Transport Of L-methionine Research Articles

Multidrug resistance plasmids commonly reprogram the expression of metabolic genes in Escherichia coli.

The increase in infections that are resistant to multiple classes of antibiotics, including those isolates that carry carbapenamases, beta-lactamases, and colistin resistance genes, is of global concern. Many of these resistances are spread by conjugative plasmids. Understanding more about how an isolate responds to an incoming plasmid that encodes antibiotic resistance will provide information that could be used to predict the emergence of MDR lineages. Here, the identification of metabolic networks as being particularly sensitive to incoming plasmids suggests the possible targets for reducing plasmid transfer.

Examining the Bacterial Methionine Transporter Utilizing Soluble Lipid Bilayer Systems

ATP‐binding cassette (ABC) transporters are ubiquitous across all kingdoms of life. These highly specific pumps translocate substrates across cell membranes using the energy from ATP binding and hydrolysis. A detailed understanding of ABC transporter mechanism could aid in the treatment of a variety of human disorders in which ABC transporters are defective (eg. Stargardt disease, adrenoleukodystrophy, Tangier disease, and cystic fibrosis). Furthermore, ABC transporters are one of the main culprits behind multidrug resistance, a principle impediment facing current cancer therapeutics. Circumventing these diseases depends largely on our ability to fully understand the mechanism of how these transporters mediate movement across the lipid bilayer.While the structural determinations of ABC transporters have provided critical insights, a detailed molecular understanding of how these proteins work has been precluded by difficulties in the functional study of transporters. This gap in our understanding reflects the experimental challenges to their study. One main issue is preserving a transmembrane (TM) protein's native structure and functionality for study. The native membrane environment is a lipid bilayer, which historically has been difficult to recreate for biochemical study. Processes used to produce membrane vesicles require repeated trial and error attempts to address each protein's unique preferences. A more universal strategy for solubilizing TM proteins is the use of synthetic detergents, however many preceding studies suggest that detergents may alter the structure and functionality of the protein. To address this issue, we have turned to a system called nanodiscs, which is a discoidal lipid bilayer encircled by a protein helical belt. Unlike liposomes, nanodiscs have been shown to be a viable system for fluorescent spectroscopic analysis. This system has been experimentally proven to be durable in a wide range of experimental conditions, including pH, temperature, and salt concentration.As part of the Yang Group at the University of San Francisco, I am probing the mechanism of the E. coli methionine transporter model system. This bacterial transporter, named MetNI, imports the amino acid methionine into the cell using energy from ATP. Importantly, MetNI shares high sequence and structural homology with human transporters known to confer multidrug resistance and disease. Over the last year, we have optimized the protocol to generate monodisperse MetNI nanodiscs in high yield. Now, I am utilizing quantitative methods to understand how the MetNI transporter, embedded in a lipid bilayer, can coordinate the binding of substrate and ATP hydrolysis to drive the transport of L‐methionine across cell membranes.Support or Funding InformationAcknowledgements to the Whitehead Fellowship, Clare Boothe Luce Program, and University of San FranciscoThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Multiple pathways for L-methionine transport in brush-border membrane vesicles from chicken jejunum.

1. The intestinal transport of L-methionine has been investigated in brush-border membrane vesicles isolated from the jejunum of 6-week-old chickens. L-Methionine influx is mediated by passive diffusion and by Na+-dependent and Na+-independent carrier-mediated mechanisms. 2. In the absence of Na+, cis-inhibition experiments with neutral and cationic amino acids indicate that two transport components are involved in L-methionine influx: one sensitive to L-lysine and the other sensitive to 2-aminobicyclo[2.2. 1]heptane-2-carboxylic acid (BCH). The L-lysine-sensitive flux is strongly inhibited by L-phenylalanine and can be broken down into two pathways, one sensitive to N-ethylmaleimide (NEM) and the other to L-glutamine and L-cystine. 3. The kinetics of L-methionine influx in Na+-free conditions is described by a model involving three transport systems, here called a, b and c: systems a and b are able to interact with cationic amino acids but differ in their kinetic characteristics (system a: Km = 2.2 +/- 0.3 microM and Vmax = 0.13 +/- 0.005 pmol (mg protein)-1 (2 s)-1; system b: Km = 3.0 +/- 0.3 mM and Vmax = 465 +/- 4.3 pmol (mg protein)-1 (2 s)-1); system c is specific for neutral amino acids, has a Km of 1.29 +/- 0.08 mM and a Vmax of 229 +/- 5.0 pmol (mg protein)-1 (2 s)-1 and is sensitive to BCH inhibition. 4. The Na+-dependent component can be inhibited by BCH and L-phenylalanine but cannot interact either with cationic amino acids or with alpha-(methylamino)isobutyrate (MeAIB). 5. The kinetic analysis of L-methionine influx under a Na+ gradient confirms the activity of the above described transport systems a and b. System a is not affected by the presence of Na+ while system b shows a 3-fold decrease in the Michaelis constant and a 1.4-fold increase in Vmax. In the presence of Na+, the BCH-sensitive component can be subdivided into two pathways: one corresponds to system c and the other is Na+ dependent and has a Km of 0.64 +/- 0. 013 mM and a Vmax of 391 +/- 2.3 pmol (mg protein)-1 (2 s)-1. 6. It is concluded that L-methionine is transported in the chicken jejunum by four transport systems, one with functional characteristics similar to those of system bo, + (system a); a second (system b) similar to system y+, which we suggest naming y+m to account for its high Vmax for L-methionine transport in the absence of Na+; a third (system c) which is Na+ independent and has similar properties to system L; and a fourth showing Na+ dependence and tentatively identified with system B.

