Susceptibility of Legionella pneumophila grown extracellularly and in human monocytes to indole-3-propionic acid.

Indole-3-propionic acid (IPA), a phytohormone derivative, is a potent inhibitor of growth of Legionella pneumophila cultivated extracellularly in a chemically defined hypotonic medium and intracellularly in human monocytes. The inhibitory activity turns into bactericidal activity with increasing concentrations. The susceptibility of the microorganism to IPA was more evident in "fast-growing" cultures (under conditions of vigorous shaking) than in static cultures growing under an atmosphere of 5% CO2-95% air, which resulted in a decreased growth rate. The MIC, after incubation with the drug for 48 h and as determined by counting of the CFU, was 1.58 microM for fast-growing cultures and 2.64 microM for those grown under static conditions. The MBCs were 5.28 and 26.43 microM, respectively. Tryptophan (Trp) at 150 microM prevented the inhibition caused by 2.64 microM IPA, increased the MIC about 3-fold, and increased the MBC by 10-fold. The effect of Trp was less remarkable in "slow-growing" cultures. The susceptibility of L. pneumophila proliferating in human monocytes was markedly lower than that when it was cultivated extracellularly in the chemically defined hypotonic medium. The MIC after incubation for 48 h was 5.28 microM, and a decrease in viable count was achieved with 105.70 microM. The lower susceptibility was apparently due (at least partially) to the presence of Trp (24.50 microM) in the RPMI 1640 medium that was used for the monocyte cultures. The effect of IPA was time dependent, and prolonged exposure enhanced the bactericidal activity and turned the inhibitory dose into a bactericidal dose. The present data demonstrate that IPA is a potent anti-L. pneumophila factor, although it has a markedly lower activity against bacteria growing intracellularly compared with its activity against extracellularly proliferating microorganisms.

Purification and characterization of uridine and thymidine phosphorylase from Lactobacillus casei.

Uridine and thymidine phosphorylases have been purified to homogeneity from crude extracts of Lactobacillus casei. Both enzymes had an apparent molecular mass of about 80 kDa. Uridine phosphorylase consisted of four identical subunits while thymidine phosphorylase was composed of two identical ones. The sequence of 23 amino-acid residues from its N-terminal end was analyzed. Uridine phosphorylase had a Km of 5.0 x 10(-3) M for uridine and 1.24 x 10(-1) M for phosphate, while thymidine phosphorylase had a Km of 1.32 x 10(-1) M for thymidine and 1.0 x 10(-1) M for phosphate. Uridine phosphorylase was equally active with uridine and 5-methyluridine, but had a low activity towards thymidine. Its activity was inhibited competitively by 3-O-methyl-alpha D-glucopyranoside, on the other hand thymidine phosphorylase activity was not affected by this compound. Thymidine phosphorylase showed specificity towards the deoxyribosyl moiety of the substrate. In addition, it required a nonsubstituted pyrimidine moiety or one which was substituted in position 5. The pattern of the double-reciprocal plots of the initial velocities vs. the concentrations of either one of the substrates, and the product inhibition kinetics, indicated that the catalytic mechanism of both enzymatic reactions is sequential rather than Ping-Pong and that the sequence of the addition of the substrates is random (rapid equilibrium). In the case of the uridine phosphorylase-catalyzed reaction, the products are also released randomly, while in the thymidine phosphorylase-catalyzed reaction deoxyribose 1-phosphate is released after thymine.

Phytohormones as specific inhibitors of Legionella pneumophila growth.

Legionellae have been found to be highly susceptible to a variety of biological products, which increases the difficulty of growing these microorganisms. We developed a hypotonic medium in which Legionella pneumophila and other legionellae grow well and multiply rapidly from small inocula. Several amino acids, mainly nonessential ones, inhibited the growth of legionellae at high concentrations (200-1,000 micrograms/ml). We describe a unique biological phenomenon of specific inhibition of growth of L. pneumophila by the plant growth hormone auxin (indole-3-acetic acid) and the closely related indole-3-propionic acid (IPA). The inhibition of growth was probably due to interference with the biosynthesis of L-tryptophan by the phytohormone or IPA. Other bacteria were found to be 50 to 100-fold more resistant to these agents. These findings may explain the peculiar ecology of legionellae. Bacterial susceptibility towards IPA (less than or equal to 5 micrograms/ml) may serve as a specific marker for the presence of legionellae.

