Analysis of root plasma membrane aquaporins from Brassica oleracea: post-translational modifications, de novo sequencing and detection of isoforms by high resolution mass spectrometry.

Plasma membrane Intrinsic Proteins (PIPs), a subfamily of aquaporins, are ubiquitous membrane channel proteins that play a crucial role in water uptake in plants. The use of high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) analysis of peptides has previously shown to be a valuable tool to differentiate among PIP homologues sharing a high sequence homology and also to characterize their post-translational modifications (PTMs). The recent introduction of mass spectrometers able to measure peptide mass with high mass accuracy, together with new alternative ways of peptide fragmentation allows the identification and characterization of proteins from nonsequenced organisms, such as broccoli. In this study, we combined three endoproteases (trypsin, Glu-C and Lys-C) with HPLC-MS/MS analysis and two types of peptide fragmentations, CID (collision induced dissociation) and HCD (higher-energy C-trap dissociation), to identify PIP isoforms and PTMs from broccoli roots. After de novo sequencing analysis, eight peptides showing homology to Arabidopsis thaliana PIPs were identified. Although Arabidopsis nomenclature of PIP isoforms has not been defined for broccoli, our results agree with the occurrence of seven AtPIP isoforms (PIP 1;1, PIP 1;2, PIP 1;3 and PIP2;2, PIP 2;3, PIP2;1 and PIP2;7) in broccoli roots, as compared to the plant model A. thaliana. To our knowledge, these results represent the deepest characterization of the PIPs isolated from the roots of broccoli, a crop with increasing agronomical interest.

Plant aquaporins: new perspectives on water and nutrient uptake in saline environment.

The mechanisms of salt stress and tolerance have been targets for genetic engineering, focusing on ion transport and compartmentation, synthesis of compatible solutes (osmolytes and osmoprotectants) and oxidative protection. In this review, we consider the integrated response to salinity with respect to water uptake, involving aquaporin functionality. Therefore, we have concentrated on how salinity can be alleviated, in part, if a perfect knowledge of water uptake and transport for each particular crop and set of conditions is available.

Long-term effects on lipid metabolism in miniature swine (Sus scrofa) of diets enriched in saturated, monounsaturated and polyunsaturated (N-6 and N-3) fatty acids.

In this paper we study the effect of long-term adaptation (twelve months) of lipidic parameters of miniature swine to diets enriched in saturated (lard; L-group), monounsaturated (olive-oil; O-group) and polyunsaturated (sunflower and fish-oil; S- and F-groups respectively). The experimental group with the highest level of total cholesterol, free cholesterol and phospholipids was the S-group. This group had as well levels of HDL-C and LDL-C significantly higher when compared with the remaining groups. The L-group had the lowest value of HDL-C. In spite of that, the index of artherogenicity (HDL/LDL+VLDL) was significantly higher in the L-group, followed by the O-, F- and S-group respectively. On the other hand, after 12 months adaptation we observed that the fatty acid composition of serum lipids clearly reflects the quality of the dietary fats. The O-group had significantly higher serum oleic acid levels than all the other groups, and its content in saturated fatty acids was the lowest. The same happens with red blood cell (Rbcs) membranes fatty acids but the effect is less marked. Membranes of the L-group were found to have the highest saturation index (SI) in Rbcs membranes, while the F-group had the highest unsaturation index (UI), followed by the O-group. Taken together, our findings show that the diet enriched in olive oil produces a lipid pattern intermediate between that obtained with the fish-oil-diet and the sunflower-oil-diet but with the advantage of Rbcs membranes with a lower amount of PUFAS. As it is known, membranes with high percentages of PUFAS are more accessible for peroxidation, and the degree of peroxidation of lipids is directly related with changes in the membranes functionality.

Long-term influence of dietary fat (sunflower oil, olive oil, lard and fish oil) in the serum fatty acid composition and in the different lipidic fractions, in miniature swine.

