A comparison of iron availability from commercial iron preparations using an in vitro digestion/Caco-2 cell culture model

The objectives of this study were to compare iron availability from commercial preparations of FeSO(4), ferrous gluconate, ferrous fumarate, and a polysaccharide-iron complex using an in vitro digestion/Caco-2 cell culture model. In addition, we sought to determine if calcium carbonate and calcium acetate (common phosphate binding agents) inhibited iron availability from an oral iron supplement when digested simultaneously. Caco-2 cell ferritin formation following exposure to simulated gastric and intestinal digests of the iron supplements was used as a measure of iron uptake and availability. Plates without cell monolayers were included in each replication of the experiment to measure the total amount of soluble iron that resulted from the in vitro digestion. Significantly more iron was taken up from the FeSO(4), ferrous gluconate, and ferrous fumarate than the polysaccharide-iron complex. Similar results comparing FeSO(4) and the polysaccharide-iron complex have been observed in humans. In addition, less iron was taken up from digests with calcium carbonate relative to calcium acetate even though similar amounts of soluble iron were observed in these experiments. The results indicate that when iron supplements and phosphate binders are consumed simultaneously, calcium acetate may be the preferred phosphate binder to maximize iron availability.     

Preparation and characterization of iron-containing liposomes: their effect on soluble iron uptake by Caco-2 cells

The aim of this work was to study the iron uptake of Caco-2 cells incubated with five different formulations of liposomes containing iron. The vesicles were also characterized before, during, and after in vitro digestion. Caco-2 cells were incubated with digested and nondigested liposomes, and soluble iron uptake was determined. Nondigested liposomes made with chitosan (CHI) or the cationic lipid, DC-Cholesterol (DC-CHOL), generated the highest iron uptake. However, these two formulations were highly unstable under in vitro digestion, resulting in nonmeasurable iron uptake. Digested conventional liposomes composed of soybean phosphatidylcholine (SPC), hydrogentated phosphatidylcholine (HSPC), or HSPC and cholesterol (CHOL) presented the highest iron-uptake values. These liposomal formulations protected iron from oxidation and improved iron uptake from intestinal cells, compared to an aqueous solution of ferrous sulphate.     

Characterization of Caco-2 and HT29-MTX cocultures in an in vitro digestion/cell culture model used to predict iron bioavailability

Cocultures of two human cell lines, Caco-2 and HT29-MTX mucus-producing cells, have been incorporated into an in vitro digestion/cell culture model used to predict iron bioavailability. A range of different foods were subjected to in vitro digestion, and iron bioavailability from digests was assessed with Caco-2, Caco-2 overlaid with porcine mucin, HT29-MTX or cocultures of Caco-2 and HT29-MTX at varying ratios. It was found that increasing the ratio of HT29-MTX cells decreased the amount of ferritin formed and resulted in an overall decline in the ability of the model to detect differences in iron bioavailability. At the physiologically relevant ratios of 90% Caco-2/10% HT29-MTX and 75% Caco-2/25% HT29-MTX, however, a mucus layer completely covered the cell monolayer and the in vitro digestion model was nearly as responsive to changes in sample iron bioavailability as pure Caco-2 cultures. The in vitro digestion/Caco-2 cell culture model correlates well with human iron bioavailability studies, but, as mucus appears to play a role in iron absorption, the addition of a physiologically realistic mucus layer and goblet-type cells to this model may give more accurate iron bioavailability predictions.     

