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Genomic profiling of digestion related enzymes in Anopheles aquasalis a major coastal neotropical malaria vector.

Digestive genes are fundamental for the development and survival of mosquitoes and can serve as a target for the development of strategies for mosquito control or vector-borne disease prevention. Genes related to digestion were identified in the genome of the neotropical malaria vector Anopheles aquasalis by similarity. We used reciprocal BLAST with annotated digestion proteins for Anopheles gambiae. Orthology and evolutionary analyses were performed using MEGA with a bootstrapped phylogenetic tree constructed by the neighbor-joining method, and copy number variation was measured by the standard deviation of the average copy number in each gene family. We identified 241 genes related to digestion in An. aquasalis: 56 genes related to carbohydrate digestion, 51 genes for lipid digestion, and 134 genes for protein digestion. Phylogenetic relationships with other anophelines show that An. aquasalis genes are closely related to those of neotropical mosquitoes Anopheles darlingi and Anopheles albimanus. Orthologous gene clusters are conserved in important families of all four species. Some of these conserved genes are of interest for studies on controlling mosquito vectors, such as larvicidal toxin receptor genes, alpha-amylase, alpha-glucosidase, and maltase; important target genes for transmission-blocking vaccines, such as aminopeptidase N1 and carboxypeptidase B; and the major intestinal serine proteases, such as trypsins and chymotrypsins, which can positively or negatively affect Plasmodium development in the midgut. These data provide a better understanding of digestion-related genes in American anopheline mosquitoes and may support further fundamental and applied studies aimed at malaria control.

Animals↗

Kinetics and dynamic modelling of batch anaerobic digestion of municipal solid waste in a stirred reactor.

A series of batch, slurry anaerobic digestion experiments were performed where the soluble and insoluble fractions, and unwashed MSW were separately digested in a 200l stirred stainless steel vessel at a pH of 7.2 and a temperature of 38 degrees C. It was found that 7% of the total MSW COD was readily soluble, of which 80% was converted to biogas; 50% of the insoluble fraction was solubilised, of this only 80% was converted to biogas. The rate of digesting the insoluble fraction was about four times slower than the rate of digesting the soluble fraction; 48% of the total COD was converted to biogas and 40% of the total nitrogen was converted to ammonia. Soluble and insoluble fractions were broken down simultaneously. The minimum time to convert 95% of the degradable fraction to biogas was 20 days. The lag phase for the degradation of insoluble fraction of MSW can be overcome by acclimatising the culture with the soluble fraction. The rate of digestion and the methane yield was not affected by particle size (within the range of 2-50mm). A dynamic model was developed to describe batch digestion of MSW. The parameters of the model were estimated using data from the separate digestion of soluble and insoluble fractions and validated against data from the digestion of unwashed MSW. Trends in the specific aceticlastic and formate-utilising methanogenic activity were used to estimate initial methanogenic biomass concentration and bacterial death rate coefficient. The kinetics of hydrolysis of insoluble fraction could be adequately described by a Contois equation and the kinetics of acidogenesis, and aceticlastic and hydrogen utilising methanogenesis by Monod equations.

Anaerobiosis↗

Immunochemical and structural analysis of pepsin-digested egg white ovomucoid.

Ovomucoid, an egg protein comprising approximately 10% egg white, was digested using the enzyme pepsin, and fragments were isolated by anion-exchange and reverse phase HPLC. Four distinct fragments were identified by analysis with SDS-PAGE, including three large fragments with molecular weights of around 24, 18, and 14 kDa. N- and C-terminal and amino acid sequencing analyses identified the fragments as V134-C186 (domain 3), V21-A133, and A1-A133 (domain 1+2). Further separation and sequencing of the fraction composed of small peptides, to determine their exact makeup and location in the protein, remained to be carried out and identified a peptide G51-Y73. All four fragments showed IgE-binding activity, as measured by ELISA, using human sera from egg-allergic individuals. Little change in the digestibility of ovomucoid by trypsin and chymotrypsin was observed following digestion with pepsin, indicating that pepsin-digested ovomucoid retains its trypsin (protease) inhibitor activities. Reduced carboxymethylated ovomucoid was prepared, and digestion with pepsin produced significantly more peptides than did the digestion of the native ovomucoid, indicating that the disulfide bonds play a significant role in the digestive resistance of ovomucoid. The reduction of ovomucoid enhanced its digestibility and lower allergenicity of the protein.