A total of 24 miniature swine (sus scrofa) was fed with four diets of 9% fat content, differing only in the quality of the fat source (sunflower oil, olive oil, lard fat and fish oil) for a 12 months period. After the experimental period, the influence of the dietary fat on the serum fatty acid composition, and the distribution of those fatty acids in the different serum lipidic fractions was studied. The olive oil group had the lowest SI value (total sum of saturated fatty acids) and the highest MUFA value (total sum of monounsaturated fatty acids) in serum. The sunflower group had higher proportions of n6 polyunsaturated fatty acids in serum, when compared with the remaining groups. The lard group and the fish oil group had both the highest values of SI and n3 polyunsaturated fatty acids. The results show that fatty acid composition in serum reflected, but not in all cases, the fatty acid composition of the diet. The highest correspondence was found with sunflower oil and olive oil diets. The existence of interactions between the different fatty acid series was evident, especially with the lard and the fish oil diets, as well as endogenous synthesis mechanisms. In lipidic fractions, higher correspondence with diet was found in EC fraction, but, as well as in serum, the existence of interactions between fatty acid series was evident, and especially activity and biosynthesis of desaturases may have been affected.

[Postoperative enteral nutrition with a high content of mono- and polyunsaturated fatty acids]. / Nutrición enteral postoperatoria con alto contenido en ácidos grasos mono y poliinsaturados.

We have done a clinical study in patients with benign biliary disease and indication for surgery, with the aim of analyzing some parameters of gastric secretion, biliary and pancreatic function, and gastrointestinal hormones in relation to the quantity and quality of the dietary fat (monounsaturated and polyunsaturated fatty acids), acting on the postoperative enteral ingestion, modifying the lipid source by its elaboration. In the present work we show the results derived from the analysis of the gastric and duodenal juices in relation to the type of fat administered, with a global description of the study being necessary. The studied sample consisted of 20 patients diagnosed with simple gallstones, who were divided into two groups (n = 10) according to the usual ingestion of fats: olive oil (Olive Group) or sunflower oil (Sunflower Group). A control group (n = 10) has also been considered, without taking into account their dietary habits. The patients were subjected to surgery, and after the immediate postoperative period they were fed, orally, with an enteral diet prepared with commercially available formulae (modular EDA); as a lipid source we have added olive oil, rich in monounsaturated fatty acids, for the Olive Group, and sunflower oil, rich in polyunsaturated oil, for the Sunflower Group. In the control group cream was added as lipid source (mono and polyunsaturated fatty acids). The caloric value of the diet is calculated based on the caloric value of its components (protein: 17%, carbohydrate: 53%, lipid 30%). The daily caloric requirements are determined based on the daily basal metabolism, according to the Harris-Benedick formula.(ABSTRACT TRUNCATED AT 250 WORDS)

The influence of dietary fat source (sunflower oil or olive oil) on LDL composition and serum lipid levels in miniature swine (Sus scrofa).

A total of 24 miniature swine (Sus scrofa) were fed with two diets of 9% fat content, differing only in the quality of the fat source (sunflower oil and olive oil). Two groups of animals were fed for a 12-week period, and the other two groups were fed for a 50-week period. After the two experimental periods, the influence of the dietary fat on serum lipids and protein and fatty acid composition of isolated LDLs was studied. In the short term, the serum cholesterol level was slightly higher in the olive oil group but, with the time of adaptation to the diet, serum levels of TC, FC and PL increased significantly in the sunflower group. In the long term, LDL and HDL were also significantly higher in the sunflower group when compared to the monounsaturated diet. In the sunflower group, PROT/TC and PROT/LIP ratios decreased significantly with the experimental period, while in the olive oil group they increased, due to the decrease in EC and TG fractions. The LDL particle in the olive group contained fewer saturated fatty acids and more monounsaturated fatty acids, specially oleic acid, than the LDL in the sunflower group. The changes found in chemical and fatty acid compositions of LDL, according to the saturation degree of the predominant fat of the diet, could alter its cellular metabolism.

Effects of adaptation to dietary fat on fatty acid composition of serum and serum lipids in miniature swine.

