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SUMMARY: The uptake and synthesis of folate polyglutamates from [3H]pteroylglutamic acid and [14C]p-aminobenzoic acid was studied in Lactobacillus casei, Streptococcus faecalis, L. plantarum and Escherichia coli. The analytical techniques were based on oxidative or hydrolytic degradation of the folate compounds followed by chromatographic separation of the resulting mixtures of pteroylglutamate derivatives or p-aminobenzoylpoly-γ-l-glutamates and comparison with appropriate synthetic standards. All four organisms synthesized numerous polyglutamate derivatives from the precursors provided. The large majority (85 to 95%) of folates of lactobacilli were polyglutamates with more than seven glutamyl residues; L. casei formed several such unidentified long-chain components. In contrast, E. coli and particularly S. faecalis synthesized mainly polyglutamates with seven or less glutamate residues; tetra-, hexa- and heptaglutamates were tentatively identified in E. coli, and tri-, tetra- and pentaglutamates in S. faecalis. While L. casei appeared to synthesize a mixture of formyl- and methyltetrahydro derivatives, formyltetrahydro derivatives predominated in E. coli and S. faecalis.

During growth in the presence of 10 ng of [3H]pteroylglutamic acid (PteGlu) per ml of medium, Lactobacillus casei (ATCC 7469) incorporated 80 to 90% and Streptococcus faecalis (ATCC 8043) 40 to 60% of the added radioactivity. Folate compounds in L. casei were identified as reduced derivatives of PteGlu9 (∼19%), of PteGlu8 (∼42%), of PteGlu7 (∼14%), and PteGlu6 (∼8%); in S. faecalis as reduced derivatives of PteGlu5 (∼24%), of PteGlu4 (∼61%), and PteGlu3 (∼4%). After treatment of cell extracts with hog kidney conjugase ∼39% of folate compounds in L. casei were converted to tetrahydrofolic acid (H4PteGlu), ∼26% to 5-methyl-H4PteGlu and ∼10% to 5- and 10-formyl-H4PteGlu; in the case of S. faecalis ∼25% were converted to H4PteGlu and ∼45% to 5- and 10-formyl-H4PteGlu. No 5-methyl-H4PteGlu derivatives could be found in S. faecalis. Simple methods for the preparation and separation of the biologically active forms of 5-methyl-[3H]H4PteGlu and 5-formyl-[3H]H4PteGlun (n = 1 to 9 glutamic acid residues) with a high specific radioactivity are described. Growth of L. casei in the presence of 100 ng of PteGlu-[U-14C]Glu-Glu or 100 ng of PteGlu3-[U-14C]Glu-Glu per ml of medium was the same as in the presence of 100 ng of PteGlu per ml. PteGlu5 (100 ng per ml of medium) did not inhibit the uptake of [3H]PteGlu (0.3 ng per ml of medium), in contrast to PteGlu3 (100 ng per ml) and PteGlu (100 ng per ml). L. casei converted PteGlu-[U-14C]Glu and PteGlu3-[U-14C]Glu-Glu to the same metabolites as [3H]PteGlu without loss of the 14C label.

Diethyl pyrocarbonate (DEPC) inhibited the intracellular growth of Lactobacillus casei phage J1. At bacteriostatic concentrations of DEPC (0.5-8mM) growth inhibition of the phage was reversible; after the decomposition of DEPC into ineffective components the phage burst began to occur with a prolonged latent period. At bactericidal concentrations of DEPC (10mM or greater) growth inhibition of the phage was completed and irreversible; the number of infective center rapidly decreased and, thereafter, did not increase.DEPC (10mM) blocked the lysis of phage-infected cells. Artificial lysis of the lysis-inhibited cells revealed no intracellular mature phage particles. DEPC blocked an early stage(s) of phage growth, resulting in an inhibition of the synthesis of phage structural proteins, including antigenic materials having serum-blocking power, and phage DNA. However, DEPC seemed not to react with the injected phage DNA. The addition of DEPC at any time during the latent period also produced an immediate inhibition of the syntheses of phage protein and phage DNA and the cellular lysis, even after these had been initiated. The results are discussed in relation to the control of phage infection in dairy fermentation.