Congenital isolated folic acid malabsorption.

We report a case of congenital isolated malabsorption of folic acid, the first in a boy. Only seven previous cases have been reported, and we discuss two aspects--namely, the tendency to infection, with evidence of impairment of both cellular and humoral immunity, and the absence of neurological disturbances.

Active extrusion of potassium in the yeast Saccharomyces cerevisiae induced by low concentrations of trifluoperazine.

Trifluoperazine (TFP), the antipsychotic drug, induces substantial K+ efflux, membrane hyperpolarization and inhibition of H+-ATPase in the yeast Saccharomyces cerevisiae. Investigations on the mechanism of these effects revealed two different processes observed at different incubation conditions. At an acidic pH of 4.5 and an alkaline pH of 7.5, K+ efflux was accompanied by substantial proton influx which led to intracellular acidification and dissipation of delta psi formed by cation efflux. The results indicated nonspecific changes in membrane permeability. Similar results were also observed when cells were incubated at pH 5.5-6.0 with higher concentrations of TFP (above 75 microM). On the other hand, low concentrations of TFP (30-50 microM) at pH 5.5-6.0 caused marked membrane hyperpolarization and K+ efflux unaccompanied by the efflux of other cations and by H+ influx. Our experiments indicate that under these conditions K+ efflux was an active process. (1) K+ efflux proceeded only in the presence of a metabolic substrate and was inhibited by metabolic inhibitors. (2) When 0.3-0.9 mM-KCl was present in the medium at pH 6.0, the concentration of K+ within the cells (measured at the end of the incubation with TFP) was much lower than the theoretical concentration of Kin+ if the distribution of K+ between medium and cell water was at equilibrium (at zero electrochemical gradient). (3) Valinomycin decreased the net K+ efflux and decreased the membrane hyperpolarization induced by TFP, probably by increasing the flux of K+ into the cells along its electrochemical gradient. (4) Conditions which led to active K+ efflux also led to a marked decrease in cellular ATP level. The results indicate that under a specific set of conditions TFP induces translocation of K+ against its electrochemical gradient.

The effect of growth temperature on the thermotropic behavior of the membranes of a thermophilic Bacillus. Composition-structure-function relationships.

The following study was carried out with the aim of widening our understanding of the thermoadaptive mechanisms of the membrane of thermophiles, using Bacillus stearothermophilus var. nondiastaticus as test-organism. The phospholipids and their acyl chain composition of this Bacillus studied in relation to the physical properties of its membrane from bacteria grown at various temperatures. Phospholipids account for 68-75 weight% of the total lipid in cells grown at 45, 55 or 65 degrees C. Phosphatidylglycerol and diphosphatidylglycerol constitute up to 90% of the total phospholipids; no amino phospholipids were found. Increasing the growth temperatures from 45 degrees to 65 degrees C caused an approximately 4-fold decrease in the proportion of the branched-chain fatty acids and a 2-fold increase in the amount of the saturated acyl chains. The reduced proportion of the branched fatty acids was mainly due to a decrease in their anteiso forms. Unsaturated fatty acids were not produced by cells grown at 65 degrees C. In accordance with the fatty acid composition, the molecular packing of phospholipids in monolayers was more expanded with phospholipids from 45 degrees C grown cells as compared with cultures grown at 55 degrees C. The thermotropic gel to liquid-crystalline phase transition of the membrane lipids was monitored by differential scanning calorimetry and fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene. With increase of the growth temperature the phase transition was progressively shifted to higher but narrower range of temperatures. Completion of the lipid melting occurred always at temperatures below those employed for growth. A constructed phase diagram enabled to relate the growth temperature, the fatty acid composition and the lipid apparent microviscosity at temperatures not used in the present study for growth of the thermophile. The minimum temperature for growth and the upper boundary temperature of the least saturated lipid crystallization were extrapolated in this manner; they correspond to the experimentally determined minimal growth temperature. The apparent microviscosity, a measure of membrane order, decreased gradually and conspicuously as the growth temperature was elevated. The delimiting apparent microviscosity values, at the maximal (65 degrees C) and minimal (41 degrees C) growth temperatures were 0.8 and 1.8 poise, respectively. This lack of rigorous homeostatic control of the bulk lipid viscosity prompted reevaluation of the physiological significance of 'homeoviscous adaptation' in Bacillus stearothermophilus.