Assessment of [11C]-L-methionine transport into the human brain.

Neutral amino acid transport into human brain was measured using a dual-probe positron detection system or positron emission tomography (PET). Rate constants (ml/min/cc) for brain accumulation of [11C]L-methionine measured with the dual detector ranged from 0.012 to 0.078 (average 0.031) under baseline conditions and from 0.010 to 0.017 (average 0.014) after administration of nonradioactive L-phenylalanine (100 mg/kg). The net rate of brain accumulation of L-methionine ranged from 0.42 to 2.89 (average 1.28) nmol/min/cc, and decreased by 27.5-91.2% (average 53.9%) after L-phenylalanine. PET-estimated accumulation rates (ml/min/cc) of [11C]L-methionine ranged from 0.004 to 0.028 (average 0.016) baseline and from 0.010 to 0.021 (average 0.017) after L-phenylalanine. Initial volumes of distribution (ml/cc) of [11C]L-methionine (dual detector) were 0.044-0.070 (average 0.058) baseline and 0.032-0.074 (average 0.051) after phenylalanine and (PET) 0.026-0.098 (average 0.051) baseline and 0.021-0.061 (average 0.042) after phenylalanine. PET permits more accurate measurement of tracer accumulation by brain, excluding noncerebral regions included in dual-detector measurements. The dual-detector system permits better temporal resolution, facilitating kinetic analysis, and requires only one-fortieth the dose of tracer needed for PET. Multiple studies in the same patient are thus possible at low cost.

Na + -Stimulated Transport of l -Methionine in Brevibacterium linens CNRZ 918

The transport of l-methionine by the gram-positive species Brevibacterium linens CNRZ 918 is described. The one transport system (K(m) = 55 muM) found is constitutive for l-methionine, stereospecific, and pH and temperature dependent. Entry of l-methionine into cells is controlled by the internal methionine pool. Competition studies indicate that l-methionine and alpha-aminobutyric acid share a common carrier for their transport. Neither methionine derivatives substituted on the amino or carboxyl groups nor d-methionine was an inhibitor, whereas powerful inhibition was shown by l-cysteine, s-methyl-l-cysteine, dl-selenomethionine and dl-homocysteine. Sodium plays important and varied roles in l-methionine transport by B. linens CNRZ 918: (i) it stimulates transport without affecting the K(m), (ii) it increases the specific activity (on a biomass basis) of the l-methionine transport system when present with methionine in the medium, suggesting a coinduction mechanism. l-Methionine transport requires an exogenous energy source, which may be succinic, lactic, acetic, or pyruvic acid but not glucose or sucrose. The fact that l-methionine transport was stimulated by potassium arsenate and to a lesser extent by potassium fluoride suggests that high-energy phosphorylated intermediates are not involved in the process. Monensin eliminates stimulation by sodium. Gramicidin and carbonyl cyanide-m-chlorophenylhydrazone act in the presence or absence of Na. N-Ethylmaleimide, p-chloromercurobenzoate, valinomycin, sodium azide, and potassium cyanide have no or only a partial inhibitory effect. These results tend to indicate that the proton motive force reinforced by the Na gradient is involved in the mechanism of energy coupling of l-methionine transport by B. linens CNRZ 918. Thus, this transport is partially similar to the well-described systems in gram-negative bacteria, except for the role of sodium, which is very effective in B. linens, a species adapted to the high sodium levels of its niche.