The synthesis of polyribitol phosphate by polyribitol phosphate polymerase from Staphylococcus aureus H proceeds by a single chain mechanism so that the enzyme completes a chain of approximately 30 units linked to lipoteichoic acid carrier, before starting a new chain. The lipoteichoic acid carrier of Staphylococcus aureus and Bacillus subtilis are interchangeable and can be used both for the synthesis of polyglycerolphosphate or polyribitol phosphate. Both of these polymers appear to be attached to the same site on the lipoteichoic acid carrier. Lipoteichoic acid carrier, active with the S. aureus polyribitolphosphate polymerase can be extracted from Bacillus licheniformis, Lactobacillus casei, Lactobacillus plantarum, Streptococcus faecalis, and Mycobacterium phlei but not from a variety of other microorganisms. No evidence has been found for the involvement of an additional carrier such as undecaprenol phosphate in polyribitol phosphate synthesis.

(l)-Tetrahydropteroyltriglutamate and (l)-tetrahydropteroylhexaglutamate were prepared and tested as substrates for thymidylate synthetase (EC 2.1.1.6) (methylenetetrahydrofolate:deoxyuridylate C-methyltransferase) from Lactobacillus casei. Both tetrahydropteroylpolyglutamates were more effective substrates than (l)-tetrahydropteroylglutamate, enhancing the reaction rate 3-fold when compared at 10-5 m. Pteroylpolyglutamates and their corresponding dihydro and (d)-tetrahydro forms were inhibitors of the enzyme, the inhibitory potency increasing with the number of glutamyl residues. The concentration for 50% inhibition with pteroylglutamate was 1.5 x 10-4 m and for pteroylhexaglutamate 6 x 10-7 m. Inhibition by pteroylhexaglutamate, but not that by pteroylglutamate, was abolished in the presence of 0.4 m NaCl. Inhibition obtained with dihydropteroylhexaglutamate and dihydropteroyltriglutamate (50% at 3.2 x 10-6 m) is sufficient to warrant consideration of these compounds as physiological regulators of thymidylate formation. p-Aminobenzoylhexaglutamate and hexaglutamate did not inhibit thymidylate synthetase at 10-2 m indicating that polyglutamates do not bind to the enzyme in the absence of the pteridine. Dihydropteroyltriglutamate and dihydropteroylhexaglutamate were no more effective than dihydropteroylglutamate as substrates for dihydrofolate reductase (EC 1.5.1.3) (5, 6, 7, 8-tetrahydropteroylglutamate:nicotinamide adenine dinucleotide phosphate oxidoreductase) from L. casei.

In a study of the genetic basis of multiple nutritional requirements in Lactobacillus casei , systematic attempts were made to isolate mutants that can grow in the absence of a specific nutrient required by the parental organism. Such mutants have successfully been isolated with respect to seven of twelve amino acids (aspartic acid, leucine, isoleucine, lysine, methionine, serine, and threonine) and three of four vitamins (pantothenic acid, nicotinic acid, and pyridoxal) tested, after extensive screenings employing various mutagens. Mutants that can grow without tryptophan were not isolated, but those that can grow on anthranilate or indole as well as on tryptophan were obtained at a frequency expected for single-step mutations. Activity of tryptophan synthetase was demonstrated in extracts of these anthranilate-utilizing mutants, but not in the parental strain. These results suggest that the multiple nutritional requirements of L. casei are often, if not always, due to one or a few small lesions such as base substitution mutations rather than large deletions affecting the genes involved in each biosynthetic pathway. The data would also imply that many of the biosynthetic pathways that are not fully functional in L. casei were active at one time and became nonfunctional during evolution of the present species.

A fully automated method for the measurement of serum pyridoxal has been developed. Acid phosphatase was used for dephosphorylation and precipitation of the serum proteins was not required. A chloramphenicol-resistant strain of L. casei was used as the test organism and this removed the need for sterilization. The method gives highly reproducible results, and is suitable for population and institutional studies.

1. [3H]Folic acid infused intravenously into patients with biliary fistulae appeared promptly in bile, coincident with 131I-labelled Rose Bengal injected simultaneously. The radioactivity was distributed among several fractions of biological folates present in bile and was associated with folic acid and with an unidentified folate which chromatographed on DEAE-Sephadex close to 10-formyltetrahydrofolyl monoglutamate. 2. Based on affinity for DEAE-Sephadex and support of growth of Lactobacillus casei, Streptococcus faecalis and Pediococcus cerevisiae, we have tentatively identified some of the folates of human bile as 10-formyltetrahydrofolate, 10-formylfolate, 5-formyltetrahydrofolate, 5-methyltetrahydrofolate and tetrahydrofolate or 5,10-methylene tetrahydrofolate. After infusion of folic acid, the formyltetrahydrofolates increased more rapidly than did 5-methyltetrahydrofolate. 3. The unidentified radioactive folate contained both the pteridine and p-aminobenzoate portions of folate. It appeared not to support growth of the test microorganisms and not to be bound to a protein or a chain of γ-glutamates. It was present in the bile of a dog injected with [3H]folic acid but was absent from extracts of liver. This material may be a transport form of folate or a special modification imposed on folic acid during transport across the liver.