Mechanism of stimulation of Ca2+ uptake by miconazole and ethidium bromide in yeasts: role of vacuoles in Ca2+ detoxification.

The antifungal antibiotic miconazole and the cationic dye ethidium bromide, both caused K+ efflux, membrane depolarization and intracellular acidification in the yeast Saccharomyces cerevisiae. Whereas miconazole inhibited the activity of the plasma membrane H+-ATPase, no such inhibition was observed using ethidium bromide at concentrations up to 600 microM. Low concentrations of both drugs caused marked stimulation of the energy dependent Ca2+ uptake. The extra Ca2+ taken up in the presence of the drugs was localized within the vacuoles, whereas K+ was lost mainly from the cytosolic pool. The ions Zn2+ and La3+ inhibited the effect of both drugs on the stimulation of Ca2+ uptake. The results indicated that both drugs caused an increase in the permeability of cell membranes to ions, leading to an increase in the influx of Ca2+ into the cytosol along its electrochemical gradient. Consequently, the concentration of Ca2+ within the cytosol increased and in turn led to the enhancement of Ca2+ uptake by the energy dependent vacuolar Ca2+ system, which functioned as a Ca2+ detoxification system.

Effects of inhibitors of plasma-membrane ATPase on potassium and calcium fluxes, membrane potential and proton motive force in the yeast Saccharomyces cerevisiae.

The plasma membrane ATPase inhibitors N,N-dicyclohexylcarbodiimide (DCCD), diethylstilboestrol and sodium orthovanadate caused inhibition of proton ejection in Saccharomyces cerevisiae at a concentration range of 0.1-0.4 mM. At this concentration K+ efflux was not observed; DCCD caused K+ efflux only at a much higher concentration (2mM). It was shown that induction of K+ efflux by DCCD was not mediated via inhibition of ATPase and that it may be the result of a direct effect of the drug on the cell membrane or on the K+ carrier. All three inhibitors also caused mild hyperpolarization of the membrane and an increase in the carrier mediated Ca2+ uptake into the cells. The increase in delta psi balanced the decrease in delta pH so that the value of delta mu H+ changed very little following incubation of the cells with the inhibitors. The respiratory deficient mutant ('petite') displayed similar phenomena to the wild type, but membrane potential values were lower than in the wild type.

Biotransformation of homofolate from a growth inhibitor to a growth-promoting derivative.

Homofolic acid (HF) was found to inhibit the growth of Lactobacillus casei ATCC 7469 in 100 ng/ml or higher concentrations in the presence of folic acid (PGA) on short (24-48 h) incubation. HF blocked the uptake of 3HPGA into L. casei cell suspensions. On prolonged (3-5 days) incubation HF (greater than 10 ng/ml) promoted growth of L. casei both in the presence and absence of PGA. No growth was obtained when both PGA and HF were omitted. Thus, HF was apparently biotransformed from an inhibitory analog into a growth-promoting HF derivative. Indeed, sterile filtrates from HF-grown cultures supported growth of L. casei in a folate-free medium on short incubation, supporting the notion that a folate-like active compound was formed from HF on prolonged incubation with L. casei. In the presence of homopteorate (10-60 micrograms/ml) no transformation of HF into folate-active compounds took place.