L-Methionine Transport During the Induction of Freezing Hardiness by Abscisic Acid in Brassica napus Cell Suspension Cultures

Transport of methionine was determined for Brassica napus suspension culture cells during the induction of freezing tolerance by ABA at room temperature. Methionine uptake was linear over one hour and methionine was concentrated 100-fold above the medium concentration. Alteration of the medium by adding or deleting ABA or BA and altering sucrose concentrations elicited marked differences in transport rates, but these were not correlated with the induction of freezing tolerance. Transport was characterized in both cells with respect to kinetics, pH optima and Ki 's of amino acids. Apparent Km in hardened cells was twice that for nonhardened cells, while Vmax was the same. Both cells exhibited a pH optimum of 6.5. Hardened cells had a greater specificity for D-methionine compared to the L-isomer. Hardened cells continued to accumulate methionine in the cytoplasm and vacuole below 5°C, whereas transport was negligible at this temperature in non-hardened cells. These results suggest that methionine transport may be altered by the hardening process.

Methionine and glutamine transport systems in D-methionine utilising revertants of Salmonella typhimurium

In Salmonella typhimurium, methionine auxotrophs such as metB can use D-methionine as a methionine source. MetP mutations prevent this growth since D-methionine can enter only via the metP high-affinity methionine transport system. D-methionine utilising revertants ( Dmu +) were selected from metB23 metP760 ( HU76 ) following nitrosoguanidine mutagenesis. The properties of two such revertants, HU206 and HU415 , indicated that reversion was not due to backmutation of the metP760 mutation. Genetic analysis indicated that each strain possessed two mutations, designated dmu and gln, in addition to the original metB23 and metP760 mutations. The dmu mutation restores ability to grow on D-methionine, partly restores D- and L-methionine transport activity, and makes the cells particularly sensitive to inhibition by L-glutamine while growing on D but not L-methionine. The growth inhibition by L-glutamine was shown to be caused by competition by L-glutamine for D-methionine transport by the high-affinity methionine system. The gln mutation greatly reduces activity of the high-affinity glutamine transport system. The Dmu + strains are also partly defective in the glutamine low-affinity transport system, possibly because the partially-restored methionine high-affinity system, or a component of tis system, functions in the transport of glutamine by its low-affinity system.

Influence of retinoic acid on protein synthesis and transport of l-methionine in cultured L cells

The effects of retinoic acid (RA) on cell proliferation, activity of acid phosphatase, protein synthesis and methionine uptake were studied in transformed murine LPA cells. Early inhibition of protein synthesis was demonstrated under experimental conditions in which the rate of cell proliferation was diminished and non-specific effects of vitamin action could be excluded. Measurements of l-methionine uptake revealed a decrease to approximately one-half of that in control cultures after treatment with RA at the concentrations of 5 × 10 −5 M and 10 −5 M.

Transport of methionine in sea-urchin sperm by a neutral amino-acid carrier.

A carrier-mediated transport for L-methionine and other neutral amino acids exists in sperm of the sea urchin Lytechinus pictus. The initial rate of L-methionine entry is a Michaelis-Menten function of the methionine concentration in the external medium. The maximum velocity is low [V = 250 pmol h-1 (10(9) sperm)-1 at 22 degrees C] and the affinity is high (Km = 6-10 microM). The initial rate of transport under steady-state exchange conditions is also a Michaelis-Menten function of the external concentration of methionine. The Km determined by this method is about 14 microM. Neutral amino acids compete with L-methionine transport as shown by initial velocity measurements. These results indicate that L-methionine transport is a carrier-mediated process. The temperature dependence of the process is approximately 84 kJ (20 kcal) mol-1 K-1, which is not compatible with a simple diffusion mechanism, but in the range of values usually found for a mediated transport. The transport is largely Na+-independent and does not depend on Ca2+, K+ or H+ gradients. It is only partially sensitive to KCN, showing it is mainly independent of oxidative phosphorylation. The steady-state internal methionine concentration is not a linear function of the external amino acid concentration. This suggests that an exit by diffusion competes with a carrier-mediated concentrative transport in a cellular compartment. This mediated transport is compared to those of higher animal cells.

Role of precursor availability in control of monoamine biosynthesis in brain.

Role of precursor availability in control of monoamine biosynthesis in brain.