An approach to assessing trace element bioavailability from milk in vitro

Both in vitro and in vivo, the use of a radioisotope can significantly enhance the sensitivity of methods for trace element studies. An essential prerequisite for this approach, however, is that the added (extrinsic) radiolabel equilibrates with the native (cold) element within all compartments of the diet. By using ultracentrifugation, ultrafiltration, and gel filtration chromatography, we have shown that the method is valid for zinc, copper, and manganese when using milks and formulas. For iron, however, extrinsic labeling does not necessarily yield results similar to the native distribution. We have used extrinsic labeling to follow the distribution of Zn, Cu, and Mn between high molecular weight compounds (proteins) and low molecular weight complexes in human and bovine milk after in vitro proteolysis. Peptic digestion at various pHs and pancreatic digestion for varying times were used to mimic digestion in the infant. After limited proteolysis, a large proportion of trace minerals in human milk was found in the low molecular weight fraction, whereas in cow's milk a large proportion was bound to incompletely digested casein. These findings may, at least in part, explain the higher bioavailability of trace elements from human milk compared to cow's milk.     

A clean, more efficient method for in-solution digestion of protein mixtures without detergent or urea

Proteolytic digestion of a complicated protein mixture from an organelle or whole-cell lysate is usually carried out in a dilute solution of a denaturing buffer, such as 1-2 M urea. Urea must be subsequently removed by C18 beads before downstream analysis such as HPLC/MS/MS or complete methylation followed by IMAC isolation of phosphopeptides. Here we describe a procedure for digesting a complicated protein mixture in the absence of denaturants. Proteins in the mixture are precipitated with trichloroacetic acid/acetone for denaturation and salt removal and resuspended in NH4HCO3 buffer. After trypsinolysis, the resulting peptides are not contaminated by urea or other nonvolatile salts and can be dried in a SpeedVac to remove NH4HCO3. When this protocol was applied to an extract of A431 cells, 96.8% of the tryptic peptides were completely digested (i.e., had no missed cleavage sites), in contrast to 87.3% of those produced by digestion in urea buffer. We successfully applied this digestion method to analysis of the phosphoproteome of adiposomes from HeLa cells, identifying 33 phosphorylation sites in 28 different proteins. Our digestion method avoids the need to remove urea before HPLC/MS/MS analysis or methylation and IMAC, increasing throughput while reducing sample loss and contamination from sample handling. We believe that this method should be valuable for proteomics studies.     

Liver Nonprotein Sulfhydryl and Taurine Concentrations,and Fat Content of Rats Fed a Low Protein Diet Supplemented with Sulfur-containing Amino Acids

The relative excess of some catabolites of sulfur-containing amino acids in the liver of rats fed a low protein diet might be one of the factors which cause the liver fat accumulation. To investigate the possibility were studied relationships between changes in concentrations of some metabolites of sulfur-containing amino acids and those in fat contents of rats fed a low protein diet consisting of heated soybean flour, casein or wheat flour with or without added methionine, threonine or lysine. The addition of 0.6% methionine to the 25% heated soybean flour diet increased the nonprotein-sulfhydryl (NP–SH) concentration and fat content in the liver. These changes were prevented by the further addition of 0.5% threonine to the diet, although the NP–SH concentration was remarkably higher than that of rats fed the unsupplemented diet. The addition of 0.6% cystine HC1 to the 25% heated soybean flour diet containing sufficient choline elevated the NP–SH concentration and fat content in the liver, which were not affected by the further addition of 0.5% threonine. The addition of 0.6% cystine HC1 to the 10% casein diet significantly increased the fat content, and NP–SH and taurine concentrations in the liver. The further addition of 0.5 % threonine completely decreased the fat content, and partially reduced the NP–SH and taurine concentrations. Effects of supplementation of 0.4% lysine HC1 to the 70% wheat flour diet on the fat content and NP–SH concentration in the liver demonstrated the trends similar to those of supplementation of cystine to the 10% casein diet. The further addition of threonine remarkably decreased the fat content, NP–SH and taurine concentrations in the liver.     