[3H]Methotrexate was metabolized mainly to 4-amino-4-deoxy-10-methylpteroic acid in resting cells of Lactobacillus casei and a small amount of methotrexate remained inside the cells after 24 hours of incubation. Preincubation of L. casei with methotrexate inhibited the incorporation of [3H]folic acid completely and l-5-formyl-[3H]tetrahydrofolic acid partially into pteroyloctaglutamate and increased the amount of pteroyltetraglutamate and lower polyglutamates. In rats, methotrexate was metabolized partially to lower methotrexate polyglutamates; however, its uptake was much lower than folic acid and no pentaglutamate of methotrexate was found. Pretreatment of rats with 100 µg of methotrexate daily for 5 days decreased the uptake of [3H]folic acid into liver considerably (∼20 to ∼8%) and the radioactivity in liver was mainly reduced derivatives of pteroylpentaglutamate. With treatment with 300 µg of methotrexate per day for 6 days, however, not only the uptake of [3H]folic acid decreased to ∼4%, but also pteroylpentaglutamate comprised only ∼50% of total radioactivity in liver and the rest was lower polyglutamate and unmetabolized monoglutamate. Column chromatography with Sephadex G-25 and DEAE-cellulose provides a good method for the identification of methotrexate-related compounds.

Physicochemical studies of the multiple forms of fructose-1,6-diphosphate (FDP) aldolase obtained from Lactobacillus casei showed that these had the same molecular weight of approximately 100,000 daltons. These isozymes appeared to be tetrameric and composed of two types of equal-sized subunits. The amino acid composition of these fractions showed only slight variations, though there were distinct differences with respect to catalytic functions, substrate binding capacity, and free thiol content. The immunological analyses showed that antibody prepared against one of them could cross-react with the other fractions. Three forms could howerver be distinguished on immunoelectrophoresis showing charge differences. The results suggest that these multiple forms of aldolase in L. casei may be conformational isozymes.

An enzyme which possesses lytic activity against cariogenic streptococci was obtained from strains of streptomyces in soil and sewage. The strains of streptomyces which produced a highly active enzyme in the culture supernatant were identified as Streptomyces griseus strain H-402 and Streptomyces gloaisporus strain 1829. The enzyme of S. globisporus strongly lysed the strains of cariogenic streptococci in the tris acid malate-NaOH buffer, pH 7.0.This study is concerned with the estimation of the bacteriolytic activity of the enzyme derived from S. globisporus on cariogenic microorganisms such as the strains of Streptococcus mutans, Lactobacillus casei and Actinomyces viscosus, and also deals with the effect of fluoride ion, sodium N-lauroylsarcosinate and sodium dehydroacetate on the activity of the bacteriolytic enzyme.Twenty seven strains of S. mutans employed in this experiment were all sensitive to the bacteriolytic enzyme. Dergrees of bacteriolysis were compared with each of 5 serological groups of S. mutans. Strains of group a and b were more sensititive to the enzyme than group, c, d and E. Cariogenic L. casei ATCC 4646 and A. viscosus T6 were also extremely sensitive, whereas 4 strains of Streptococcus sanguis were less sensitive to the enzyme.Strains of S. mutans and S. sanguis which showed the least sensitivity to the enzyme were significantly lysed by prolonged incubation with the enzyme.The bacteriolytic activity was apparently enhanced by the presence of 20 μg/ml of lysozyme or 0.3% of sodium N-lauroylsarcosinate. 0.2% of sodium dehydroacetate was also effective in enhanceing of the enzyme activity. The enzyme activity was not influenced by fluoride ions with a concentration of less than 200 ppm, whereas 40% of the enzyme activity was supressed in the presence of fluoride ions with a concentration of more than 400 ppm. The enzyme activity was also supressed by halide ions such as F-, Cl-, Br-, and I-.