Digestion↗

Sodium copper chlorophyllin: in vitro digestive stability and accumulation by Caco-2 human intestinal cells.

Sodium copper chlorophyllin (SCC), a mixture of water-soluble chlorophyll derivatives, is used as both a food colorant and a common dietary supplement. Although the potential antimutagenic and antioxidant properties of this commercial preparation have been demonstrated, limited information is available on its digestion and absorption by humans. Stability of SCC was examined during simulated gastric and small intestinal digestion. Three preparations were subjected to in vitro digestion: SCC in water, SCC in water + 10% corn oil, and SCC in applesauce. SCC components from raw material preparations and in digested samples were analyzed by C(18) HPLC with photodiode array detection. Cu(II)chlorin e(4), the major chlorin component of SCC, was relatively stable during simulated digestion. In contrast, greater than 90% of Cu(II)chlorin e(6) was degraded to undetermined products during digestion. Recovery of Cu(II)chlorin e(6) after digestion was increased by incorporation of SCC into applesauce, suggesting a protective role of the inclusion matrix for stabilization of labile SCC components. Accumulation of SCC derivatives was investigated by using differentiated cultures of the TC7 clone of the Caco-2 human intestinal cell line. Cellular accumulation from media containing 0.5 to 60 ppm SCC was linear with intracellular content ranging between 0.2 and 29.6 microg of total SCC per mg of cellular protein. Uptake of SCC by Caco-2 cells was significantly (p < 0.01) lower in cultures incubated at 4 degrees C than in those incubated at 37 degrees C. Although intracellular SCC was transported into both apical and basolateral compartments when Caco-2 cells were grown on inserts, apical efflux was significantly greater (p < 0.01) than basolateral efflux. Stability of Cu(II)chlorin e(4) during in vitro digestion and effective uptake by Caco-2 enterocyte-like cells support the likelihood that a portion of this SCC component or its metabolites is absorbed from the human intestine.

Caco-2 Cells↗

Bioavailability of cadmium from in vitro digested infant food studied in Caco-2 cells.

The solubility and bioavailability of cadmium (Cd) in infant foods, three cereal- and milk-based diets and two ready-to-use baby dishes, were studied after in vitro digestion and by using human intestinal Caco-2 cells. The solubility of Cd after in vitro digestion varied between diets; liver casserole had the highest solubility and was lower after infant as compared to adult digestion conditions. Generally, more Cd was soluble in infant intestinal than gastric juice in contrast to the results from the adult digestion. Caco-2 cells were incubated with supernatants of infant digests that had been equilibrated with (109)Cd during the in vitro digestion procedure, and cellular uptake and transport of (109)Cd were measured after 180 min. Statistically significant differences in both uptake and transport of Cd were detected between some of the diets and a control solution containing only digestive enzymes and (109)CdCl(2). Uptake of soluble Cd in the cells varied between diets from 4 to 6%, and the transport over the monolayers was 1-2% of the dose. We conclude that age specific digestion conditions as well as composition of diets affect both solubility and bioavailability of Cd.

Biological Availability↗

Phospholipid interactions protect the milk allergen alpha-lactalbumin from proteolysis during in vitro digestion.

Interactions with food components may alter the resistance of food proteins to digestion, a property thought to play an important role in determining allergenic properties. The kinetics of breakdown of the bovine milk allergen alpha-lactalbumin during in vitro gastrointestinal digestion was found to be altered by interactions with physiologically relevant levels of phosphatidylcholine (PC), a surfactant that is abundant both in milk and is actively secreted by the stomach. Breakdown during gastric digestion was slowed in the presence of PC and accompanied by small alterations in the profile of resulting peptides, with little effect being observed during subsequent duodenal digestion. alpha-Lactalbumin was found to unfold at gastric (acid) pH, giving a CD spectrum similar to that obtained for the partially folded state it is known to adopt at pH values below its isoelectric point. Fluorescence polarization studies performed at low pH indicated that this partially unfolded form of the protein was able to penetrate into the PC vesicles. These interactions are probably responsible for the slowing of gastric digestion by reducing the accessibility of the protein to pepsin. These findings show that interactions with other food components, such as lipids, may alter the rate of breakdown of food proteins in the gastrointestinal tract. It underlines the importance of the food matrix in affecting patterns of food allergen digestion and hence presentation to the immune system and that in vitro digestion systems used for assessing digestibility of allergens must take account of surfactants.

