocalization of MCH/neuropeptide-glutamic acid-isoleucine immunoreactivity (-IR) and catecholamine indicated that MCH-expressing cells are probably antigen-presenting cells forming part of the enterochromaffin cell system. Third, we performed reverse phase HPLC coupled to RIA to characterize MCH-like materials in different portions of the rat gut. Crude acidic extracts of rat intestine contained about 2-3 pmol/g tissue of MCH-IR, close to the values found in brain extracts. Reverse phase HPLC of MCH-IR in the GI tract revealed that only 10-30% of the immunoreactivity corresponded to mature MCH, whereas the rat brain contained 94% mature peptide. Finally, we compared the effect of MCH and ANF on water and electrolyte secretions at different levels of the GI tract by using the in situ ligated loop technique. Similar effects were noted for ANF and MCH; both stimulated water, Na, and K fluxes at the proximal colon level and increased Na and K fluxes in the duodenum. However, only ANF increased water and Cl fluxes in the duodenum and decreased bicarbonate secretion in the ileum, whereas MCH increased bicarbonate absorption in the jejunum. The dose required was 10 nmol/100 g.h for MCH, i.e. 10 times more than for the ANF. These studies strongly suggest that MCH produced by antigen-presenting cells of the lamina propria may have an important role, similar to that of ANF at the colonic level, in the physiology of the GI tract.

Dual effect of bombesin and gastrin releasing peptide on gastric emptying in conscious cats.

The effect of bombesin (BBS) and gastrin releasing peptide (GRP) on gastric emptying was studied in conscious cats. This effect was measured simultaneously with antral motility. Acid and pepsin secretions as well as blood hormonal peptide release were additionally measured. A dual effect was observed. First, BBS and GRP slowed gastric emptying of liquids, while antral motility was decreased, then after 60 minutes of continuous intravenous infusion, antral motility returned to basal values and gastric emptying effect reversed. The mechanism of this peculiar action is independent of gastrin, pancreatic polypeptide, somatostatin and motilin release and most probably connected with a cholinergic stimulation induced by the peptides, the late predominance of which counterbalances the inhibitory effect of bombesin-like peptides on antral motility.

Coding region of the sorbin gene in different species.

The coding region of 153 amino-acid sorbin, isolated from porcine intestine has been cloned and sequenced in pig, human and rat. The coding region includes 459 bases comprising the 5' region of 24 bases, the middle region named "sorbin-like sequence" (25-432) and the 3' region (433-459). The peptidic C-terminal segment presents the biological activity: absorption of water and electrolytes from the intestine and gall-bladder. The cDNA homology between the three species was 95%. Three forms of mRNA were found, two major forms (6.5 and 8 Kb) and one minor (4.5 Kb).

Effect of sorbin derivatives on cholera toxin-induced intestinal secretion in rat in vivo.

The effect of synthetic sorbin derivatives was determined on cholera toxin-stimulated jejunal secretion in anesthetized rats in vivo, using both perfused and ligated loop. An inhibitory effect on water secretion induced by cholera toxin was shown with C-terminal sorbin peptides: C20 (YEPGKSSILQHERPVTKPQA-amide), C10 and Dala 7 heptapeptide-amide of sorbin, given by subcutaneous (SC) or intraduodenal administration. When perfused intravenously, C20-sorbin inhibited the cholera-induced stimulation of net flux of water, Na+ and K+, in the jejunum and at the same time the net flux of water and Cl- in the duodenum, which was not in contact with the toxin. 5-hydroxytryptamine was not significantly changed in plasma or fluid. Prostaglandin E2 release in jejunal as well as duodenal fluid was significantly stimulated by cholera toxin, but was not significantly different from basal value after C20 administration.

Effect of porcine gastrin releasing peptide on gastric secretion and motility and the release of hormonal peptides in conscious cats.

The effect of porcine gastrin releasing peptide (GRP) was compared to those of bombesin (BBS) and pentagastrin (PG) in conscious cats. GRP and BBS augmented acid and pepsin secretions, as well as antral motility with an early effect comparable to that produced by pentagastrin with an elevation of low amplitude contractions and a diminution of high amplitude contractions. BBS and GRP increased plasma gastrin and pancreatic polypeptide (PP) levels and decreased motilin levels measured by a C terminus-directed antiserum. In all cases, BBS and GRP displayed parallel dose-response curves. PG showed slight differences in the slopes of the dose-response curves slopes of the dose-response curves except for acid secretion stimulation where no difference was noted (PG was the most effective) and for pepsin stimulation where the difference was large (PG was much less effective). According to the different targets studied, BBS was 4 to 9 times more potent than GRP, 6 to 200 times more than PG. Gastrin release, elicited by the lowest ED50 of both BBS and GRP, should be considered as their primary effect in the cat.