Effects of estradiol on uptake of folate in isolated intestinal epithelial cells.

Estradiol 17 beta caused significant changes in the time course of 3H-folate uptake into isolated chick-intestinal epithelial cells. The radioactivity within the cells initially increased reaching a level of 1.5-2 times that of the control thereafter decreased to somewhat below the control levels. These changes were prevented by sodium azide but not by inhibitors of protein synthesis.

Nystatin effects on cellular calcium in Saccharomyces cerevisiae.

The primary effects of nystatin, a polyene antibiotic, on the yeast Saccharomyces cerevisiae were investigated. Though K+ leakage was observed shortly after the addition of nystatin, Ca2+ leakage was delayed 2-3 h after its application and it occurred only at an acidic pH and in the absence of K+, Na+ or Mg2+ from the medium. However, within 4 min after application nystatin induced a passive influx of Ca2+ into the cells even at a concentration of 1 microM in the medium. These results led to the conclusion that the primary membranal lesion induced by nystatin is not restricted to monovalent cations but is also manifested by increased permeability to Ca2+. The delayed leakage of Ca2+ is explained by the assumption that the bulk of cellular calcium is sequestered so that the concentration of free Ca2+ in the cytoplasm is very low. The sequestered calcium may be liberated 2-3 h after the addition of nystatin as a consequence of secondary damage to the cells such as intracellular acidification and loss of cations.

Effect of phenytoin on folic acid uptake in isolated intestinal epithelial cells.

The anticonvulsant drug phenytoin was found to inhibit the uptake of folic acid into isolated chick intestinal epithelial cells. At a concentration of 100 micrograms/ml the drug inhibited the cellular accumulation of folic acid by 60%. The efflux of folic acid from preloaded cells was not affected by the drug. The inhibition was observed at an acidic pH of 5.8 and at a neutral pH of 7.4. Both the Na+-dependent and the Na+-independent components of folate uptake were inhibited. Phenytoin had no effect on the uptake of the glucose analog 3-O-methylglucose. Thus it was concluded that the effect of phenytoin on folate uptake was not due to changes in Na+ fluxes, concentration gradients or energy metabolism, and suggested instead a direct effect on the folic acid uptake system.

Physiological and enzymatic properties of a thymidine-requiring Pediococcus cerevisiae mutant.

We describe the isolation and characterization of a Pediococcus cerevisiae thymidine-requiring mutant and its thymidine-independent revertant. The mutant strain lacked thymidylate synthetase activity and had an absolute requirement for low concentrations (2 micrograms/ml) of thymidine in addition to a requirement for N-5-formyl tetrahydrofolic acid (folinate). Even at high concentrations (up to 500 micrograms/ml), thymine could not replace thymidine. In contrast to its wild-type parent, which grows only on folinate, the thymidine-requiring mutant (Thy- Fol+) was able to take up and grow on picogram quantities of unreduced folic acid. When both strains were grown on folinate, the Thy- Fol+ strain was at least 10(3)-fold more resistant to the folic acid analogs aminopterin and methotrexate than the wild-type strain. On the other hand, when grown on folic acid, the Thy- Fol+ strain was as sensitive to the folic acid analogs as the Thy+ Fol+ strain and was 10(2)-fold more sensitive than the wild-type strain grown on folinate. The thymidine-independent revertant (Thy+ Fol+) regained the wild-type level of thymidylate synthetase activity, but maintained the ability to take up and grow on unreduced folic acid like its Thy- Fol+ parent.

Effect of insulin on folic-acid transport in cultured fibroblasts.