Iron uptake by Caco-2 cells following in vitro digestion: Effects of heat treatments of pork meat and pH of the digests

The present in vitro studies report on iron uptake by Caco-2 cells from pepsin and pepsin + pancreatin-digested pork meat proteins at pH values between 4.6 and 7 mimicking conditions in the duodenum and the proximal jejunum, respectively. Heat treatment of the pork meat resulted in increased iron uptake from pepsin-digested samples to Caco-2 cells at pH 4.6. The major enhancing effects on iron uptake by Caco-2 cells were observed after pepsin digestion in the pH range 4.6–6.0, whereas the pepsin + pancreatin-digested samples resulted in negligible iron uptake in Caco-2 cells at pH 7. Thus, the results emphasize the importance of separating pepsin-digested and pepsin + pancreatin-digested proteins during in vitro studies on iron availability. Furthermore, the present results showed the pH dependency of iron uptake anticipated. The enhancing effect of ascorbic acid was verified by increased iron uptake from pepsin-digested pork meat samples at pH 4.6, while no effect of ascorbic acid was observed at pH 7 in pepsin + pancreatin-digested samples.     

C-terminal ladder sequencing of peptides using an alternative nucleophile in carboxypeptidase Y digests

A method for improved sequence coverage in C-terminal sequencing of peptides, based on carboxypeptidase digestion, is described. In conventional carboxypeptidase digestions, the peptide substrate is usually extensively degraded and a full amino acid sequence cannot be obtained due to the lack of a complete peptide ladder. In the presented method, a protecting group is introduced at the C terminus of a fraction of the peptide fragments formed in the digest, and thereby further degradation of the C-terminally modified peptides are slowed down. The protecting group was attached to the C-terminal amino acid through a carboxypeptidase-catalyzed reaction with an alternative nucleophile, 2-pyridylmethylamine, added to the aqueous digestion buffer. Six peptides were digested by carboxypeptidase Y with and without 2-pyridylmethylamine present in the digest buffer, and the resulting fragments subsequently were analyzed with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Comparison of the two digestion methods showed that the probability of successful ladder sequencing increased, by more than 50% using 2-pyridylmethylamine as a competing nucleophile in carboxypeptidase Y digests.     

Dietary oxidized fatty acids: an atherogenic risk?

Previous studies have suggested that heated fat that contains oxidized fatty acids in the diet might contribute to the presence of oxidized components in circulating lipoproteins. On the other hand, studies in our laboratory showed that cultured cells such as smooth muscle cells take up oxidized fatty acids poorly. Because intestinal cells are morphologically quite distinct, we studied the uptake of oxidized linoleic acid by Caco-2 and smooth muscle cells (control). When 16-day-old Caco-2 cells were incubated with oxidized linoleic acid (ox-linoleic acid), its uptake was comparable to that of unoxidized linoleic acid (unox-linoleic acid) or that of oleic acid (40;-58, 70, and 55%, respectively). In contrast, the uptake of ox-linoleate by smooth muscle cells was about 3%. To determine whether the brush border structure of Caco-2 cells was responsible for increased uptake of oxidized fatty acids, we compared uptake in 4- and 16-day-old cells. The uptake of unox-linoleate and oleic acid (18:1) was comparable for the 4- and 16-day cells. In addition, saturation and competition experiments showed that the uptake of ox-linoleate by Caco-2 cells is not saturable even at 150 microm and that this uptake is diluted in the presence of unox-linoleate. In esterification experiments utilizing rat intestinal microsomes, we show that both ox- and unox-linoleate are esterified equally well.In summary, dietary oxidized fatty acids can be absorbed by the intestine and incorporated into lipoproteins and could potentially impose an oxidative stress and exacerbate atherogenesis.     