The distribution of the endogenous folates and the forms biosynthesized from parenterally administered [3′5′9(n)- 3 H] pteroylglutamate (PteGlu) were examined in monkey tissues by column chromatography and microbiological assay. Tissue extracts were either subjected to “conjugase” (γ-glutamylcarboxy-peptidase) treatment yielding pteroylglutamates or to KMnO 4 H 2 O 2 oxidation yielding p-aminobenzoyl-poly-γ- l -glutamates. Components of these mixtures were then separated by column chromatography with Sephadex QAE-A25 and Whatman DE52 anion exchangers respectively. The liver incorporated the largest amount (26%) of tritium activity. A greater proportion of labelled polyglutamate compounds were found at three days following administration than at one day. The predominant forms observed at three days contained five and six glutamyl residues and substitutions were primarily methyltetrahydro (i.e. 5-CH 3 -H 4 PteGlu 5 ,6 ) with small quantities of 10 and/or 5 formyltetrahydro (10-CHO, 5-HCO-H 4 PteGlu 5 ,6 ). Estimation of endogenous distribution by microbiological assay for pteroyloligoglutamates ( Lactobacillus casei ) and p -aminobenzoate ( Escherichia coli ) indicates that exogenous tritiated compounds are not fully equilibrated with the endogenous pools at periods of up to three days. Polyglutamate biosynthesis occurred more slowly in kidney with unmetabolized [ 3 H] PteGlu still present after one day, but closely resembled liver distribution at three days. The biosynthesis of a complex mixture of labelled polyglutamates has also been demonstrated in intestinal in intestinal mucosal cells.

It has been shown that a bacterolytic enzyme derived from Streptomyces globisoporus strain 1829 was able to lyze rapidly glucose-grown cells of many odontopathic plaque microorganism and that the enzyme inhibited the formation of human dental plaque in vivo. The present report described the effects of the bacteriolytic enzyme on the plaque-like deposits produced on human enamel pieces by cariogenic plaque bacteria cultivated in Brain Heart Infusion broth supplemented with 5% sucrose.The susceptibilities to the bacteriolytic enzyme of the sucrose-grown cells were estimated by following the reduction in optical density at 550nm, of the cell suspensions. The extents of lysis of the bacterial strains tested were in the following order from high to low: Streptococcus mutans BHT>Lactobacillus casei ATCC 4646>Streptococcus sanguis 10557>Actinomyces viscosus 15987 (T9) >>S. mutans AHT≈IB≈P4≈B14≈S. salivarius HHT>>>S. mutans K1R. The sucrose-grown cells of each strain were less sensitive than their glucose-grown cells.All streptococcal strains except S. sanguis formed plaque-like deposits on the enamel pieces after cultivation in the 5% sucrose broth at 37°C for 4 days. The artificial plaque forming ability of strain K1R was show to be the most marked and that of strain BHT the least, and those of the other five strains were intermediate between the two strains. S. sanguis, L. casei and A. viscosus failed to produce “atrificial plaque”, and only a few bacterial cells were scattered on the enamels surface.The microbial deposits were treated at 37°C for 60 min. with the bacteriolytic enzyme, freeze-dried and examined with a scanning electron microscope. The bacterial cells in the plaque were lyzed on the enamel surface in situ and losing their original forms became amorphous masses, membranous strings of fibrils which intersected each other and formed a network or honey-comb and sponge-like caves. The intensity of the morphological changes of a plaque was roughly consistent with the susceptibility to the enzyme of the strain which formed the plaque.

1. Bei drei Stammen vonLactobacillus casei, die auf Apfelsaure als C-Quelle wuchsen, wurde durch Kultur mit Apfelsaure entweder Malatenzym oder durch Kultur mit Apfelsaure und Glucose Malo-Lactat-Enzym induziert. Zwei Stamme vonStreptococcus faecalis bildeten nur Malatenzym,Streptococcus lactis, der Glucose zum Wachstum braucht, nur Malo-Lactat-Enzym. 2. Durch aufeinanderfolgende Induktion wurden Zellen vonLactobacillus casei M40 mit Malatenzym und Malo-Lactat-Enzym erhalten. 3. Malatenzym und Malo-Lactat-Enzym unterscheiden sich im Induktionsverhalten, im pH-Optimum, in der Affinitat zum Substrat, in den Endprodukten und im Molekulargewicht.