Na +/solute symport in membrane vesicles from Bacillus alcalophilus

The characteristics of α-aminoisobutyric acid translocation were examined in membrane vesicles from obligately alkalophilic Bacillus alcalophilus and its non-alkalophilic mutant derivative, KM23. Vesicles from both strains exhibited α-aminoisobutyric acid uptake upon energization with ascorbate and N, N, N′, N′-tetramethyl- p-phenylenediamine. The presence of Na + caused a pronounced reduction in the Km for α-aminoisobutyric acid in wild-type but not KM23 vesicles; the maximum velocity ( V) was unaffected in vesicles from both strains. Passive efflux and exchange of α-aminoisobutyric acid from wild-type vesicles were Na +-dependent and occurred at comparable rates (with efflux slightly faster than exchange). This latter observation suggests that the return of the unloaded carrier to the inner surface is not rate-limiting for efflux. The rates of α-aminoisobutyric acid efflux and exchange were also comparable in KM23 vesicles, but were Na +-independent. Furthermore, in vesicles from the two strains, both efflux and exchange were inhibited by generation of a transmembrane electrochemical gradient of protons, outside positive. This suggests that the ternary complex between solute, carrier, and coupling ion bears a positive charge in both strains even though the coupling ion is changed. Evidence from experiments with an alkalophilic strain that was deficient in l-methionine transport indicated that the porters, i.e., the solute-translocating elements, used by non-alkalophilic mutants are not genetically distinct from those used by the alkalophilic parent; that is, the change in coupling ion cannot be explained by the expression of a completely new set of Na +-independent, H +-coupled porters upon mutation of B. alcalophilus to non-alkalophily.

Methionine requirement of normal and leukaemic haemopoietic cells in short term cultures

The in vitro l-methionine requirement of bone marrow cells from patients with leukaemia was compared with that from patients with non-malignant diseases by means of incorporation of (methyl 3H) thymidine into acid-insoluble material. After 24 h in a medium in which l-methionine was substituted by l-homocysteine there was a lower incorporation of (methyl 3H) thymidine by leukaemic marrow at either 0.1 mM (normal 84 ± 15; leukaemic 60 ± 11%, p < 0.05) or 0.4 mM l-homocysteine (normal 76 ± 13; leukaemic 58 ± 9%, p > 0.01). The response of bone marrow cells from leukaemic patients in regression was similar to that of normal marrow. Bone marrow cells from non-leukaemic patients required a lower concentration of l-methionine in the culture media for optimal incorporation of (methyl 3H) thymidine than did those from leukaemic patients. Bone marrow cells from leukaemic patients had a decreased maximal initial rate of l-methionine transport ( V max) when compared with marrow cells from normal subjects, though in all cases the Km value for l-methionine transport was lower in leukaemic cells. Saturation studies suggested that the size of the cytoplasmic pool for l-methionine was larger (about two times) in marrow cells from normal subjects. These results suggest a therapeutic strategy based on a reduction of the plasma l-methionine concentration.

Transport of l-methionine in human diploid fibroblast strain WI38

The transport of L-methionine in human diploid fibroblast strain WI38 was investigated. The uptake of l-methionine was measured in sparse cell cultures in a simple balanced salt solution buffered with either Tris·HCl of N-2-hydroxyethylpiperazine- N′-2-ethanesulfonic acid (HEPES). Similar results were obtained with these two buffers. Cultures were allowed to equilibrate with the buffered saline before transport was measured. The presence of glucose in the buffered saline results in a slight reduction in the initial rate of transport for the first 2 h of equilibration in part buffered saline. l-Methionine is actively transported in WI38 by saturable, chemically specific mechanisms which are temperature, pH and, in part, Na + dependent, and are reactive with both l- and d-stereoisomers. Kinetic analysis of initial rates of transport at substrate concentrations from 0.0005 to 100 mM indicated the presence of two saturable transport systems. System 1 has an apparent K M of 21.7 μM and an apparent V of 3.57 nmol/mg per min. System 2 has an apparent K M of 547 μM and an apparent V of 22.6 nmol/mg per min. Kinetic analysis of initial rates of transport in Na +- free media or after treatment with ouabain suggested that system 1 is Na + independent and that system 2 is Na + dependent. Preloading of cells with unlabeled l-methionine greatly increases the initial rate of uptake. Efflux of transported methionine is temperature dependent, and is greatly increased in the presence of unlabeled l- or d-methionine or l-phenylalanine, but not in the presence of l-arginine. l-Methionine transport is strongly inhibited by other neutral amino acids, and is very weakly inhibited by dibasic amino acids, dicarboxylic amino acids, proline or glycine.