Dephosphorylation of sodium caseinate, enzymatically hydrolyzed casein and casein phosphopeptides by intestinal alkaline phosphatase: implications for iron availability

Clusters of phosphoserine residues in casein bind iron with high affinity. Casein inhibits iron absorption in humans but partial hydrolysis of casein prior to ingestion diminishes this inhibition. The objective of this study was to test two hypotheses: 1. Partial hydrolysis of the peptide bonds in casein exposes phosphoserine residues to attack by intestinal alkaline phosphatase (IAP). 2. Hydrolysis of the phospho-ester linkage in phosphoserine residues in casein by IAP releases bound iron or inhibits iron chelation, thereby allowing its absorption. Test of hypothesis 1: Suspensions of sodium caseinate (SC), enzymatically hydrolyzed casein (EHC), and casein phosphopeptides (CPP) were subjected to an in vitro pepsin/pancreatin digestion and subsequently incubated in the presence of calf IAP. The rate of release of inorganic phosphate was measured with the following results (expressed as &mgr;mol phosphate released/unit of IAP/min): 0.081, 0.104, 0.139 for SC, EHC, and CPP, respectively. These results are consistent with hypothesis 1. Test of hypothesis 2: (59)Fe-citrate or (59)Fe-citrate + CPP in minimum essential media were spiked with a Na(2)WO(4) solution or water (Na(2)WO(4) is a known inhibitor of IAP) and placed on Caco-2 cell monolayers. Uptake of (59)Fe by the cells was used as an index of iron bioavailability. Na(2)WO(4) did not affect (59)Fe uptake from samples containing only iron but did slightly inhibit (by 10%) uptake from samples containing iron + CPP. These results are consistent with hypothesis 2 and provide a possible explanation for the observation that partial hydrolysis of casein improves iron bioavailability.     

Caffeine enhancement of digestion of DNA by nuclease S1.

The activity of Aspergillus orzae nuclease S1 on DNA has been investigated under varying pH and metal ion conditions. Nuclease S1 was found to preferentially digest denatured DNA. With native DNA as substrate the enzyme could only digest the DNA when caffeine was added to the reaction mixture. The enzyme was more active in sodium acetate buffer (pH 4.5), than in either standard saline citrate (PH 7.0) or sodium phosphate buffer (pH 6.8). Caffeine was also found to affect the thermal stability of DNA, resulting in a melting profile characterized by two transitions. The first transition (poorly defined) was below the normal melting temperature of the DNA, while the next transition was at the normal melting temperature of the DNA, while the next transition was at the normal melting temperature of the DNA. The susceptibility of caffeine-treated DNA to nuclease digestion seems to be a result of the local unwinding that caffeine causes in the regions of DNA that melt in the first transition. This selective destabilization presumably sensitizes the unwound regions to nuclease hydrolysis. The hydrolysates of the DNA digested by nuclease S1 were subjected first to ion exchange chromatography followed by paper chromatography. The results from this partial characterization of the digestion products showed that they contain mononucleotides as well as oligonucleotides of varying lengths. The base composition of the mononucleotide digests suggests that caffeine has greater preference for interacting with A-T base-pairs in DNA.     

Effects of dietary factors on iron uptake from ferritin by Caco-2 cells

Biofortification of staple foods with iron (Fe) in the form of ferritin (Ft) is now possible, both by conventional plant breeding methods and transgenic approaches. Ft-Fe from plants and animals is absorbed well (25-30%) by human subjects, but little is known about dietary factors affecting its absorption. We used human intestinal Caco-2 cells and compared Fe absorption from animal Ft and FeSO4 to determine the effects of inhibitors and enhancers, such as phytic acid, ascorbic acid, tannic acid, calcium and heme. When postconfluent cells were coincubated with 59Fe-labeled (1 microM) FeSO4 and dietary factors, at different molar ratios of dietary factor to Fe (phytic acid:Fe, 10:1; ascorbic acid:Fe, 50:1; tannic acid:Fe, 50:1; calcium:Fe, 10:1 and hemin:Fe, 10:1), all inhibited uptake from FeSO4, except ascorbate, confirming earlier studies. In contrast, these dietary factors had little or no effect on Fe uptake from undigested Ft or Ft digested in vitro at pH 4, except tannins. However, results after in vitro digestion of Ft at pH 2 were similar to those obtained for FeSO4. These results suggest that Fe uptake occurs from both undigested as well as digested Ft but, possibly, via different mechanisms. The Fe-Ft stability shown here could minimize Fe-induced oxidation of Fe-supplemented food products.     