The demonstration of partial immunological identity between the fructose diphosphate aldolases of Streptococcus faecalis, Lactobacillus casei, and Pediococcus cerevisiae indicated that the three proteins are structurally related. Although the enzymes differ markedly in molecular weight and electrophoretic mobility, the purified aldolases were shown to be composed of subunits of similar or identical size. The S. faecalis aldolase, which had been characterized previously, is composed of two 28,000 molecular weight subunits as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis while the immunologically cross-reactive aldolases of L. casei and P. cerevisiae are composed of four and six subunits, respectively, with a molecular weight of 29,500 each. L. casei possesses a second fructose diphosphate aldolase which is not antigenically related to the enzymes described above. This enzyme was also purified and found to differ markedly from the immunologically related enzymes. Unlike the latter group of enzymes which appear to be typical class II (metallo) aldolases, the second L. casei aldolase exhibits the characteristics of a class I (Schiff-base) aldolase. The class I enzyme is a dimeric protein with a molecular weight of 82,000.

SummaryA study was made of the utilization of citrate in milk by some lactic acid bacteria. WhenStreptococcus diacetilactis1007 was grown alone or with eitherStreptococcus cremoris924 orLactobacillus caseiB 142/C or with both these latter organisms, > 99% of the milk citrate was utilized within 5 days.L. caseiB 142/C andL. casei/Str. cremorisutilized 57 and 14% of the citrate, respectively. WhenL. caseiC 2 andL. caseiC 5 were grown in milk in whichStr. cremoris924 had been previously grown, 94 and 64%, respectively, of the citrate was utilized after 7 days at 30°C.Cheeses were made using a citrate-fermenting and a non-citrate-fermenting starter and citrate concentrations of the milks, wheys and curds were determined during cheese-making. WithStr. cremoris924, citrate was preferentially retained in the curd at pressing, the concentration in the curd moisture being 2·9 times that in the whey. With the mixed starterStr. cremoris924/Str. diacetilactis1007, the curd at pressing and from the press contained only 27 and 5%, respectively, of the citrate present in theStr. cremoriscurd at these times.Cheeses were made usingStr. cremoris924, combinations ofStr. cremoris/Str. diacetilactis1007, or with δ-gluconic acid lactone instead of starter, with and without the addition ofL. caseiC 5, in order to examine the ability of the latter organism to produce ‘blowing’ in the sense of distension of the Cryovac wrapping of film-wrapped cheeses.L. caseiC 5 neither accelerated the decrease in cheese citrate nor produced blowing of the film-wrapping. Possible reasons for this behaviour are discussed.

Saccharomyces cerevisiae and a riboflavin assay strain of Lactobacillus casei have been propagated anaerobically in mixed culture. Both batch and continuous culture techniques were used. By varying the concentrations of glucose and riboflavin in the growth medium, it was possible to produce symbioses of commensalism + competition, competition, and mutualism + competition. In short, the interaction prevailing is determined by the medium as well as by the genetic characteristics of the organisms. The behavior of the mixed cultures in these situations was predicted from data taken on pure cultures of the organisms.

Bacillus subtilis elaborates a series of PGA-related compounds apparently similar to those synthesized by Lactobacillus plantarum, but in larger quantities.Paper chromatography of the broth, or of concentrates derived by ion exchange and carbon treatment of the broth, reveals the presence of six factors having activity for Lactobacillus casei, Streptococcus faecalis, or Pediococcus cerevisiae.Separation of the six active components by standard isolation techniques permitted the identification of four known folic acid related compounds and of two materials not previously reported and presumed therefore to be novel.The four known compounds are pteroic, folic, folinic, and N10-formylfolic acids. Pteroic and folic acids are not produced per se by B. subtilis but arise as degradation products. Both of the novel compounds are unstable. One of these has an Rfvalue of 0.35 in 5% aqueous Na2HPO4and is active for S. faecalis but not for L. casei. This compound decomposes to yield pteroic acid. The second novel material has an Rfvalue of 0.55 and is active for both S. faecalis and L. casei. Decomposition of this material yields folic acid.

Summary The types and sequence change of lactic acid bacteria in 30 orchardgrass and alfalfa silages have been determined. The 3,142 strains studied were representative of silage quality, different cuttings, and stages of fermentation. Both cocci and lactobacilli were found on the forages and in the silages. It was generally true that better quality silages were obtained when the early bacterial flora consisted predominantly of cocci. Most cocci, except the pediococci, disappeared a few days after ensiling. In the good quality silages, three species of lactic acid bacteria emerged as the dominant population. These included Lactobacillus brevis , Lactobacillus plantarum , and pediococci. L. brevis was usually found in the later stages of fermentation and was more prevalent in the alfalfa silages. In the poor quality silages, four species were found, including the three mentioned above and Lactobacillus casei (variable), an attenuated strain of lactobacilli. The results do not directly explain why the low acid producing strains reached such high numbers in poor quality silages. It is suggested, however, that a series of interacting factors may be responsible for selecting certain organisms. Among these are included entrance of air into the forage mass, increased temperature, and amino acid imbalance. These factors assume an important role in determining the course of the fermentation, especially in the conflict between the lactic acid bacteria and the spore-forming anaerobes.