Allergens↗

Subcellular distribution of cadmium and its cellular ligands in mussel digestive gland cells as revealed by combined autometallography and X-ray microprobe analysis.

Autometallography (AMG) and electron probe X-ray microanalysis (EPXMA) were applied in combination to determine the subcellular distribution of Cd and its subcellular ligands in the digestive gland cells of Cd-exposed mussels Mytilus galloprovincialis. Black silver deposits (BSD), which reveal the presence of metals when AMG is applied, were only localized in digestive cell lysosomes. Digestive cell cytoplasm and basophilic cells were devoid of BSD. EPXMA (static probe and X-ray mapping) indicated that Cd, S (possibly associated with metallothioneins or metallothionein-like proteins) and autometallographical Ag ions are co-localized within digestive cell lysosomes. In addition, Cd and S co-occur in the absence of Ag in the cytosol of digestive cells. AMG does not reveal the presence of the Cd 'pool' strongly bound to cytosolic Cd-metallothionein complexes; only 'free' Cd or Cd supposedly loosely bound to (semi)digested metallothionein within lysosomes was revealed. The levels of lysosomal Cd were indirectly quantified by stereology as the volume density of BSD (V(v)BSD). Significantly higher values were recorded in Cd-exposed mussels compared with controls at all exposure times. However, V(v)BSD values were lower at days 7 and 21 than at day 1. This relative decrease in V(v)BSD reflected another (and confounding) response elicited by Cd-exposure in the digestive epithelium: the volume density of basophilic cells (V(v)BAS) increased significantly as exposure progressed. Due to this cell-type replacement, the net accumulative capacity of the digestive epithelium decreases at long exposure times.

Animals↗

Casein enhances stability of peptides in intestinal lumen: role of digested products of casein.

PURPOSE: To investigate the inhibitory activity of casein on proteases in detail, the effect of digested products of casein itself on trypsin and chymotrypsin in rat small intestine was examined. METHODS: Male Sprague-Dawley rats weighing 200-300 g were used as the animal model. The luminal content of the jejunum was prepared, and the enzymatic activities of trypsin and chymotrypsin were determined using a specific substrate for each protease. Then, the effect of enzymatic digested products of casein on them was examined. RESULTS: The inhibitory activity of trypsin-digested casein against trypsin decreased as its digestion proceeded, but its inhibitory activity against chymotrypsin came to be more effective. On the other hand, the inhibitory activity of chymotrypsin-digested casein against chymotrypsin decreased with the degree of digestion, but no change in the inhibitory activity against trypsin was observed. Even the completely digested products of casein with trypsin or chymotrypsin showed inhibitory activities against the two proteases. CONCLUSIONS: It was suggested that not only the intact casein but also the products digested with trypsin or chymotrypsin contribute to the inhibitory effect of casein on the proteases in the intestinal lumen.

Animals↗

Drug solubilization behavior during in vitro digestion of simple triglyceride lipid solution formulations.

PURPOSE: The purpose of this study was to characterize the solubilization and precipitation characteristics of a range of poorly water-soluble drugs during digestion of either long-chain or medium-chain triglyceride (TG) lipid formulations. METHODS: TG solution formulations of five selected drugs (griseofulvin, diazepam, danazol, cinnarizine, and halofantrine) were digested in ritro and drug distribution/solubilization behavior in the resulting digests assessed. RESULTS: For the less lipophilic drugs, the mass of drug dissolved in either medium or long-chain TG was low and the drugs partitioned rapidly into the aqueous digestion phase. For the higher log P drugs, drug transfer to the aqueous phase was limited by accumulation in undigested long-chain TG. In contrast, medium-chain TG was digested completely producing a dispersed aqueous phase that was capable, at least in the case of the high log P drugs, of supporting supersaturated drug concentrations. CONCLUSIONS: The solubilization behavior of lipophilic drugs on digestion of simple TG lipid formulations is a function of the lipophilicity of the drug (which dictates the drug dose and the partitioning behavior), the nature of the colloidal phases produced on digestion of the different formulation lipids, and the kinetics of drug transfer between the digesting formulation and the colloidal phases produced.

