The effect of nisin on the physiology of Bifidobacterium thermophilum.

The effects of nisin on lactate accumulation, growth, and Fe(III) binding by Bifidobacterium thermophilum (ATCC 25866) and Bifidobacterium breve (ATCC 15700) were investigated. Nisin inhibited lactate production by B. thermophilum at concentrations of less than 1 microg/ml, but this effect could be largely eliminated by pretreatment of the organism with 100 to 400 microM Al(III) or La(III). Nisin also inhibited the growth of B. thermophilum at concentrations of 2 to 3 microg/ml, with lower concentrations showing lag periods and/or slower rates of growth. However, Al(III) could not negate these effects, most likely because of Al(III) chelation by the trypticase-proteose-yeast extract medium. Nisin was able to increase instantaneous Fe(III) binding by both B. thermophilum and B. breve, though prolonged-time experiments (up to 120 min) with B. thermophilum indicated no difference in total Fe(III) bound. Nisin was thus able to increase the free radical reaction rate with bifidobacteria and the resultant rate of Fe(III) binding. It was concluded that nisin will normally inhibit the metabolic activity of B. thermophilum along with that of certain bacterial pathogens; however, this effect may in some instances, be abated by a pretreatment with Al(III). Moreover, by accelerating free radical action and the binding of iron by bifidobacteria, nisin may be able to potentiate their normal probiotic action.

Probiotics: determinants of survival and growth in the gut.

Bifidobacteria and lactobacilli are purportedly beneficial to human health and are called probiotics. Their survival during passage through the human gut, when administered in fermented milk products, has been investigated intensely in recent years. Well-controlled, small-scale studies on diarrhea in both adults and infants have shown that probiotics are beneficial and that they survive in sufficient numbers to affect gut microbial metabolism. Survival rates have been estimated at 20-40% for selected strains, the main obstacles to survival being gastric acidity and the action of bile salts. Although it is believed that the maximum probiotic effect can be achieved if the organisms adhere to intestinal mucosal cells, there is no evidence that exogenously administered probiotics do adhere to the mucosal cells. Instead, they seem to pass into the feces without having adhered or multiplied. Thus, to obtain a continuous exogenous probiotic effect, the probiotic culture must be ingested continually. Certain exogenously administered substances enhance the action of both exogenous and endogenous probiotics. Human milk contains many substances that stimulate the growth of bifidobacteria in vitro and also in the small intestine of infants; however, it is unlikely that they function in the colon. However, lactulose and certain fructose-containing compounds, called prebiotics, are not digested in the small intestine but pass into the cecum unchanged, where they are selectively utilized by probiotics. Beneficial effects may thus accrue from exogenously administered probiotics, often administered with prebiotics, or by endogenous bifidobacteria and lactobacilli, whose metabolic activity and growth may also be enhanced by the administration of prebiotics.

Binding of ferric iron to the cell walls and membranes of Bifidobacterium thermophilum: effect of free radicals.

Bifidobacterium thermophilum (ATCC 25866) was incubated with 100-120 microM (59)FeSO(4) and 300 microM excess of H(2)O(2) for up to 120 min in the presence and absence of glucose. Samples were withdrawn after 5, 30, 60, and 120 min. These were protoplasted and (59)Fe(III) measured in the supernatant fraction (cell walls) and protoplasts (cell membranes). These experiments were also repeated in the presence of 400 microM Al(III), which, in whole cells, caused an increase in Fe(III) binding. The amount of iron in the cell wall fraction was constant regardless of time of incubation, was unaffected by Al(III), and was reduced by approximately 20% by glucose. On the other hand, the amount of iron on the protoplasts increased with time and was affected by both Al(III) (upward) and glucose (downward). Scatchard plots indicated that the number of Fe(III) binding sites on the cell walls was 37.6 nmol/mg of dry cell weight at zero time, whereas that of cell membranes was (1)/(10) of that. It was concluded that Fe(III) binding by bifidobacterial cell walls was instantaneous and marginally dependent on free radical action. That of the cell membranes was time-dependent and was due to lipid peroxidation initiated by free radicals. Bifidobacteria can therefore function in the intestinal tract as probiotics by making Fe(OH)(3) unavailable to pathogens and by moderating free radical activity in the intestinal tract.

Iron uptake by Bifidobacterium thermophilum protoplasts.