The effect of dietary protein on the growth and digestive physiology of larval Heliothis zea and Spodoptera exigua

We compared the ability of larval H. zea (Boddie) and S. exigua (Hubner) to digest and utilize dietary protein by: (a) determining the ability of different concentrations of dietary protein to support larval growth, and (b) determining the effect of different concentrations of dietary protein on the digestive physiology of the organisms, as measured by in vivo digestion of protein and proteolytic activity. Using an artificial diet containing casein as the primary source of protein, we found that H. zea was able to grow at very low levels of casein (≤0.6%), while optimal growth occurred at 1.2% casein. For S. exigua, dietary casein levels of >0.6% were required for growth, and optimal growth occurred at ≥1.2% casein. However, optimal growth in both species was not correlated with the degree of in vivo digestion of protein. The level of in vivo digestion of protein and tryptic activity in S. exigua was proportional to the concentration of dietary protein (under both acute and chronic exposure), and not the amount of food in the gut, suggesting that enzyme synthesis and/or secretion is controlled by a secretagogue mechanism. H. zea only demonstrated a secretagogue mechanism of control of tryptic activity while under acute exposure to different concentrations of casein; under chronic exposure, tryptic activity was uniform regardless of the concentration of dietary casein. When comparing the two species of noctuid, H. zea, which is the larger of the two species, produced less tryptic activity on a unit weight basis, and also digested less of the available dietary protein than S. exigua. Hence, these closely related organisms are processing dietary protein at different efficiencies.     

Upregulation of oxidative stress markers in human microvascular endothelial cells by complexes of serum albumin and digestion products of glycated casein

The extent of absorption of dietary advanced glycation end products (AGEs) is not fully known. The possible physiological impact of these absorbed components on inflammatory processes has been studied little and was the aim of this investigation. Aqueous solutions of bovine casein and glucose were heated at 95°C for 5 h to give AGE‐casein (AGE‐Cas). Simulated stomach and small intestine digestion of AGE‐Cas and dialysis (molecular mass cutoff of membrane = 1 kDa) resulted in a low molecular mass (LMM) fraction of digestion products, which was used to prepare bovine serum albumin (BSA)‐LMM‐AGE‐Cas complexes. Stimulation of human microvascular endothelial cells with BSA‐LMM‐AGE‐Cas complexes significantly increased mRNA expression of the receptor of AGE (RAGE), galectin‐3 (AGE‐R3), tumor necrosis factor alpha, and a marker of the mitogen‐activated protein kinase pathway (MAPK‐1), as well as p65NF‐κB activation. Cells treated with LMM digestion products of AGE‐Cas significantly increased AGE‐R3 mRNA expression. Intracellular reactive oxygen species production increased significantly in cells challenged with BSA‐LMM‐AGE‐Cas and LMM‐AGE‐Cas. In conclusion, in an in vitro cell system, digested dietary AGEs complexed with serum albumin play a role in the regulation of RAGE and downstream inflammatory pathways. AGE‐R3 may protect against these effects. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:364–372, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20301     

Effects of gastric digestive products from casein on CCK release by intestinal cells in rat

We investigated the ability of gastric digestive products from casein to stimulate cholecystokinin release by intestinal cells using the isolated vascularly perfused rat duodenojejunum. Casein digests were prepared with an in vitro system simulating gastric digestion and emptying.