Cinnarizine↗

Magnesium chloride slows gastric emptying, but does not affect digestive functions.

BACKGROUND: An inverse relationship has been established between serum magnesium and serum lipid levels. By means of breath tests, we tested the hypothesis that magnesium inhibits intraluminal lipid digestion and subsequently causes changes in lipid metabolism. We also investigated the influence of the administration of magnesium chloride on protein digestion and gastric emptying. METHODS: Five healthy volunteers performed simultaneous breath tests for gastric emptying and intraluminal lipid digestion, and six others for gastric emptying and protein digestion. Each test was performed in basal conditions and after the intake of 800 mg of magnesium chloride dissolved in water. Breath samples were taken at regular time intervals and analysed for 13CO2 and 14CO2 enrichment in order to calculate gastric emptying and lipid and protein digestion rates. RESULTS: The oral administration of a single dose of magnesium chloride resulted in a diminished rate of intraluminal lipid and protein digestion. The most pronounced effect of magnesium chloride, however, was a decreased gastric emptying rate of both test meals. After correction for gastric emptying, no differences were noted in intraluminal lipid or protein digestion. Therefore, the lower lipid levels noted after magnesium supplementation are unlikely to be the result of altered lipid assimilation. CONCLUSION: Magnesium chloride slows gastric emptying but does not influence lipid digestion.

Breath Tests↗

Reduction of the energy density of cat foods by increasing their fibre content with a view to nutrients' digestibility.

Indigestible compounds are useful for reducing the energy density of food for obese cats, but the acceptance and digestibility of such components must also be taken into consideration. In this trial, dried apple pomace was investigated as a compound having sweet taste, aromatic flavour and high fibre content (207 g/kg dry matter). It was mixed in a meat-based diet at a ratio of 10, 20, and 40%. These diets were fed to adult cats in digestibility trials. As could be predicted, by increasing the percentage of apple pomace in the mixtures the digestibility of nutrients significantly decreased (p < 0.01; p < 0.001). However, the digestibility of ether extract decreased much less than that of the other nutrients. The addition of apple pomace to the diets at an inclusion rate of 10 or 20% reduced their energy density but only slightly changed the digestibility of fat. However, the digestibility of crude protein decreased considerably. According to the present findings, the energy density of mixed diets decreases proportionally to the percentage of apple pomace added. However, due to the much lower digestibility, the protein supply to the cat will be worse. It was concluded that the addition of palatable fibrous components is the best method for reducing the energy content of food for obese cats, as it maintains a normal level of food intake. When calculating the required protein content of a diet supplemented with a fibrous component, in addition to the degree of dilution the reduced protein digestibility must be taken into account.

Animal Feed↗

The rat as a model animal for the growing pig in determining ileal amino acid digestibility in soya and milk proteins.

The growing pig is an established model animal for humans to determine protein digestibility to the end of the small intestine. An ileal amino acid digestibility assay based on the laboratory rat offers an alternative. True ileal amino acid digestibility in high quality refined protein sources was determined and compared between the laboratory rat and the growing pig. Both species of animals were fed their daily ration in nine separate meals. Ileal digesta were collected at slaughter and chromic oxide was used as an indigestible marker. For several of the amino acids examined, there were no significant (p < 0.05) differences between the two species for true ileal amino acid digestibility, but for aspartic acid, serine, cysteine, methionine, tyrosine, phenylalanine and histidine, significant (p < 0.05) interspecies differences were found. The overall average amino acid digestibility, for all amino acids and all diets, was 95.8% for the pig and 96.1% for the rat. Cysteine digestibility was highly variable between individual animals fed the same diet. The extent of amino acid digestion and absorption appears to be broadly similar across the two species, but some important differences in digestibility were recorded.