Protoplasts of Bifidobacterium thermophilum were prepared by a combination of lysozyme and protease digestion, and ferrous iron uptake studies were carried out. Little, if any, iron was internalized by the protoplasts, although large amounts of iron were bound to the protoplast surface. This binding was much greater than that of intact cells, which prefer to internalize iron by an energy-dependent process. It was also found that the binding of iron by protoplasts of cells grown in an iron-deficient medium was much more extensive than that of cells grown in an iron-sufficient medium. Soluble and particulate fractions of protoplasts were prepared by grinding them in a glass homogenizer, and the particulate fraction was also subjected to iron binding studies. The amount of iron bound was the same as that in intact protoplasts, indicating that the particulate fraction membrane fragments bound iron on their outer surface only. Nevertheless, when iron-preloaded cells were protoplasted and their surface cleared of iron, their particulate fraction contained considerable amounts of iron, indicating that the inner surface of the membranes is capable of binding iron as long as the cell is intact. The amount of iron so bound was dose-dependent on the amount of iron entering the cell. The failure of the outer and inner surface iron pools to mix was confirmed by the fact that when iron-preloaded protoplasts were incubated with additional iron, only the latter (surface-bound) was elutable with nonradioactive 2 mM FeSO4. It is concluded that increasing bifidobacterial iron load increases the amount of iron bound to the inner surface of the membrane; the procedure, which is effective in forming bifidobacterial protoplasts, destroys their iron transport mechanism while uncovering surface iron-binding sites; and that such iron-binding sites may be of significance in the cellular iron metabolism processes.

Effects of Mg2+ and Ca2+ on Fe2+ uptake by Bifidobacterium thermophilum.

Ferrous iron uptake was investigated in Bifidobacterium thermophilum (B. thermophilum) in the presence of Mg2+ and Ca2+ with the following findings: 1. Mg2+ inhibited Fe2+ accumulation in the cells in a dose-dependent manner at 37 degrees, but not at 0 degrees. Removal of Mg2+ from the medium resulted in a resumption of rapid iron uptake. 2. Mg2+ had no effect on the binding of Fe2+ by B. thermophilum protoplasts, its cellular particulate fraction, or distribution between the particulate and soluble fractions. 3. Ca2+ exerted a stimulatory effect on iron uptake by B. thermophilum, but was not able to reverse the inhibitory effects of Mg2+. 4. It was concluded that Mg2+ has no effect on the binding of iron on the surface or interior of B. thermophilum and that it affected the Fe2+ transport mechanism (permease) in a reversible manner. It is possible that iron and magnesium share the same permease in this microorganism.

Iron accumulation by bovine aortic endothelial cells.

Bovine aortic endothelial cells in monolayers were used to study iron and transferrin binding and transport mechanisms. Diferric bovine transferrin labeled with 59Fe was used as an iron donor. We have shown the presence of saturable iron uptake when cells were incubated with varying concentrations of diferric transferrin. This uptake decreased when the cells were treated with trypsin, ammonium chloride and methylamine. The effects of the latter two could be reversed by the addition of 2.0 mM Ca2+. Energy dependence was shown by using various electron transport/oxidative phosphorylation inhibitors. The presence of transferrin receptors on the cell surface was confirmed by their isolation, SDS-PAGE and autoradiography. There were approximately 1.5 x 10(6) transferrin receptors per cell with a Kd of 9.1 x 10(-7) M in the physiological iron range. Iron was also taken up when the cells were incubated with radioactive ferrous iron without transferrin. Uptake was not affected by receptor-mediated endocytosis inhibitors. Calcium increased ferrous iron uptake and overcame the effects of metabolic inhibitors on iron uptake from transferrin. A ferrireductase was detected in cell membranes. It is proposed that iron is transported by bovine endothelial cells by two mechanisms: one is receptor-mediated endocytosis from transferrin, and the other involves a non-endocytic mechanism from transferrin and Fe2+, which is possibly promoted by Ca2+.

Ferrous iron uptake by Bifidobacterium breve.

Bifidobacterium breve transports ferrous iron in preference to the ferric form in a saturable, concentration-dependent manner with an optimum pH of 6. Iron transport is highly temperature sensitive. Two transport systems with apparent Km's of 86 +/- 27 and 35 +/- 20 microM (p greater than 0.01) were distinguished, one operating at high iron concentrations, the other at low iron concentrations. Iron uptake could not be accounted for by surface binding. Uptake of iron was inhibited by iron chelators, a protein ionophore, and ATPase inhibitors, and it was stimulated by potassium ionophores. The presence of a ferri reductase in the insoluble cell fraction of B. breve and its "spent" growth medium was demonstrated. The hypothesis is presented that iron uptake by bifidobacteria is related to the nutritional immunity phenomenon.

Biochemistry of nonheme iron in man. II. Absorption of iron.