Mechanism of chlorpromazine action on rat intestinal transport processes

In a previous study, it was observed that chlorpromazine (CPZ) inhibited the transport of l-methionine in the rat intestine. The present experiments were designed to delineate the possible mechanism of this effect. Pre-incubation studies with CPZ demonstrated that the tissue concentration of CPZ was much more important than the medium concentration in inhibiting l-methionine transport. This seemed to favor an ‘indirect’ or ‘within-cell’ effect (metabolic inhibition or inhibition of sodium and potassium ion-stimulated adenosine triphosphatase [(Na + + K +)-ATPase]) over a ‘direct’ or ‘surface’ effect (an effect on passive membrane permeability or at the carrier level). CPZ did not affect diffusional entry of l-methionine at the intestinal brush border. The inhibition of non-diffusional mucosal transport of l-methionine by CPZ was not competitive; CPZ inhibited d-galactose transport in the same time-dependent manner as it inhibited l-methionine transport. No CPZ effect on carrier-mediated diffusion of d-xylose was observed. Thus, CPZ effect on active transport processes in the intestine seems to be mediated predominantly within the cell; from the present study it is not clear whether this effect is through inhibition of metabolic energy or inhibition of (Na + + K +)-ATPase.

Multiple transport systems for branched-chain amino acids as studied by mutants of Salmonella typhimurium.

The transport systems for entry of branched-chain amino acids in the wild-type strain of Salmonella typhimurium LT2 have been separated into two components by detailed kinetic analysis; the Km values for L-isoleucine are 1μM and 12μM, for L-valine 1.2μM and 29μM, and for L-leucine 0.3μM and 11μM, respectively. Two mutant strains, KA261 (ilvC8, ilvT3, ilv-261) and KA265 (ilvC8, ilvT3, ilv-265), having multiple lesions in the transport systems have been isolated from strain CE4 (ilvC8, ilvT3), defective in the low-affinity-(1) system. Kinetic analysis of the transport for L-isoleucine shows that KA266 (ilvT3, ilv-261) and KA267 (ilvT3, ilv-265), which are ilvC8+ transductants of KA261 and KA265, respectively, are defective in both the high-affinity and the low-affinity-(1) systems, but retain the third (low-affinity-(2)) system (Km: 88 to 118μM) which is not detected in the wild-type and KA203 (ilvT3) strains. Strains KA261 and KA265 require glycyl-L-isoleucine, glycyl-L-valine, glycyl-L-leucine and Ca-pantothenate for growth, whereas the parental strain CE4 requires L-isoleucine and glycyl-L-valine. Glycyl-L-isoleucine and glycyl-L-valine can be replaced by high concentration of L-isoleucine and L-valine in strains, CE4, KA261 and KA265. Transport of L-methionine, L-proline and L-threonine remains normal in the KA261 and KA265 mutants.

Effect of chlorpromazine on l-methionine transport in the rat intestine

The inhibitory effect of chlorpromazine on l-methionine transport was studied in the everted sac preparation of the rat intestine. Chlorpromazine inhibits only the sodium-dependent active component of l-methionine transport, without affecting the sodium-independent diffusion component. Inhibition of methionine transport by chlorpromazine is time dependent and concentration dependent. Half-maximal inhibition was observed at 5.2 × 10 −4 M chlorpromazine in the medium. No significant metabolism of chlorpromazine was observed during the experiment, suggesting that the unchanged chlorpromazine was the active inhibitor. Studies on a few chlorpromazine metabolites showed that demethylation in the aminopropyl side chain or ring hydroxylation at the 7-position in the chlorpromazine molecule did not affect the inhibitory activity on methionine transport, while oxidation at the sulfur atom completely abolished it.

Transport of L-methionine in rat intestine and kidney.