Inhibitory effect of sorbin on pepsin secretion in conscious cats and rabbits.

Sorbin, a 153 amino acid polypeptide isolated from porcine upper small intestine and its shortest synthetic derivative, the C-terminal heptapeptide (C7-sorbin), substituted by D alaninamide in the last position (D7-sorbin), have proabsorptive and antisecretory effect in the different parts of the intestine. We showed that labeled C7-sorbin accumulated not only in the enterocytes and the enteric nervous system but also in the gastric chief cells in the rat. The chief cell secretion of pepsin was then studied in two other species, the cat and the rabbit, simultaneously with the acid secretion of parietal cells. Lipase secretion was studied in the rabbit because lipase is exclusively secreted by the upper cells of the fundic glands, which do not secrete pepsin. The animals were equipped with a gastric fistula, fully innervated, and a Heidenhain pouch, vagally denervated, during a continuous perfusion of pentagastrin (PG) 2 microg/kg. h and vasoactive intestinal peptide (VIP) 4 microg/kg. h. D7-sorbin (100 pmol/kg. h) inhibited cat and rabbit pepsin secretion from the innervated gastric fistula secretion and from the cat denervated Heidenhain pouc secretion, but was without effect on acid secretion and lipase secretion. These data indicate that the inhibitory effect of sorbin is specific on chief cells because the acid parietal cell secretion in both species and lipase upper cell secretion of the fundic glands, in the rabbit, are not implicated.

Pharmacokinetics and organ distribution of the sorbin C-terminal peptides.

Sorbin is a 153 amino acid peptide isolated from porcine small intestine. The heptapeptide-amide is the minimal active site of the natural molecule. A comparison of the distribution of C-7 and C-20 sorbin, which have been shown to share the activity of sorbin in increasing intestinal absorption of electrolytes, was undertaken by radioimmunoassay, after perfusion of 200 micrograms/kg/h. A longer half-life in plasma was observed for C-20 sorbin than for C-7 sorbin, with a clearance rate of 18 +/- 4 ml/min/kg vs. 40.6 +/- 13.5 ml/min/kg and a distribution volume of 192 +/- 35 ml/kg vs. 286 +/- 123 ml/kg. The accumulation of tritiated C-7 sorbin was observed in enterocytes, serosal acini of the salivary glands, and fundus chief cells. The recovery of intact peptide in the intestine was 0.06% per gram of tissue. Eighteen percent of the peptide was detected in urine.

Pharmacokinetic, metabolic, and antidiarrheal properties of (D and L) heptapeptides of sorbin in rodent.

The C-terminal heptapeptide-amide (C7-sorbin) is the minimal biologically active fragment of sorbin inducing an increase in intestinal hydroelectrolytic absorption. An analogue (D7-sorbin), characterized by the replacement of the ultimate C-terminal amino acid L-alanine-amide by D-alanine-amide, was synthetized. For pharmacokinetic studies, D7-sorbin and C7-sorbin were tritium labeled. After IV injection, clearances were 10.6 and 30.2 ml-1 for D7-sorbin and C7-sorbin, respectively, and MRT were 34 and 18 min. After SC administration, Cmax attained 0.41% and 0.12% of the dose/ml, respectively. The IP route showed a 45-min delay before Cmax and a 100% bioavailability for both peptides. D7-sorbin was principally excreted in urine, as shown by balance study, and in part in intact form, as controlled by mass spectrometry. D7-sorbin induced a significant decrease of the VIP-induced ileal secretion, previously observed with C7-sorbin. The change of L-Ala to D-Ala increased the stability of the synthetic C-terminal peptide of sorbin whereas its biological activity, bioavailability, and route of elimination were unchanged.

Comparative effects of vasoactive intestinal peptide and secretin on exocrine pancreatic secretion in the cat.

