Enzymatic reduction and methylation of folate following pH-dependent,carrier-mediated transport in rat jejunum

Intestinal transport of [³H]folate was studied using everted sacs of rat jejunum. The proximal small intestine transports folate against a concentration gradient by a system which is saturable, pH-dependent, energy-dependent, sodium-dependent, sensitive to temperature, and appears to be a common transport system for folate' compounds. Chromatographic analysis of folate compounds in the serosal compartment after a 60 min incubation with folate in the mucosal medium in sodium phosohate buffer indicated that metabolism of folate to 5-methyltetrahydrofolate was extensive at pH 6.0 and negligible at pH 7.5. The percent conversion of folate to 5-methyltetrahydrofolate at pH 6.0 was reduced by increasing the concentration of folate in the mucosal medium, thus indicating saturation of the reduction and methylation process. These findings indicate that folate transport in rat jejunum occurs by an energy-dependent, carried-mediated system and that both folate transport and intestinal conversion of folate to 5-methyltetrahydrofolate are pH-dependent.     

Metabolite profiling of natural substances in human: in vitro study from fecal bacteria to colon carcinoma cells (Caco-2)

Flavonoids, including anthocyanins, are polyphenolic compounds present in fruits, vegetables and dietary supplements. They can be absorbed from the intestine to the bloodstream or pass into the large intestine. Various bacterial species and enzymes are present along the entire intestine. The aim of the present work was to investigate the intestinal metabolism of selected dietary polyphenol and polyphenol glycosides (quercetin, cyanidin-3-O-glucoside, cyanidin-3-O-galactoside, and delphinidin-3-O-galactoside) by human fecal bacteria. Moreover, the metabolism of metabolites formed from these compounds in human colon carcinoma cells (Caco-2) was also point of the interest. Test compounds were added to fresh human stool in broth or to Caco-2 cells in medium and then incubated for 6 or 20 h at 37°C. After incubation, samples were prepared for LC/MS determination. Main metabolic pathways were deglycosylation, hydrogenation, methylation, hydroxylation, and decomposition. 2,4,5-trihydroxybenzaldehyde, as a metabolite of cyanidin glycosides, was detected after incubation for the first time. Metabolites formed by fecal bacteria were further glucuronidated or methylated by intestinal enzymes. This metabolite profiling of natural compounds has helped to better understand the complex metabolism in the human intestine and this work also has shown the connection of metabolism of natural substances by intestinal bacteria followed by metabolism in intestinal cells.     

Alkaline phosphatase of the calf intestine hydrolyzes phospholipids

Pure alkaline phosphatase of the calf intestine is able to hydrolyze phosphatidylinositol 4,5-diphosphate (TPI) to phosphatidylinositol and Pi and to dephosphorylate phosphatidic acid. This phosphomonoesterase activity shows a considerably high specific activity when an incubation medium at neutral pH containing 3 mM deoxycholate is used. The activity is inhibited by low concentrations of Ca2+. The enzyme has no detectable phosphodiesterase activity under the conditions tested.     

Influence of Urea on the Growth of T-Strain Mycoplasmas

T-strain mycoplasmas require urea for propagation, but urea metabolism also occurs in nonpropagating viable cultures. Ammonia results from this metabolism and alkalinizes the medium. Ammonium ions and an alkaline pH both inhibit the multiplication of T strains and reduce the viability of T strains in broth. These toxic effects of urea metabolism currently limit the growth of T strains in broth. Stock T-strain cultures are optimally maintained in continuous culture if the routine medium at pH 6.0 is supplemented with 0.05% urea and 0.002% phenol red, but an incubation temperature of 30 C is preferable to 37 C for subculture at 24-hr intervals.     

Static and dynamic pH-dependent components of calcium exchange in the fraction of smooth muscle sarcolemma vesicles

It is proved that in the fraction of inverted vesicles of the myometrium sarcolemma there are two components of calcium metabolism which depend on the proton concentration in the incubation medium. The first component, a static one, identified under alkalization of the incubation medium from pH 6.0 up to pH 8.0 under equilibrium conditions (Ca2+ concentration inside and outside vesicles is the same) is manifested as an increase of the calcium capacity of vesicles at the expense of Ca2+-binding centres of the inner surface of membrane vesicles. The second component, a dynamic one, is represented as a passive transmembrane flow of Ca2+ outflowing from the vesicles induced by alkalization of the extravesicle space. Alkalization-stimulated Ca2+ release from vesicles is analyzed kinetically. Possible functional role of two components of pH-dependent metabolism of Ca2+ in providing the electrical and pharmacological-mechanical conjugation in the smooth-muscular tissue is under discussion.     