Extract: L-Methionine transport was studied in vitro in rat kidney cortex slices and intestinal segments. In both tissues L-(C14) methionine at concentrations of 0.065 mmol was accumulated against a chemical concentration gradient by processes which obeyed saturation kinetics. In both tissues anaerobic conditions (incubation in 95% N2 and 5% O2 atmosphere) inhibited methionine accumulation (P < 0.001 and P < 0.025, respectively). The processes were sodium dependent. Addition of 0.3 Msucrose produced significant (P < 0.001) inhibition of L-methionine accumulation in the kidney. L-Methionine transport was essentially pH independent. A series of amino acids, including representatives of the neutral, dibasic and imino acid groups at 2.4 DIM concentrations, failed to inhibit methionine transport with the exception that valine proved a potent inhibitor of transport in the intestine (P < 0.01) and ethionine in the kidney (P < 0.025). Methionine inhibited glycine accumulation in the kidney (P < 0.025) and intestine (P < 0.001), but failed to affect lysine transport in either tissue. Speculation: The data presented indicate that methionine uptake in the gut and the kidney has the characteristics of an active transport process which is unaffected by most other amino acids. These findings suggest that methionine is either transported by an independent mechanism and/or by a transport system which is shared with the other neutral amino acids but for which it has a much higher affinity.

Transport systems for L-methionine in Escherichia coli.

The transport of l-methionine into cells of Escherichia coli is described. The transported methionine undergoes a fairly rapid conversion to other cellular metabolites, primarily polyamines, but within 2 min, at least 65% of the intracellular label remains as methionine. The uptake process, which is temperature dependent, allows the accumulation of methionine against a concentration gradient. The dependence of the initial rate of uptake on the extracellular substrate concentration indicates the presence of multiple transport systems, whose kinetic behavior can be approximated by two systems, one with K(T) = 7.5 x 10(-8) M and V(T) = 200 pmol per muliter of cell water per min, and the other with K(T) = 40 x 10(-6) M and V(T) = 1,550 pmol per muliter per min. Both systems are highly specific for l-methionine. Methionine derivatives substituted on the amino or carboxyl group were somewhat effective as inhibitors of l-methionine uptake, whereas d-methionine, ethionine, or other amino acids were poorly inhibitory, if at all. The uptake process is dependent on metabolic energy, but apparently this energy can be derived either from glycolysis or from oxidative phosphorylation. Efflux of methionine was demonstrable, and both the influx and efflux process were susceptible to inhibition by N-ethylmaleimide. The intracellular pool of l-methionine was estimated to be 0.1 to 0.3 mM. The transport in two mutant strains defective in methionine uptake (metD and metP) showed that the high-affinity transport system was lost, whereas the low-affinity system remained more or less intact.

Effects of neomycin and penicillin administration on mucosal proliferation of the mouse small intestine. With morphological and functional correlations.

The effects of an oral neomycin and penicillin regimen on intestinal bacteriology and on morphology and function of the small intestine of mice were investigated. Quantitative and qualitative stool cultures on selective media of the treated animals revealed only growth of yeast organisms. The treated animals developed enlargement of the ceca with fluid contents and watery stools, resembling characteristics of germfree animals. Radioautography with tritiated thymidine revealed an increased epithelial cell migration rate in the mice treated with the antibiotics for 3 to 5 wk. A slight increase in villus height was also noted. The treated male mice showed greater variance than the treated females in epithelial cell migration rates. Histochemical staining reactions showed a decrease in nonspecific esterase and in NADH dehydrogenase activity in the proximal gut of the antibiotic animals. Stains of distal gut and those for acid and alkaline phosphatase, NADPH dehydrogenase, lactic dehydrogenase, and succinic dehydrogenase were similar to the controls. A slight increase in sucrase activity and a slight decrease in lactase activity in the antibiotic animals was observed in contrast to control animals. Germfree mice, however, had greater sucrase and lactase activity. Transport of L-methionine was slightly reduced in the distal segment of the treated animals. Since the direction of these changes is away from the intestinal state observed in germfree animals, they are probably the result of the direct action of the antibiotics on the gut.

Inhibitory Effects of Alcohol on Intestinal Amino Acid Transport in vivo and in vitro

The effect of ethanol on the active transport of amino acids in the rat intestine was studied in vitro and in vivo. It was shown that 0.5 and 2.0% ethanol significantly inhibited the active transport of L-phenylalanine in everted sacs in vitro by 60 and 84%, respectively. The latter concentration completely abolished the active transport of L-methionine. At this concentration ethanol reduced tissue respiration by only 19%. In studies in vivo, 250 mg alcohol/100 g body weight given by stomach tube significantly inhibited the intestinal absorption of L-phenylalanine by about 50% but did not modify absorption of D-phenylalanine, an amino acid that is not actively transported. The possible significance of these findings is discussed in relation to the nutritional deficiencies and fatty liver that occur in chronic alcoholism.