The stimulatory effect of vasoactive intestinal peptide (VIP) and secretin has been compared on exocrine pancreatic secretion in anaesthetized cats. Both peptides were given by bolus intravenous injection and continuous intravenous infusion. After bolus injection, VIP stimulated pancreatic secretion only weakly. On the contrary, during intravenous infusion, the maximal effect of VIP did not differ significantly from that of secretin. Therefore, while the potency of VIP is always lower than that of secretin, its efficacy appears to be strictly dependent on the mode of administration.

Effect of somatostatin on the secretin-induced gastric secretions of pepsin and mucus in cats.

The effect of somatostatin 14 on gastric stimulation produced by secretin was determined in 6 conscious cats equipped with a gastric fistula and a denervated fundic pouch. Somatostatin strongly inhibited the basal and secretin-induced pepsin secretion. It did not, however, inhibit the secretin-induced mucus secretion, even though it decreased the basal mucus secretion. During somatostatin administration, the secretagogue effect of secretin on mucus secretion might be dissociated from its stimulatory action on pepsin secretion.

Distant intestinal stimulation by cholera toxin in rat in vivo.

Cholera toxin (16 microg/rat) locally administered in the jejunum of anesthetized rats stimulated jejunal secretion and also distant duodenal secretion, as determined with the ligated loop technique. The release of prostaglandin E2 in both jejunal and duodenal secretions and in plasma was increased by cholera toxin, while the release of 5-hydroxytryptamine (5-HT) was unchanged in the early phase of secretion (2 h). The inhibitor of prostaglandin E2 release, indomethacin (10 mg/kg, s.c.), and the 5-HT3 subtype receptor antagonist, granisetron (30 microg/kg i.v.), inhibited the jejunal secretion but had no effect on distant duodenal secretion. However, indomethacin statistically significantly decreased prostaglandin E2 release in both jejunal and duodenal secretions as well as in plasma. The vasoactive intestinal peptide antagonist (VIP-(6-28), 1.2 nmol/100 g h) did not modify jejunal and duodenal secretions. Our study confirmed the local involvement of 5-HT and prostaglandin E2 in choleraic jejunal secretion but not in distant duodenal secretion.

Guanylin-, heat-stable enterotoxin of Escherichia coli- and vasoactive intestinal peptide-induced water and ion secretion in the rat intestine in vivo.

The heat-stable enterotoxin of Escherichia coli binds to an intestinal receptor, guanylyl cyclase-C, and produces cGMP to induce diarrhea. Guanylin is an endogenous ligand of this receptor. In the present in vivo study, the intestinal water and ion secretion induced by mucosal application of 2 nmol/ml guanylin or 5 or 10 units/ml heat-stable enterotoxin into closed loops was compared in the rat. The characteristics of secretion induced by cAMP following intravenous perfusion of 1.2 nmol/100 g per h vasoactive intestinal peptide were compared to those induced by cGMP. Unidirectional Na+ and Cl- fluxes were estimated by addition of 22Na into the loop and i.v. injection of 36Cl. Guanylin induced less water and ion secretion than that produced by heat-stable enterotoxin in the colon, confirming the results of in vitro studies, and also in duodenum and ileum. The cAMP- or cGMP-mediated response had a similar pattern, i.e., an inhibition of Na+ absorption and an increase in anion secretion.

Circannual and circa-hemiannual rhythms of basal and stimulated gastric secretion in conscious cats.

Conscious cats equipped with a gastric fistula and a denervated Heidenhain pouch were submitted to weekly measurements of the basal and pentagastrin-stimulated gastric secretion for 1 to 14 years. Rhythms of basal secretion were documented in 37 cats for the group studies, in 25 cats only for the individual studies which required at least whole year data. Twelve-month or 6-month rhythms were detected for each variable studied, i.e. volume, acid, pepsin, fucose and uronic acid outputs in the group studies, with peaks for volume, acid and pepsin in Winter, peaks for uronic acid in Spring and Fall indicating different rhythms for oxyntic, chief and mucous cells. Individual studies detected rhythms in 25% of the analyses, and demonstrated male and female and cat to cat differences. Spectral analysis in 3 cats confirmed the differences in the individual rhythms with prominent peaks differing from 365 days in 50% of the cases. Chronopharmacological responses to pentagastrin were documented for volume, acid and pepsin outputs in 5 male and 6 female cats. Group analysis detected a Winter acrophase for volume and acid secretion and a Summer acrophase for pepsin secretion. Analysis of the stimulated response data showed interindividual variation but a higher percentage of detection for rhythms, i.e. 38% for all variables and 50% for pepsin secretion. Different rhythms in acid and pepsin secretion documented in individual studies could provide the basis of a better understanding of the discrepancies reported in the literature concerning the seasonal incidence of peptic ulcer disease.