Effect of pH on the metabolism and fertility of turkey spermatozoa

Oxygen uptake during a 4-h incubation period at 37 degrees C, and the motility of the spermatozoa before and after incubation, increased significantly with increasing pH from 6.3 to 8.8. No interaction between buffer and pH was noticed. In a second series of experiments on the aerobic metabolism of turkey spermatozoa, the effect of the pHs 6.8, 7.3 and 7.8 was studied. Fructose was formed from glucose without regard to the pH of the medium. The glucose consumption, i.e. the glucose disappearance minus fructose formation, the lactic acid accumulation, and the oxidation of glucose and of other substances, were higher, although not always statistically, at pH 7.8 than at pH 6.8. The percentage of fertile eggs during the 3rd week of collection after insemination with fresh semen diluted in the pH 7.8 medium was significantly lower than that with semen diluted in the pH 6.8 or 7.3 media. After 4 h of storage at 15 degrees C, the decrease in the fertility of spermatozoa in the high pH medium was apparent from the 1st week of collection.     

Steady-state metabolism and transport of d-glucose by rat small intestine in vitro

1. Conditions of incubation of everted sacs of rat small intestine were selected to ensure that absorption of d-glucose by mucosal tissue from the incubation medium, intracellular metabolism of the absorbed glucose and transport of glucose through the intact intestinal tissue proceeded linearly with respect to time of incubation within stated time intervals. 2. Under these experimental conditions, steady intracellular concentrations of glucose and lactate were demonstrated. 3. The quantitative translocational and metabolic fate of absorbed glucose was determined under these steady-state conditions. About 25% of glucose absorbed from the external mucosal solution was accumulated (temporarily) within mucosal tissue and about 25% transported through the intact tissue into the external serosal solution; the remainder (about 50%) of the absorbed glucose was metabolized, 90% to lactate and 10% to CO₂. Concomitant respiration rates were comparable with those reported for several other preparations of intestine and were stoicheiometrically in excess of the O₂ metabolism required to account for the production of CO₂ from the absorbed glucose. 4. Water transport through the everted sacs proceeded at an optimum rate under the experimental conditions selected. 5. Some other observations are recorded which influenced the design of the experiments and the interpretation of results; these include the initial physiological state of the animal, the anaesthetic used and the ionic composition of the incubation medium.     

Enzymatic reduction and methylation of folate following pH-dependent, carrier-mediated transport in rat jejunum.

Intestinal transport of [3H] folate was studied using everted sacs of rat jejunum. The proximal small intestine transports folate against a concentration gradient by a system which is saturable, pH-dependent, energy-dependent, sodium-dependent, sensitive to temperature, and appears to be a common transport system for folate compounds. Chromatographic analysis of folate compounds in the serosal compartment after a 60 min incubation with folate in the mucosal medium in sodium phosohate buffer indicated that metabolism of folate to 5-methyltetrahydrofolate was extensive at pH 6.0 and negligible at pH 7.5. The percent conversion of folate to 5-methyltetrahydrofolate at pH 6.0 was reduced by increasing the concentration of folate in the mucosal medium, thus indicating saturation of the reduction and methylation process. These findings indicate that folate transport in rat jejunum occurs by an energy-dependent, carried-mediated system and that both folate transport and intestinal conversion of folate to 5-methyltetrahydrofolate are pH-dependent.     

The characteristics of protein hydrolysis on the digestive-transport surfaces of the cestode Eubothrium rugosum and of the intestines of its host--the burbot]

The functioning of different proteinases hydrolysing proteins in a wide pH range, most of which display activity in the alkaline zone of pH, on the digestive-absorptive surfaces of the parasite and host has been investigated. The dynamics of desorption of these proteinases from the intestine of fishes and tegument of cestodes has been studied. It has been shown that the worms possess less proteolytic activity and less capacity for adsorption of proteinases as compared to the intestines of their hosts. The dependence of proteolytic activity of desorbed fractions on the incubation medium temperature has been noted: with the increase in temperature the enzymes, bound closely with the membranes, increase their capacity to hydrolyse proteins. The predominance in cestodes, as compared to the intestine, of easily desorbed fractions D1 and D2 (in the percent ratio of the total proteolytic activity of all fractions) has been detected.     