The luminal infusion of the digesta emptied from the artificial stomach for the first 10 minutes produced a sharp rise of portal cholecystokinin-like immunoreactivity to 300% of basal, followed by a well-sustained plateau secretion until the end of the infusion. The residual casein fraction of this digest brought about a modest cholecystokinin secretion, while the peptide component was as strong a stimulant as total digest. The peptide responsible for this effect was the glycomacropeptide that is a glycosylated fragment (106–169) of κ-casein. Only the slightly glycosylated forms of the peptide originating from variant A of κ-casein were active. The carbohydrate-free peptide did not alter basal cholecystokinin. The highly glycosylated forms of the peptide and the slightly glycosylated peptide from κ-casein variant B induced only a transient and low rise of portal cholecystokinin. The removal of N-acetylneuraminic acid from the active peptide suppressed its effect, while the infusion of an N-acetylneuraminic acid solution induced only a very low response.

It is concluded that the glycomacropeptide released from dietary casein during gastric digestion can stimulate cholecystokinin release by intestinal cells in the rat. A well-defined structure is required for the peptide activity. A part of the peptide chain and some glycosidic chains containing N-acetylneuraminic acid, especially those bound to the amino acid residue threonyl 31 of caseinomacropeptide variant A, would be involved in this structure.     

Domain-specificity of Cd2+ and Zn2+ binding to rabbit liver metallothionein 2. Metal ion mobility in the formation of Cd4-metallothionein alpha-fragment.

The yield of the alpha-fragment of rabbit liver metallothionein 2 was used to test the domain-specificity and mobility of Cd2+ and Zn2+ when bound to metallothionein. Increasing molar ratios of Cd2+ were added to either Zn7-metallothionein or the metal-ion-free apo-metallothionein. The enzyme subtilisin was used to digest those parts of the peptide chain that were not bound to Cd2+. Analysis of the digestion products was carried out by separation by polyacrylamide-gel electrophoresis. The chelation agent EDTA was used as a competitive chelator. It was found that the presence of excess EDTA greatly enhances the formation of the Cd4-metallothionein alpha-fragment, and catalyses the complete digestion of all other the metal-ion-containing peptides, so that even Cd7-metallothionein, formed when 7 molar equivalents of Cd2+ are added to Zn7-metallothionein, is digested to the alpha-fragment. These results suggest that the Cd2+ bound in the beta-sites is very labile, much more labile than the kinetics of the off-reaction would suggest. The observation of significant amounts of alpha-fragment on the gels, even when the stoichiometry of the metal ions initially present in the protein should not have resulted in much concentration of Cd4-alpha-fragment clusters, indicates that as the digestion proceeds the metal ions move to sites that form complete clusters and therefore selectively protect that part of the peptide chain from digestion. We also find that rabbit Cd4-metallothionein 2 alpha-fragment stains near to the top of the gel, in complete contrast with the location of rat Cd4-metallothionein 2 alpha-fragment. This difference in the mobilities suggests that the alpha-fragment prepared from rabbit metallothionein 2 is much less negatively charged than the analogous protein fragment prepared from rat liver metallothionein 2.     

Proteolytic activity associated with human erythrocyte membranes. Self-digestion of isolated human erythrocyte membranes.

At least two kinds of enzymes are active in the proteolytic self-digestion of erythrocyte membranes. The specific activities of these enzymes do not decrease with repeated washings of purified stroma. The effects of a variety of inhibitors on the membrane preparation's capacity to digest 125-I-labelled casein, covalently linked to latex beads, have been examined. Pepstatin-inhibitable enzyme, active at low pH, digests the membrane extensively to small polypeptide fragments. Spectrin, located at the internal part of the membrane, is readily degraded. Diisopropylfluorophosphate-inhibitable enzyme, active at pH 8-9, has only limited digestive capacity. Some of the membrane components, such as the small molecular weight glycoproteins, are resistant to digestion. The restricted capacity of digestion is due to the membrane molecular arrangement; increased disaggregation removes the restriction and increases the activity. Spectrin is not digested unless the membrane topography is disrupted by NP-40 neutral detergent. These observations suggest that the enzymes active at basic pH are located external to the cell. Intact cells do possess a limited capacity to degrade 125-I-labelled casein when their surfaces are brought into contact with substrate-coated beads.     