Uptake of folic acid was measured in secondary cultures of skin fibroblasts from fetal rats. The cultures were made quiescent by 24 hours preincubation in medium containing 1% serum and subsequent 3 hours preincubation in phosphate buffered saline. The uptake of 3H-folic acid was linear with time during 15 seconds and reached a plateau level at 2-3 minutes. There was no further increase in the intracellular radioactivity until the end of the experiments at 10 minutes. The uptake of folic acid in fibroblasts was not concentrative and proceeded until equilibration with the extracellular concentration. Intracellular metabolic conversion of folic acid was not significant during the time of experiments (up to 10 minutes). Insulin caused a two-fold increase in the initial rate of folate uptake as determined from the 15 second uptake values. The dose response curves for the insulin effect showed that 85% of the maximal effect was exerted by 1 microM insulin. A lag period of 7-10 minutes was observed after the addition of insulin and before the effect on folic acid uptake was manifested. Thereafter the effect increased with the time of preincubation with insulin. The concentration dependence of folate uptake yielded non homogeneous curves. At low concentrations of substrate, saturable components were observed while at high concentrations (above 5 X 10(-6) M) a linear component was observed. Insulin increased the slope of the linear component and the Vmax of the saturable component while the Km remained unaltered.

Diagnosis of iron deficiency anemia in a rural population of children. Relative usefulness of serum ferritin, red cell protoporphyrin, red cell indices, and transferrin saturation determinations.

The diagnostic usefulness in iron deficiency anemia of serum ferritin, red cell protoporphyrin (Epp), mean corpuscular volume, mean corpuscular hemoglobin (MCH), and transferrin saturation measurements has been studied in a population of 294 children aged 1 to 6 yr. Of the children studied 19% had hemoglobin below 11 g/dl. Iron deficiency, diagnosed by at least two abnormal independent laboratory parameters, was the cause of anemia in all except two cases. The Pearson correlation coefficient for hemoglobin was highest with MCH, followed in decreasing order of magnitude by MCV, Epp, transferrin saturation, and finally by ferritin. Sensitivity and specificity were highest for MCH and lowest for ferritin. Of anemic, iron deficient individuals 97 to 100% could be identified by low MCH, 88 to 100% by transferrin saturation, 66 to 83% by ferritin, and 61 to 74% by Epp. In contrast, only 0 to 6% of normal, nonanemic individuals had low MCH, 0 to 4% had high Epp, but 21 to 39% had low transferrin saturation and 25 to 39% had low ferritin. Although reduced serum ferritin in anemic individuals is good evidence of iron deficiency, a significant proportion of anemic iron-deficient patients is missed by this procedure rendering it less useful than other, less expensive laboratory methods.

Cryoprotected Lactobacillus casei: an approach to standardization of microbiological assay of folic acid in serum.

Folate-depleted, cryoprotected preparations of Lactobacillus casei are stable for at least eight months at -18 degrees C, capable of reproducible growth and suitable as a ready source of inoculum for measurement of folates in physiological fluids. Cryoprotected microorganisms can be a commercially available laboratory reagent, thus simplifying and further standardizing the microbiological assay of various nutriments. Standard folate growth curves of cryoprotected L. casei, prepared at intervals over eight months, are superimposable, have a low blank, and should eliminate the variations encountered with the continuous passages of microorganisms required for the classic microbiological assays. Serum folate values obtained by use of the cryoprotected L. casei fall into the same diagnostic groups as determined by the classic microbiological assay.

On the mechanism of folate transport in isolated intestinal epithelial cells.

The mechanism of folic acid (FA) uptake was studied in isolated intestinal epithelial cells prepared from 2- to 6-wk-old chicks. The cells accumulated FA, reaching a level of three- to fivefold that at equilibrium. In the presence of the metabolic inhibitors, NaN3 or KCN, FA was taken up only until equilibration while accumulation of FA was inhibited. Addition of these inhibitors at a steady state of FA accumulation caused a release of intracellular FA. The kinetics of FA uptake were found to be saturable (Km = 3.5 x 10(-5) M), indicating a carrier-mediated mechanism. The steady-state level of FA accumulation was higher as the concentration of NA+ in the medium increased from 0 to 120 mM. This stimulation of FA uptake by Na+ was not due to the stimulation of glucose uptake, because in experiments carried out in the presence of phlorizin, a glucose-transport inhibitor, FA accumulation was not diminished. It is suggested that FA is taken up by a Na+-coupled transport system.