Eva Kot

P‐31 Nuclear Magnetic Resonance Analysis of Brain: The Perchloric Acid Extract Spectrum

Abstract: Perchloric acid (PCA) extracts were prepared from liquid‐N2‐frozen guinea pig brains and their organophosphate profiles examined by P‐31 nuclear magnetic resonance (NMR) spectroscopy. Thirty‐two phosphorus‐containing brain metabolites were characterized and quantitated. A distinctive feature of brain tissue metabolism relative to that of other tissues probed by P‐31 NMR is its pronounced ribose 5‐phosphate content. Comparison of brain metabolite levels following control or sublethal cyanide treatment (4 mg/kg) revealed specific cyanide‐induced changes in brain metabolism. Brains from cyanidetreated animals were characterized by a reduced phosphocreatine content and elevated α‐glycerolphosphate and inorganic orthophosphate contents relative to control. P‐31 NMR spectra of brain PCA extracts at pH 7.2 were also obtained under conditions that approximate those used for in vivo and intact tissue in vitro P‐31 spectroscopic analyses. The spectra reveal nine separate resonance bands corresponding to: sugar phosphates, principally ribose 5‐phosphate (3.7δ); inorganic orthophosphate (2.2δ); glycerol 3‐phosphorylethanolamine (0.3δ); glycerol 3‐phosphorylcholine (−0.1δ); phosphocreatine (−3.2δ); adenosine tri‐(β‐ATP) and di‐(β‐ADP) phosphate ionized end‐groups (−6.2δ); α‐ATP, α‐ADP, and nicotinamide adenine dinucleotides esterified end‐groups (−11.1δ); uridine diphosphohexose, hexose esterified end‐groups (−13.0δ); and β‐ATP ionized middle group (−21.6δ). Knowledge of the phosphatic molecules that contribute resonances to the brain P‐31 NMR spectrum as well as understanding their magnetic resonance properties is essential for the interpretation of in vivo brain spectroscopic data as well as brain extract data, since these same compounds contribute to the intact brain P‐31 spectrum.

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.

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.

Ferrous iron uptake by bifidobacteria in the absence of glucose

Abstract Bifidobacterium breve and Bifidobacterium thermophilum, grown in metal-poor media under anaerobic conditions, were used to investigate ferrous iron uptake in their non-energized (glucose-free) states at pH 5.0. Considerable uptake was observed in the absence of glucose, which was relatively unaffected by Mg 2+ and was decreased by increasing the medium pH to 6.5. Surface-bound iron accounted for 20% to 30% of total iron taken up. By determining soluble iron concentrations inside the cells, it was calculated that iron was concentrated some 14–16-fold with respect to exterior (100 and 200 μM). Ferric iron failed to enter the cell in any significant amounts as did ferrous iron when both the energized and nonenergized cells had been grown on iron-replete medium or heated. It was concluded that ferrous iron enters the nonenergized bifidobacterial cell as a result of a potential difference across the bacterial membrane (negative inside) created by a proton concentration difference. This provides an opportunity for bifidobacteria to participate in the nutritional immunity phenomenon even when a carbon source is lacking in their environment.

Iron Uptake by Bifidobacterium thermophilum Protoplasts

Protoplasts ofBifidobacterium 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.

Effect of Al(III) on surface properties of Bifidobacterium thermophilum as a function of temperature

The effects of Al(III) on surface properties and lactate accumulation by Bifidobacterium thermophilum were investigated. Bacteria were treated with Al(III) at 37°C and 4°C, then exposed to free radicals or nisin. When exposed to Al(III) at 37°C, the organism exhibited spreading on hydrophobic surfaces and showed high susceptibility to free-radical alteration as indicated by Fe(III) binding, but showed little effect on lactate production in the presence or absence of nisin, even after washing with 2 mM EDTA. At 4°C, there was no increased surface spreading or binding of Fe(III), but protection against nisin action was present. This, however, was abolished after washing with EDTA. It was concluded that membrane fluidity is required to affect membrane lipid rearrangement, resulting in surface spreading and increased susceptibility to peroxidation, whereas only loose binding of Al(III) to membrane surfaces is sufficient to prevent transmembrane channel formation by nisin.