Amino Acids↗

Age-related changes in apparent digestibility in growing kittens.

The ability of the growing kitten to digest protein, fat, carbohydrate, dry-matter and energy were assessed. Kittens were divided into two groups; one group was fed a wet diet, the other a dry diet. Both groups were allowed ad libitum access to food. Apparent digestibility of the two diets, and kitten bodyweights were measured over a 24-week period. There were no significant differences in mean bodyweight between the two groups. Digestible energy intake of the kittens decreased with increased age; regression analysis of the data generated two fitted models which appeared to accurately predict the digestible energy intake between 39 and 41 weeks of age. There was a significant (P < 0.05) effect of diet on the apparent digestibility of protein, organic-matter and dry-matter. Age had a significant (P < 0.05) effect on the apparent digestibility of all the parameters measured. Newman-Keuls multiple range tests showed that apparent digestibility of carbohydrate, organic-matter and total energy significantly (P < 0.05) increased in kittens older than 19 weeks. This suggests that the digestive capacity of the younger cat may be affected by the physiological development of the gut, and dietary induced enzyme modulation.

Aging↗

The digestion of yeast cell wall polysaccharides in veal calves.

1. The digestibility of the cell wall polysaccharides of an alkane-grown yeast in different parts of the digestive tract of two veal calves fitted with re-entrant cannulas at the end of the ileum was studied by replacing part of the skim-milk powder of their 'normal', milk-substitute (all-milk-protein) diet by yeast (yeast diet). 2. The lactose and glucose of both the all-milk-protein diet and the yeast diet were almost completely digested before the end of the ileum. During this digestion a small amount of oligosaccharides composed of galactose and glucose was synthesized. These oligosaccharides were digested again in the large intestine. 3. The constituent sugars of the water-soluble fraction of the yeast cell wall carbohydrates were glucose and mannose. The 0-5 M-sulphuric acid-hydrolysate of the water-insoluble fraction contained glucose and mannose and the 12 M-H2SO4-hydrolysate only glucose. 4. Digestibilities measured at the end of the ileum varied considerably between the two animals and averaged only about 0-40. 5. These findings suggest that the cell wall polysaccharides of yeast are digested very little by the normal digestive enzymes of the calf's small intestine, but are used as a substrate by the bacterial flora which are mainly concentrated in the large intestine.

Animal Feed↗

Utilization of low-quality roughage by Bos taurus and Bos indicus cattle. 1. Rumen digestion.

Six Hereford and six Brahman steers were fed ad lib. Pangola grass (Digitaria decumbens) and Spear grass (Heteropogon contortus) hay alone and supplemented with rumen-degradable nitrogen and sulphur and minerals. The rumen digestion of the two feeds was determined by reference to the disappearance of substrate from nylon bags suspended in the rumen and withdrawn after intervals ranging from 8 to 120 h. The digestion of the unsupplemented Pangola grass diet occurred more rapidly in Brahmans than in Herefords and was associated with higher rumen ammonia concentrations in Brahmans (40 v. 16 mg/l). The rumen NH3 concentrations were increased to over 100 mg/l by supplementation. The digestion rate increased in both breeds after supplementation and the breed difference disappeared. Increases in digestion rate were not achieved above NH3 concentrations of 60-80 mg/l. Spear grass, especially the cell-wall-constituent fraction, was more resistant to digestion than Pangola grass. Digestion of the unsupplemented Spear grass diet proceeded more rapidly in Brahmans than in Herefords. The digestion rate in Brahmans were similar irrespective of whether the diet was supplemented or not. Supplementation increased digestion rate in Herefords.

Ammonia↗

Utilization of ileal digestible amino acids by pigs: lysine.