The currently accepted concept of iron absorption proposes first the entry of iron into the intestinal mucosal cell through the brush border membrane. It is a relatively slow process. In the cell, the iron may be transferred to plasma or become sequestered by ferritin. The latter becomes unavailable for transfer to plasma and is exfoliated and excreted. In iron deficiency and idiopathic hemochromatosis, the rate of iron uptake into the intestinal mucosal cell is increased and entry into ferritin is decreased, whereas the rate of transfer to plasma remains constant. The reverse occurs in case of secondary iron overload. It is currently accepted that a transferrin, whose levels increase in iron deficiency, enters the intestinal lumen from the liver via bile, where it may sequester iron and bring it into the cells by the process of endocytosis. Iron presented as inorganic ferric or ferrous salts may also be absorbed, though the more soluble ferrous salts are adsorbed much more rapidly. Heme iron is absorbed very effectively, though it is not subject to regulation by the individual's iron status to the same extent as is inorganic iron absorption. Brush border membranes apparently contain saturable iron receptors for inorganic iron, but whether or not the absorption process requires energy is an open question. Absorption of iron may also be affected by its availability; different food components affect iron absorbability to a different extent.

Biochemistry of nonheme iron in man. I. Iron proteins and cellular iron metabolism.

Total plasma iron turnover in man is about 36 mg/day. Transferrin is the iron transport protein of plasma, which can bind 2 atoms of iron per protein molecule, and which interacts with various cell types to provide them with the iron required for their metabolic and proliferative processes. All tissues contain transferrin receptors on their plasma membrane surfaces, which interact preferentially with diferric transferrin. In erythroid cells as well as certain laboratory cell lines, the removal of iron from transferrin apparently proceeds via the receptor-mediated endocytosis process. Transferrin and its receptor are recycled to the cell surface, whereas the iron remains in the cell. The mode of iron uptake in the hepatocyte, the main iron storage tissue, is less certain. The release of iron by hepatocytes, as well as by the reticuloendothelial cells, apparently proceeds nonspecifically. All tissues contain the iron storage protein ferritin, which stores iron in the ferric state, though iron must be in the ferrous state to enter and exit the ferritin molecule. Cellular cytosol also contains a small-molecular-weight ferrous iron pool, which may interact with protoporphyrin to form heme, and which apparently is the form of iron exported by hepatocytes and macrophages. In plasma, the ferrous iron is converted into the ferric form via the action of ceruloplasmin.

Growth-enhancing supplements for various species of the genus Bifidobacterium.

Various biological materials were tested for their growth-promoting activity of several bifidobacterial species in a synthetic medium containing ample sources of inorganic salts, vitamins, nitrogen, and carbon. It was found that only Bifidobacterium adolescentis and B. longum (ATCC 15708) grew optimally or near optimally in the synthetic medium. All the other bifidobacteria tested grew optimally only in the synthetic medium supplemented with a growth promoter. The best growth promoters for all bacteria were bovine casein digest and yeast extract rather than human milk whey. Other growth promoters, including human and bovine milk wheys, hog gastric mucin, and bovine serum albumin digest were effective with some bacterial species but not with others. Bifidobacteria also grew well when the bovine casein digest (20 mg/ml) was used as the nitrogen source. Only the yeast extract was able to improve growth under these circumstances. The nature of these growth factors has not yet been determined.

Transport of ferrous iron and lactate production in Bifidobacterium bifidum var. pennsylvanicus.

Initial rates of ferrous iron transport into Bifidobacterium bifidum var. pennsylvanicus were measured at low and high iron concentrations. The low affinity system (LAFIUS) had an apparent Km of 167 microM, the high affinity system (HAFIUS) had a Km of 50 microM. Iron removal from preloaded bifidobacteria revealed the existence of a labile and an inert iron pool in the bacterial cells. Iron uptake by the bifidobacteria was associated with lactate production, though lactate production could continue without iron uptake. Cessation of iron uptake and lactate production was not because of an exhaustion of any nutrient nor the accumulation of fermentation end products in the medium. It was apparently the result of an inactivation of the cellular enzyme machinery without replacing it through normal biosynthetic processes.

Ferrous iron uptake by Bifidobacterium bifidum var. pennsylvanicus: the effect of metals and metabolic inhibitors.