The influence of dietary fat on the serum fatty acid composition and distribution in different serum lipids was studied in 24 miniature swine (Sus scrofa) divided into 4 groups. Three groups were fed a low-fat chow with different kind of fats added (sunflower oil, olive oil and bovine lard) and the other group was fed a control diet with no fat added. The animals were fed the four diets for an experimental period of 12 weeks. Afterwards blood samples were taken to obtain serum, and the serum lipids were separated into the following fractions: phospholipids, triglycerides, and esterified cholesterol. The fatty acid composition of the serum and fractions was analyzed by gas chromatography. Under our experimental conditions, the fatty acid composition of the diets was reflected in serum, but not in all cases. In the triglyceride fraction the fatty acid composition was more evident. Our results show the interaction of the fatty acids in the different series and even within a single series, along with the endogenous synthesis processes.

Effects of adaptation to diets enriched with saturated, monounsaturated and polyunsaturated fats on lipid and serum fatty acid levels in miniature swine (Sus scrofa).

A total of 37 miniature swine (Sus scrofa) was fed with three diets of 9% fat content, differing only in the quality of the fat source (sunflower oil, olive oil and lard fat). Three groups of animals were fed for a 3 month period, and three other groups were fed for a 12 month period. After the two experimental periods, the influence of the diet on serum lipids and serum fatty acids was studied. In the short term, the serum cholesterol level was higher in the lard group, but with time, it undergoes a significant increase in the sunflower group, due to increases both in HDL-C and in LDL-C. In the olive oil group, LDL-C hardly varies with time, while HDL-C tends to decrease. In the lard group, LDL-C increases and HDL-C decreases. The atherogenic index is, in the long term, lower in the lard group and higher in the polyunsaturated fat group. The fatty acid composition of serum lipids in the long term shows a greater saturation index (SI) for the sunflower group and lard group, an increase in monounsaturated fatty acids in the olive oil group, and a higher percentage of linoleic acid in the sunflower group.

Distal ileum resection totally suppresses the late pancreatic postprandial hypersecretion in conscious dogs.

Exocrine pancreatic secretion and serum levels of secretin, vasoactive intestinal peptide (VIP) and gastrin within postprandial 12 hours have been studied in dogs that have undergone a partial ileum resection (last 25 cm) 4 weeks before. In these conditions the following effects were observed: 1) the flow of pancreatic juice secreted after the food intake (1-4h period) was strictly similar to the flow described in intact dogs which did not suffer that surgical manipulation but it came back to its basal value more rapidly; 2) there were no effects regarding the postprandial hormonal pattern; 3) the late pancreatic hypersecretion period that is usually observed in intact dogs within the postprandial 8-12 h period, completely disappeared. These results allow to conclude that the mechanisms inducing the late hypersecretion process that usually takes place within the postprandial 8-12 h in dogs are generated in the distal ileum.

Effect of dietary fat composition on biliary cholesterol saturation index in dogs.

In dogs, the effect of dietary fat on bile cholesterol saturation index and the evolution of molar percentages of biliary lipids have been studied in both fasting and postprandial periods after a long-term adaptation period to diets which only differ in their lipidic source (olive oil and sunflower oil). It has been observed that for similar bile cholesterol saturation indexes in both groups, dietary fat altered differently biliary lipid composition through a double mechanism which involved bile acids and phospholipids. Dietary fat is postulated to affect differently the lipidic composition of bile as well as the biliary tree motility both during interdigestive and postprandial periods.

Role of vasoactive intestinal polypeptide (VIP), secretin and gastrin in the genesis of the late exocrine pancreatic hypersecretion, food intake dependent in conscious dogs.

Plasma levels of vasoactive intestinal polypeptide (VIP), secretin and gastrin were studied in four saphenous vein-catheterized dogs during the first 12 h after ingestion of a standard solid meal. Under these conditions we found significant postprandial increases in secretion only, which rose from a basal value of 219 +/- 27 pg/ml to 449 +/- 66 pg/ml 60 min postprandial (p < 0.001), and remained elevated until 4 h after food intake. However, no increase was seen from 8 to 12 h in any of the hormones studied, indicating that they are not directly involved in the enhancement of exocrine pancreatic secretion during this period.

Adaptation of biliary response to dietary olive oil and sunflower-seed oil in dogs.