Two experiments were conducted to determine the utilization of ileal digestible lysine by pigs. In the first, the apparent ileal digestibility of amino acids in cottonseed meal, meat-and-bone meal and soya-bean meal was determined in pigs fitted with 'T'-shaped cannulas. In the second experiment, three lysine-deficient diets were formulated to 0.36 g ileal digestible lysine/MJ digestible energy (DE), with lysine contributed from the three protein concentrates as the only source of lysine in sugar-based diets. An additional three diets were formulated with supplements of lysine to verify that lysine was limiting in the first three diets. The growth performance and retention of lysine by pigs given the six diets over the 20-45 kg growth phase were then determined. The apparent ileal digestibility of lysine in the three protein concentrates (proportion of total) was: cottonseed meal 0.74, meat-and-bone meal 0.78, soya-bean meal 0.89. Growth rates (g/d) of the pigs given the three diets formulated to 0.36 g ileal digestible lysine/MJ DE were significantly different (P less than 0.001): cottonseed meal 377, meat-and-bone meal 492, soya-bean meal 541. The response of pigs to the addition of lysine confirmed that lysine was limiting in these diets. Crude protein (nitrogen x 6.25) deposited by the pigs was significantly higher (P less than 0.001) for those given soya-bean meal (77 g/d), relative to meat-and-bone meal (66 g/d) and cottonseed meal (38 g/d). The proportion of ileal digestible lysine retained by pigs given the three protein concentrates was: cottonseed meal 0.36, meat-and-bone meal 0.60, soya-bean meal 0.75. The results indicate that values for the ileal digestibility of lysine in protein concentrates are unsuitable in dietary formulations as the assay does not reflect the proportion of lysine that can be utilized by the pig. It appears that, with heat-processed meals, a considerable proportion of the lysine is absorbed in a form(s) that is (are) inefficiently utilized.

Animal Feed↗

Daily food intake and digestibility in rats.

The present work with growing rats was undertaken to study the effect of daily food intake (DFI) on true protein digestibility (TD), and apparent digestibility of dry matter (DM), energy (DE), starch, soluble dietary fibre (SDF) and insoluble dietary fibre (IDF). The design involved two different dietary combinations, barley + rapeseed meal (diet 1) and oats + wheat bran (diet 2). A slight but significantly negative relationship was seen between DFI and TD on diet 1 while no such relationship was found on diet 2. Although significant, DFI influenced DM digestibility of both diets only slightly. A similar situation could also be seen for the effect of DFI on DE. The digestibility of starch was significantly affected by DFI on both diets even though the lowest values were as high as 0.994. SDF digestibility (fermentability) was not influenced by DFI when the rats were given diet 1, while there was a significant negative effect of DFI on digestibility of SDF when diet 2 was given. The digestibility (fermentability) of IDF was not affected by DFI on either of the two diets. The results confirm the existence of a weak negative relationship between DFI and digestibility of a range of nutrients although the effect seems to be only marginal and of no importance under practical feeding conditions.

Animals↗

The effect of dietary crude protein content on apparent and true ileal nitrogen and amino acid digestibilities.

The effect of dietary protein content (25, 60, 95, 130, 165 and 200 g crude protein (N x 6.25)/kg diet) on the apparent and true ileal digestibilities of N and amino acids in meat-and-bone meal given to the growing rat was investigated. Semi-synthetic diets in which meat-and-bone meal was the sole protein source were given to 180 g body-weight rats for 14 d. On the fourteenth day the rats were fed and then killed 4 h after the start of feeding and digesta were sampled from the terminal 200 mm ileum. Endogenous amino acid excretion was determined for eighteen rats given an enzymically hydrolysed casein (EHC)-based diet and with subsequent treatment of the digesta using ultrafiltration. The EHC-fed rats were killed 3 h after the start of feeding and digesta were collected from the terminal 200 mm ileum. True ileal digestibility values determined with references to Cr as a marker were higher than the corresponding apparent estimates. Apparent digestibility values of N and amino acids increased significantly (P < 0.001) with increasing dietary protein level; however, dietary protein content had no significant (P > 0.05) effect on the true ileal digestibilities of N and amino acids. The mean apparent ileal digestibility of N in meat-and-bone meal ranged from 65.6 to 75.3%. The corresponding range for the true ileal digestibility of N was 76.9 to 78.2%. True ileal digestibility, unlike apparent digestibility, appears to be independent of dietary protein level and may allow feed ingredients to be compared accurately even if they are ingested in different quantities.

Amino Acids↗