Inhibition of D-glucose uptake by isatin in rat intestine: effect of harmaline and various sulfhydryl reagents

The effect of isatin (indole-2,3-dione) on D-glucose uptake has been studied in rat intestine. Isatin at 6 mM concentration significantly inhibited both the sugar uptake and transmural (mucosal to serosal side) transport in the intestine. The suppression of glucose uptake by isatin was irreversible. Similar to the action of various SH-group-reacting agents, isatin inhibited the sugar uptake, presumably by binding to membrane sulfhydryl groups through a covalent linkage. Isatin-induced reduction in glucose uptake was unaffected by pH (between 5.5 and 8.4) and by DTT addition to incubation medium. Inhibition of sugar uptake by isatin and harmaline was additive in nature; this suggested that these compounds interact at different sites on the microvillus membrane surface.     

pH-dependent glycine uptake in the presence and absence of sodium ions from rat small intestine

Intestinal uptake of glycine in rats was stimulated 15-20% in the presence of 120 mM Na at pH 6.0 and below but around neutral pH, the amino acid uptake was augmented to 60% compared to that in the Na-free medium. Glycine uptake was 30% more at pH 5.5 compared to that at pH 7.3 in the absence of Na. Kinetic analysis revealed a decrease in Kt for glycine uptake (9.62 mM) at pH 5.5 compared to that at pH 7.3 (Kt = 16.67 mM) with no change in maximal velocity (1.51 mumole/10 min/g tissue) in Na-free buffer. Addition of -SH group reacting reagents to the incubation medium produced 36-58% inhibition of glycine uptake in the presence of Na. However, in absence of Na, inhibition of the order of 21-35% and 8-23% was observed at pH 5.5 and 7.0, respectively. These findings suggest that glycine uptake in rat intestine is influenced by pH and -SH groups are implicated in the process(es).     

Secretary sialidase activity and GM3 ganglioside

The sialidase activities with GM3 ganglioside and sialyllactitol were demonstrated in the conditioned medium of human fibroblasts. pH versus activity profiles of conditioned medium with GM3 as substrate suggested the presence of two sialidases with optimal activities at pH 4.5 and pH 6.5. The GM3 sialidase activity at pH 6.5 was suppressed in the medium of contact-inhibited cells. This sialidase may function in the metabolism of cell surface GM3 since there was a selective loss of labeled sialic acid from GM3 at different times of incubation after pulse-labeling with a radioactive sialic acid precursor ([3H]N-acetyl-mannosamine) and a radioactive ceramide precursor ([14C]serine). In addition, a sialidase inhibitor, 2-deoxy-2, 3-dehydro-N-acetyl-neuraminic acid (NeuAc-2-en) resulted in a reversible growth inhibitory effect and the suppression of the sialidase activity in the medium. We have speculated that GM3 hydrolysis on the cell surface by the sialidase may be coordinated with the cell cycle and may be at its maximum during early in the G1 phase.     

Effects of Codonopis bulleynana forest ex diels on Deferribacteres in constipation predominant intestine tumor: Differential analysis

As a complicated micro-ecosystem, gut microbes are closely related to metabolic disease, immune disease and tumor (such as constipation. Long-term constipation would cause intestinal mucosal injury, enteritis, ileus, etc., thus inducing intestine cancer). In this research, intestine cancer model group and Codonopsis foetens treatment group were successfully constructed, and the variation of intestinal microbes were analyzed by 16S rRNA sequence. Results showed that there were changes in bacteria abundance of Firmicutes, Bacteroidetes, Proteobacteria, Deferribacteres, Tenericutes, and Actinobacteria, etc. Codonopsis foetens could directly or indirectly affect the growth and metabolism of Deferribacteres by altering the nutritional ingredient and pH value of intestine “medium”, thus affecting the occurrence and development of intestinal microbes.     