Rapid enrichment of phosphopeptides from tryptic digests of proteins using iron oxide nanocomposites of magnetic particles coated with zirconia as the concentrating probes

Iron oxide nanocomposites of magnetic particles coated with zirconia were used as affinity probes to selectively concentrate phosphopeptides from tryptic digests of alpha- and beta-caseins, milk, and egg white to exemplify the enrichment of phosphopeptides from complex samples. Phosphopeptides, in quantities sufficient for characterization by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS), were enriched by the affinity probes within only 30 s. The affinity probe-target species conjugates were separated from the sample solution simply by applying an external magnetic field. The detection limit for tryptic digest of beta-casein using this approach is approximately 45 fmol. Furthermore, we combined this enrichment method with a rapid enzymatic digestion method, that is, microwave-assisted enzymatic digestion using magnetic particles as the microwave absorbers, to speed up the tryptic digest reactions. Thus, we alternatively enriched phosphoproteins on the zirconia-coated particles followed by mixing with trypsin and heated the mixture in a microwave oven for 1 min. The particles remaining in the mixture were used as affinity probes to selectively enrich phosphopeptides from the tryptic digestion product by pipetting, followed by characterization using MALDI MS. Using the bifunctional zirconia-coated magnetic particles as both the affinity probes and the microwave absorbers could greatly reduce the time for the purification and characterization of phosphopeptides from complex samples.     

Overproduction of an α-Amylase/Glucoamylase Fusion Protein in Aspergillus oryzae Using a High Expression Vector

The systemic immune response against orally administered antigens is suppressed (oral tolerance), and this has been postulated to avoid excess immunity against dietary constituents which are present in large amounts in the gastrointestinal tract. Taking into consideration that such orally administered protein antigens are subjected to enzymatic degradation in the gastrointestinal tract, we examined whether an enzymatic digest of milk proteins could induce oral tolerance. A tryptic digest of casein, containing mainly fragments smaller than 6000 Da, was fed to mice as a constituent of their diet. Mice fed with the casein-digest diet responded poorly to subsequent immunization with casein, indicating that oral tolerance to casein was induced in these animals. The results suggest the presence of immunosuppressive fragment(s) in the casein digest, which may be of use for preventing milk allergy.     

Accumulation and storage of Zn2+ by Candida utilis.

Starved cells of Candida utilis accumulated Zn2+ by two different processes. The first was a rapid, energy- and temperature-independent system that probably represented binding to the cell surface. The cells also possessed an energy-, pH-, and temperature-dependent system that was capable of accumulating much greater quantities of the cation than the binding process. The energy-dependent system was inhibited by KCN, Na2HAsO4, m-chlorophenyl carbonylcyanide hydrazone, N-ethylmaleimide, EDTA and diethylenetriaminepenta-acetic acid. The system was specific inasmuch as Ca2+, Cr3+, Mn2+, Co2+ or Cu2+ did not compete with, inhibit, or enhance the process, Zn2+ uptake was inhibited by Cd2+. The system exhibited saturation kinetics with a half-saturation value of 1.3 muM and a maximum rate of 0.21 (nmol Zn2+) min(-1) (mg dry wt(-1)) at 30 degrees C. Zn2+ uptake required intact membranes since only the binding process was observed in the presence of nystatin, toluene, or sodium dodecyl sulphate. Cells did not exchange recently accumulated toluene, or sodium dodecyl sulphate. Cells did not exchange recently accumulated 65Zn following the addition of a large excess of non-radioactive Zn2+. Similarly, cells pre-loaded with 65Zn did not lose the cation during starvation, and efflux did not occur when glucose and exogenous Zn2+ were supplied after the starvation period. Efflux was only observed after the addition of toluene or nystatin, or when cells were heated to 100 degrees C. Cells fed a large quantity of Zn2+ contained a protein fraction resembling animal cell metallothionein. In batch culture, cells of C. utilis accumulated Zn2+ only during the lag phase and the latter half of the exponential-growth phase.