Ferrous iron uptake studies in Bifidobacterium bifidum var. pennsylvanicus were carried out in a well-defined salt solution termed "modified Hanks solution" at both high iron concentrations (LAFIUS conditions) and low concentrations (HAFIUS conditions). Various divalent metals, Mn2+, Zn2+, Ni2+ and Cu2+, inhibited iron uptake under HAFIUS conditions in a non-competitive manner, and in a pseudo-competitive manner under LAFIUS conditions. Cr2+ had no effect. Co2+ inhibited iron uptake competitively under HAFIUS conditions. Metabolic affectors that inhibited iron uptake both under HAFIUS and LAFIUS conditions were: tetraphenylphosphonium chloride, diethylstilbesterol, vanadate, carbonylcyanide-m-chlorophenyl-hydrazone, and a mixture of valinomycin and nigericin. Substances that stimulated iron uptake were KCl, valinomycin, and nigericin. Iron uptake under LAFIUS conditions in piperazine-buffered modified Hanks solution was higher than that in the acetate-buffered solution, and acetate inhibited iron uptake in the piperazine buffer. HAFIUS showed no difference. It is concluded that iron uptake in bifidobacteria is driven by an ATPase-dependent proton-motive force and that both the pH gradient and membrane potential are involved in this process. Mn2+, Zn2+, Ni2+, and Cu2+ may be transported via LAFIUS, but not HAFIUS. HAFIUS may transport only Co2+ in addition to Fe2+.

Mechanisms of ferric and ferrous iron uptake by Bifidobacterium bifidum var. pennsylvanicus.

Iron uptake studies in Bifidobacterium bifidum var. pennsylvanicus were carried out using ferric citrate at iron concentrations above 0.01 mM and pH 7, ferrous iron at concentrations less than 0.01 mM at pH 5. Two ferric iron transport systems were distinguished: the temperature-insensitive polymer, and the temperature-sensitive monomer uptake. Both showed a saturation phenomenon. The transport of ferrous iron at concentrations below 0.01 mM was temperature-dependent, and its affinity for iron was higher than that of a system operating at iron concentrations higher than 0.01 mM. The use of various metabolic inhibitors indicated that ferrous iron transport at pH 5 at both high and low iron concentrations was mediated by transport-type ATPase. Proton gradient dissipators abolished ferrous iron uptakes as well as the ferric monomer uptake. Uptake of the ferric polymer was insensitive to metabolic inhibitors. The functional significance of the various types of iron transport systems may be related to the nutritional immunity phenomenon.

Cellular iron uptake from transferrin: is endocytosis the only mechanism?

Receptor mediated endocytosis has been proposed as the method of cellular iron uptake from transferrin (TF). However, the experimental evidence for endocytosis in every situation is found wanting. This is particularly true for the hepatocyte where an alternative mechanism of iron release at the cell surface can account for all iron uptake. It may be, that under appropriate physiological conditions (e.g. degree of iron saturation of TF) cells may take up iron by either an endocytotic or nonendocytotic mechanism.

Iron uptake by the microaerophilic anaerobe Bifidobacterium bifidum var. pennsylvanicus.

A system was designed to investigate ferrous iron transport into Bifidobacterium bifidum var. pennsylvanicus. It involved the incubation of the organisms with labeled ferrous iron in the Norris medium at pH 5, in which the bacteria had grown. Iron uptakes were similar under aerobic and anaerobic conditions. Ferrous but not ferric iron was taken up by the organisms. Iron uptake showed saturation kinetics and a marked temperature dependence. 2,4-Dinitrophenol and thenoltrifluoroacetate but not azide or trypsin treatment inhibited iron uptake. Zinc inhibited iron uptake competitively. Iron uptake from used medium was much greater than that from fresh medium at the same pH. It is concluded that ferrous iron uptake by the microorganisms is a carrier-mediated active phenomenon, inhibited by zinc, which may involve a substance elaborated into the medium by the organism.

Effect of various metals and calcium metabolism inhibitors on the growth of Bifidobacterium bifidum var. pennsylvanicus.

In view of the facts that the normal intestinal flora exerts beneficial effects and that bifidobacteria are a more important component in the breast-fed than in the bottle-fed infant, factors affecting the growth of the latter microorganisms are of interest. A series of transition and other metals were shown to be growth inhibitors of Bifidobacterium bifidum var. pennsylvanicus. Such inhibition could be reversed fully or partially by 0.5-1.0 mM Fe2+ in the case of Zn2+, Cu2+, Au3+, Pt4+, La3+, Cr3+, Mn2+, Ni2+, and Cd2+, but not with Ag+, Hg2+, and VO2+. In addition, 2-4 mM Ca2+ substantially relieved the inhibitory effects of Zn2+, Mn2+, and La3+, and partially relieved the effects of Cd2+. Mg2+ was ineffective in relieving Zn2+ inhibition, but Ba2+ and Sr2+ could replace Ca2+ to some extent. The calcium metabolism antagonists verapamil, ruthenium red, 2-chloroadenosine, lasalocid, Ca-ionophore A-23187, and calmodulin inhibitors W-5 and W-7 inhibited microbial growth. Inhibition could be relieved fully or partially with 0.5-1 mM Fe2+. Mg2+ relieved the inhibition by lasalocid, Ca-ionophore A-23187, and verapamil, whereas Ca2+ was effective only in the case of Ca-ionophore A-23187. We conclude that calcium and magnesium fluxes play an important role in the physiology of the bifidobacteria and that several metal growth inhibitors interfere with iron metabolism.