STUDIES ON EPITHELIAL CELLS ISOLATED FROM GUINEA PIG SMALL INTESTINE

Sheets of mucosal epithelial cells were released from guinea pig small intestine after incubation with ethylenediaminetetraacetate. Cells in sheets retained their columnar shape for 24 hr at room temperature, and exclusion of nigrosine suggested they had intact plasma membranes. When sheets were disaggregated individual cells had normal morphology for at least 4 hr. During isolation 16% of the total protein and 24% of the total lactic dehydrogenase were lost from the cells, but subsequent enzyme leakage was low. Leakage increased with shaking, incubation at 37°C, or increasing the oxygen tension of the suspending medium, but was minimal when the Na⁺:K⁺ ratio in the medium was 8:1 and the osmolarity was high. Losses of particulate enzyme activities were negligible. Respiration was constant for up to 4 hr and was insensitive to calcium, bicarbonate, oxygen tension, and pH. It was inhibited by cyanide and iodoacetate and varied with the Na⁺:K⁺ ratio of the extracellular fluid and the structural integrity of the cells. All preparations concentrated potassium and excluded sodium, but lost this ability if ouabain was added or cells were broken. Potassium-42 uptake was also sensitive to temperature, ouabain, and structural integrity. The preparations are being used to study cell metabolism in the intestinal epithelium.     

Water balance and its relation to fermentation acid production in the intestinal parasites Hymenolepis diminuta (Cestoda) and Moniliformis moniliformis (Acanthocephala).

Water balance and its relation to carbohydrate metabolism was examined in Hymenolepis diminuta in parallel with the putative osmoconformer Moniliformis moniliformis. Worms were removed from rat intestines, weighed, and incubated (37 C) 1 hr in rat serum and various salines, some with mannitol to vary osmotic concentration from 150 to 400 mOsm/L. Worms were removed at 15-min intervals, weighed, and returned to the test solution. Rat serum and a Ringer's saline (pH 7.4 and 300 mOsm/L) with or without 5 mM glucose were isotonic to M. moniliformis, which behaved like an osmometer, shrinking, or swelling in proportion to external osmotic changes. Hymenolepis diminuta rapidly lost 20-25% wet weight in these solutions and regained lost water when 5 mM glucose was added to the saline. Tapeworms maintained constant body weight between 210 and 335 mOsm/L, but they rapidly gained or lost water outside of this range. Glucose metabolism and uptake of [3H]glucose from the medium increased progressively between 210 and 310 mOsm/L, whereas uptake rates of [3H]leucine, 22Na+, and 36Cl- were not affected. Unbuffered saline (initial pH 6.5 and 300 mOsm/L) had a lower pH (5.0) and higher osmolality (307 mOsm/L) after a 1-hr incubation with tapeworms. Such saline was less hypertonic than unconditioned saline to freshly obtained worms. A Ringer's saline (300 mOsm/L) containing 50 mM acetate- was also hypertonic (greater than 20% weight loss) to tapeworms at pH 7.4, but it was hypotonic (greater than 20% weight gain) at pH 5.0. Isotonicity at 300 mOsm/L was achieved with pH 5.0 and 20 mM acetate-, the approximate pH and fermentation acid concentration in an infected rat intestine. Rats infected with tapeworms (12 days old) were fasted for 2 days. Starved worms were smaller but had the same percentage of body water and internal osmolality as controls. These results show that H. diminuta can regulate its body water content and that water balance is closely related to the fermentation acid concentration and pH of the bathing medium.     

Active pyrimidine absorption by chicken colon

Pyrimidine absorption by chicken large intestine was investigated employing the everted sac and flux chamber techniques. 3H-labelled uracil was used as substrate. The small intestine and the colon unlike the caecum, transported uracil from the mucosal to the serosal surface against a concentration gradient in the everted sac experiments. Furthermore, there was a net transport of uracil from the mucosal to the serosal side of the colon and jejunum in the flux chamber experiments. Uracil transport by the everted colon sacs against a concentration gradient was inhibited when the purine hypoxanthine was present in the incubation medium. Uracil transport by the everted colon sacs was also inhibited under anaerobic conditions and when 2,4-dinitrophenol was present in the incubation medium. Replacing the Na+ ions of the incubation medium by Li+ ions also caused an inhibition of uracil transport. It is concluded from these results that uracil (and probably other pyrimidines) are absorbed from the chicken colon by a Na+ ion-dependent active transport process having also an affinity for purines.     