The effects of adaptation to dietary fat of different degrees of unsaturation (olive oil and sunflower oil) on bile secretion were studied in dogs at rest and after food intake. The animals were prepared with a bidirectional biliary cannula and a duodenal cannula to provide bile return. The two experimental groups were fed on diets containing 150 g fat/kg in the form of either olive oil (O) or sunflower-seed oil (S). The flow-rate under resting conditions and the patterns of response to food were similar in both experimental groups, although postprandial hypersection were significantly greater in volume and more prolonged in group O. No appreciable differences in concentration and output of biliary cholesterol or phospholipids were noted between the two groups. In contrast, the concentration and output of bile acids differed significantly both at rest and after food: concentration and output of bile acids were greater at rest in group S. However, after food intake, these responses were increased only in group O. The results suggest that the type of dietary fat affects biliary response to food, probably through differences in the contribution of the gall bladder in the two experimental groups.

Late postprandial pancreatic secretion periods in conscious dogs. Effect of vagotomy.

It is well established that, in the dog, the exocrine pancreatic secretion in response to food intake is a two-phased mechanism with a first phase during 0-4 h period and a second one during 8-12 h period. In the present study we have investigated the role played by the vagus nerve in the genesis of this late pancreatic hypersecretion (second phase) in dogs with truncal vagotomy and pyloroplasty. Truncal vagotomy totally suppressed the first phase of the pancreatic secretion; it did not abolish the second postprandial phase but it increased its latency by delay of 4 hours. In fact, during the 12-18 h period a pancreatic hypersecretory response was evidenced after vagotomy which appeared to be statistically significant as compared to basal values (P less than 0.001). Our results indicate that the vagus nerve does not play a role in the genesis of the late hypersecretory second phase.

Effect of dietary fat on biliary lipids composition of gallbladder bile in miniature swine (Sus scrofa).

1. Weaning animals (34 days old) were fed for 6 months with four diets that differed only in the quantity and quality of fat sources (control group 3.2% (w/w), lard-fed group 7.6% (w/w), olive-oil-fed group 10.4% (w/w), and sunflower-oil-fed group 10.5% (w/w). Samples were collected and assessed after animals were killed at 6 months of age. 2. Our results show that the quality of fat did not alter biliary lipids composition of the gallbladder bile composition in miniature swine after 6 months adaptation to the diet, except for a significant increase of the phospholipid concentration in the lard-fed group with regard to the control group. 3. The highest and lowest bile cholesterol saturation indices were observed in sunflower and lard-fed groups, respectively showing in all cases saturation indices far from the saturation point.

Protein digestibility in dog. Effect of the quantity and quality of dietary fat (virgin olive oil and sunflower oil).

Protein digestibility has been estimated in dog when fed on two similar diets except their lipid quality (virgin olive oil and sunflower oil) from weaning up to 6 months of life. Experiments were carried out at 60, 105 and 150 days of adaptation to either diet. In dog, age does not seem to affect the protein digestive and metabolic utilization when fed on a high lipid content diet. A higher dietary fat percentage led to an increase of the mentioned parameters which could be caused to a delayed gastric emptying due to fat. As far as the fat quality is concerned, an improved protein digestive and metabolic utilization was evidenced in adult animals fed on olive oil. On the other hand, the weight progress was normal in both experimental groups. These results suggest that olive oil may have beneficial effects on protein digestibility as compared to sunflower oil.

Long-term adaptation of pancreatic response by dogs to dietary fats of different degrees of saturation: olive and sunflower oil.

Mongrel dogs were fed, from weaning to 9 months of age, on one of two diets that differed only in the type of fat content (virgin olive oil or sunflower oil) to compare the composition of exocrine pancreatic secretion in the basal period and in response to food. In resting pancreatic flow, electrolytes and the specific activities of amylase, lipase and chymotrypsin were similar in both experimental groups. However, lipase and amylase outputs, and amylase and protein concentrations were significantly higher in the group fed on the diet rich in sunflower oil. Food intake was not followed by any change in flow-rate or electrolyte or protein content in the group given the diet rich in olive oil. Amylase activity and output were also lower in this group, as was lipase output, whereas activity and specific activity of chymotrypsin were lower in dogs fed on the diet containing sunflower oil. The differences traceable to the composition of the two types of dietary fat supplied may be related to the balance between factors that stimulate and inhibit pancreatic secretion.