The behavior of transferrin receptors in rat hepatocyte plasma membranes.

The liver serves as a storage organ for iron, 2-5 mg of Fe being exchanged between plasma and liver daily in the human being. Such iron transfer from serotransferrin (TF) to hepatocytes involves a TF receptor. We are reporting some of the properties of this receptor using isolated rat hepatocyte membranes as the receptor source. Iron-saturated 125I-rat TF was used for these studies. Specific versus nonspecific TF binding was evaluated by labelling the membranes with 125I-TF and then displacing specific binding with excess unlabelled TF. A mean residency time of 18-20 min was calculated for the TF on each receptor molecule. There were 31,000 +/- 17,000 receptors/cell with a dissociation constant of 0.3 X 10(-7) mol/l. The binding obeyed simple Michaelis-Menten kinetics. Specifically bound iron-saturated 125I-TF could best be eluted with cold Fe-saturated TF; cold apo- and 50% saturated TFs were less effective. The binding of apo- and 50% saturated TF was much less pronounced than that of Fe-saturated TF. The membrane receptor was moderately heat stable, extractable with detergent, and was trypsin sensitive. Studies with 125I/59Fe-TF and whole cells suggested that Fe-TF is not in a major way internalized during iron uptake. It is concluded that there are TF receptors present on the rat hepatocyte plasma membrane that may have a role in uptake by the cells without internalization of the TF molecule.

Iron metabolism pathways in the rat hepatocyte.

A small to moderate inhibitory effect of iron uptake by isolated rat hepatocytes in short-term studies was seen with oxidative phosphorylation and electron transport inhibitors, and no inhibition by agents affecting pinocytosis. Intracellular transferrin was able to donate iron to the small-molecular weight iron pool, and the latter was able to transfer, by a process not requiring energy or movement of serum transferrin, iron to ferritin. Serum transferrin was not able to lose iron to any cytosol components. Reducing agents were not able to abstract iron from rat serum transferrin to any great extent. It is concluded that iron is taken up by the rat hepatocyte from serum transferrin by a process not requiring energy or movement of serum transferrin into the cell interior; and that intracellular transferrin is involved in acquiring iron from serum transferrin at the cell surface, with iron then being transferred to the small-molecular weight iron pool and hence to ferritin. It is also proposed that intracellular transferrins may have the general function of interacting with serum transferrin at cell surfaces.

Protein and electrolyte changes in experimental cerebral edema.

Analysis of protein and electrolyte data in cryogenic cerebral edema in the rhesus monkey has led to the conclusion that, in the first 24 hours (h) after injury, the edematous process is not homogenous, but compartmentalized. This involves, first of all, a division into intra- and extracellular compartments. The intracellular compartment is further divided into a compartment containing water, electrolytes, and serum proteins, and a compartment containing only excess sodium. The extracellular compartment is also subdivided into a compartment containing albumin, globulin, and electrolytes, and a compartment containing only albumin and electrolytes. Anatomically, the latter is most likely the pre-existing normal extracellular space.

The effect of metal chelators and other metabolic inhibitors on the growth of Bifidobacterium bifidus var. Pennsylvanicus.

Bifidobacterium bifidus var. Pennsylvanicus, a microaerophilic anaerobe, was grown in the presence of several potential growth inhibitors with the aim of defining its growth requirements and metabolic peculiarities. The following had no effect on its growth: citrate, serum transferrin, serum albumin, colchicine, fluoro-acetate, malonate, and rotenone. The following substances inhibited the growth: fluoride, azide, arsenite, 2, 4-dinitrophenol, hemin, hemoglobin, lactoferrin, alpha, alpha'-bipyridyl, and 8-hydroxyquinoline. Ferrous iron was able to negate the inhibition achieved by alpha, alpha'-bipyridyl, and 8-hydroxyquinoline. It is concluded that iron, probably in its ferrous state, is an obligatory nutrient for the microorganism, and that iron-porphyrin system(s) may be essential for the metabolism of this organism. Because the microorganisms contained in addition to iron large quantities of Mn, Zn, and Cu, it is likely that these metalloelements are crucial for the normal growth of the organism. Growth inhibition by fluoride indicates that Mg-dependent enzymes may also be present in the microorganism.