Sodium/proton antiport in brush-border-membrane vesicles isolated from rat small intestine and kidney.

Studies on proton and Na+ transport by isolated intestinal and renal brush-border-membrane vesicles were carried out to test for the presence of an Na+/H+-exchange system. Proton transport was evaluated as proton transfer from the intravesicular space to the incubation medium by monitoring pH changes in the membrane suspension induced by sudden addition of cations. Na+ transport was determined as Na+ uptake into the vesicles by filtration technique. A sudden addition of sodium salts (but not choline) to the membrane suspension provokes an acidification of the incubation medium which is abolished by the addition of 0.5% Triton X-100. Pretreatment of the membranes with Triton X-100 prevents the acidification. The acidification is also not observed if the [K+] and proton conductance of the membranes have been increased by the simultaneous addition of valinomycin and carbonyl cyanide p-trifluoromethoxyphenylhydrazone to the K+-rich incubation medium. Either valinomycin or carbonyl cyanide p-trifluoromethoxyphenylhydrazone when added alone do not alter the response of the membranes to the addition of Na+. Na+ uptake by brush-border microvilli is enhanced in the presence of a proton gradient directed from the intravesicular space to the incubation medium. Under these conditions a transient accumulation of Na+ inside the vesicles is observed. It is concluded that intestinal and renal brush-border membranes contain a NA+/H+ antiport system which catalyses an electroneutral exchange of Na+ against protons and consequently can produce a proton gradient in the presence of a concentration difference for Na+. This system might be involved in the active proton secretion of the small intestine and the proximal tubule of the kidney.     

The metabolism of the insecticide carbaryl (1-naphthyl N-methylcarbamate) by fat body of the blowfly larva Calliphora erythrocephala

1. Carbaryl is metabolized more rapidly by fat body of the blowfly larva than by gut, muscle, cuticle or haemolymph. 2. Metabolism of carbaryl by the fat body is affected by the age of the larva, the pH of the incubation medium, and the concentration of magnesium chloride in the incubation medium. 3. Chloramphenicol, 2,4-dinitrophenol and 5-dimethylamino-6-nitro-1,3-benzodioxole (a carbaryl synergist) inhibit carbaryl metabolism by the fat body. 4. Subcellular fractionation of the fat body indicates that the pellet sedimenting at 30000g is the most reactive with carbaryl. 5. Probable metabolites of carbaryl formed by the fat body include the 4- and 5-hydroxy derivatives, and, possibly, the N-hydroxymethyl and 5,6-dihydrodihydroxy derivatives.     

pH effect on cellular uptake of Sn(IV) chlorine e6 dichloride trisodium salt by cancer cells in vitro

The effects of pH value and presence of serum in an incubation medium on photosensitizer drug cellular uptake in MCF7 cancer cells have been investigated. The results showed that the presence of serum in an incubation medium reduced the drug cellular uptake at all pH values. It has been found that decreasing on pH values of the incubation medium increased the cellular uptake of the drug, demonstrating selective uptake of the sensitizer. The HepG2 liver cancer cells exhibited more drug cellular uptake than CCD-18CO normal colon cells, which assessed the selectivity uptake of photosensitizer on cancerous cells. The concentration of photosensitizer measured in 10⁶ cells showed a good correlation to the incubation time. Fluorescence and absorption spectroscopy been have used to examine the cells.     

Conditions Influencing the Synthesis of Acid Protease by Mucor pusillus Lindt

Protease synthesis by Mucor pusillus Lindt, in a wheat bran medium under submerged conditions, was influenced by substrate concentration, initial pH of the medium, and temperature of incubation. A 4% wheat bran (dry weight) concentration was satisfactory for enzyme production. The initial pH of the medium had a substantial effect on enzyme synthesis; adjustment of the enzyme production medium to pH 5.0 prior to sterilization was desirable. Incubation at 35 C resulted in the best enzyme yields. Under optimal conditions of enzyme production, maximal activity was detected after 5 days of incubation. The enrichment of the medium with glucose increased the yield of mycelia but lowered the amount of enzyme produced.