Effects of proximal gastric vagotomy on gastric secretory and plasma hormonal responses to sham feeding in patients with duodenal ulcers.

The effects of proximal gastric vagotomy on the gastric secretion of acid and pepsin, and on the release of gastrin and pancreatic polypeptide in response to sham feeding were assessed comparatively within 1-4 months after surgery in 32 male duodenal ulcer patients. Each test comprised three successive periods: basal, modified sham feeding (MSF) and pentagastrin stimulation. In each test period the acid output was strongly correlated with the corresponding pepsin output, both parameters being reduced to similar extents after vagotomy. The percentage of postoperative reduction of MSF-induced acid and pepsin outputs was positively correlated with the preoperative values. MSF resulted in a limited but significant release of gastrin, the response being significantly greater after surgery. The MSF-induced release of pancreatic polypeptide was significantly reduced by proximal gastric vagotomy, the reduction percentage being negatively correlated with the time elapsed since surgery. Neither pre- nor post-operatively did the gastrin and pancreatic polypeptide responses bear any relationship to the other parameters tested. We conclude that the study of sham feeding responses of pepsin, gastrin and pancreatic polypeptide provides no further information than does the measurement of acid secretion for the segregation of duodenal ulcer patients, especially with respect to follow-ups for ulcer recurrence.

[Effect of C-terminal derivatives of sorbin on ileal ion transport stimulated by VIP in rats]. / Effet des dérivés C-terminaux de la sorbine sur les flux ioniques iléaux stimulés par le VIP chez le rat.

OBJECTIVES AND METHODS: Sorbin, a peptide isolated from porcine intestine and composed of 153 aminoacids, has been purified because its specific action is to increase water and ion absorption in the intestine and the gall bladder. We showed that synthetic peptides containing the amidated C-terminal part of sorbin had the same activity as the natural molecule in increasing duodenal absorption. In order to characterize the site of action of sorbin, the effect of two C-terminal derivatives were determined in ileal ligated loops in situ in anaesthetised rats, following VIP-induced water and electrolyte secretions. Their effect was compared to those of metenkephalinamide, NPY and somatostatin. Unidirectional fluxes were studied to analyze the mechanism of action of sorbin, by means of 22Na, administered into the intestinal loop, and 36Cl, injected into blood. RESULTS: Results show that C20-sorbin and C7-sorbin decreased the VIP-stimulated net flux of water (inhibition of 40 and 37%, respectively), Na (inhibition of 31 and 30%), C1 (inhibition of 80 and 63%) and HCO3 (inhibition of 15 and 25%). These effects are evidently greater than those produced by equimolar doses of NPY, somatostatin, and 32 times higher dose of metenkephalinamide. Sorbin acts as a potent anti-secretor, anti-VIP, in rat ileum.

[Effect of C-terminal derivatives of sorbin on duodenal ion transports in rats]. / Effet des dérivés C-terminaux de la sorbine sur les flux ioniques duodénaux chez le rat.

OBJECTIVES AND METHODS: Synthetic derivatives of sorbin have been shown to inhibit VIP stimulated fluxes in the ileum in decreasing plasma-to-mucosa Na and Cl effluxes. The effect of this group of new peptides, without homology with any known peptides, was determined in rat duodenum where ion transport mechanisms differ. The improved technique of ligated loops in situ, was used, permitting the simultaneous measurement of net fluxes, influxes and effluxes for Na and Cl, in an integrated in vivo model. To determine the minimal active fragment of sorbin, synthetic C5, C7 and C20 peptides were tested and compared with known anti-secretor drugs such as loperamide, neuropeptide Y, somatostatine and metenkephalinamide. RESULTS: C7-sorbin was the minimal peptide able to decrease duodenal VIP-stimulated fluxes of water, Na and bicarbonate. It intervenes in increasing Na influx and more slightly Cl influx, which have been decreased by VIP. It does not modify much Na and Cl effluxes stimulated by VIP. Sorbin effect is in contrast with those of known antidiarrheic agents like somatostatine, loperamide, NPY and metenkephalinamide which chiefly decrease Cl efflux. CONCLUSIONS: Sorbin acts like an activator of absorption in the duodenum, in contrast to the other peptides or drugs and to its own anti-secretor effect in the ileum.