SummaryTwenty-five strains of lactobacilli isolated from dairy products, includingLactobacillus casei, Lactobacillus plantarumandLactobacillus brevisspecies, were grown in semi-defined media and examined for their ability to ferment citrate and produce formate. Of 7 strains ofL. casei, all utilized citrate to varying extents, as did 9 of 10 strains ofL. plantarumand 3 of 8 strains ofL. brevis.L. caseiproduced 19–35% of the theoretical yield of formate from the citrate utilized,L. plantarum0–11% andL. brevis0–28%. Of 2 strains ofL. caseitested for their abilities to ferment citrate in the presence of 2% lactose, strain C 5 was unaffected by lactose whereas strain C 2 showed a 45% decrease in the citrate utilized. However, lactose used at 2% concentration greatly reduced formate production byL. caseiC 5, which produced 62% of the theoretical yield of formate from citrate after 35 days in the absence of lactose and only 8% in its presence. The yield of diacetyl produced byL. caseiC 5 from citrate plus lactose (both at 2% concentration) was 145 times that produced from citrate alone, and 800 times that from lactose alone.

The increase in Lactobacillus casei active compounds in serum was measured after oral administration of 0.68 μmole (300 μg) of the various pteroylmonoglutamate compounds and pteroic acid to 21 normal subjects. The increases exhibited were statistically significant as were the quantitative differences between certain of the compounds tested. Compounds showing small increases ( 10 ng per ml) were elicited by 5,10-methenyltetrahydropteroylglutamate, dihydropteroylglutamate, and 10-formyltetrahydropteroylglutamate. Although the subjects varied considerably with regard to their fasting levels of L. casei activity, this had little effect upon the magnitude of the observed increases due to the administered compounds.

SNELL, Strong, and Peterson (1938SNELL, Strong, and Peterson (1939) reported pantothenic acid to be an essential growth factor for Lactobacillus casei. They pointed out, in making these reports, that an assay had been developed which appeared to be a specific and quantitative method for determining pantothenic acid and which might prove useful in view of the growing biological importance of this substance. Except for certain essential changes, this method is similar to the Snell and Strong (1939) microbiological method for determining riboflavin. In view of the success of Snell, Strong, and Peterson (1939) in using Lactobacillus casei as a means of determining the pantothenic acid content of concentrates of this vitamin, it was decided to adapt the procedure used by them to a study of the pantothenic acid content of feedstuffs, as there was a need for this information in developing diets adequate in this vitamin. While this study was in progress, a . . .

N 4 -( l -Valyl- l -valyl)sulfanilamide was synthesized and found to be an effective inhibitor of growth of Pediococcus cerevisiae , an organism which was not inhibited by sulfanilamide. The experimental data indicated that the mode of action of the compound was different from that of sulfonamides. Similar findings were obtained with the use of Lactobacillus plantarum, Leuconostoc mesenteroides , and Lactobacillus casei .

The turnover of dihydrofolate reductase has been studied in rapidly dividing cells of mouse lymphoma L1210 and Lactobacillus casei. Cells in culture were exposed to [14C]leucine for 24 hr and the subsequent decrease in radioactivity of the enzyme was followed as a function of time. The L1210 enzyme was isolated in pure form by subjecting the cell sonicate to affinity chromatography on amethopterin-Sepharose. The L. casei cells were processed by a multistep procedure which yielded the pure enzyme in both of its principal forms: (I), without TPNH; and (II), containing an equimolal amount of noncovalently bound TPNH. The half-lives (t12>) of dihydrofolate reductase in the amethopterin-sensitive L1210 cells (L1210/S) and in the cells of a partially resistant subline (L1210/R2), characterized by an 8-fold increase in enzyme level, were 18 and 19 hr. When these cells were grown in the presence of sublethal concentrations of amethopterin, t12 values were increased to 39 and 90 hr. These results suggest that the transient increase in dihydrofolate reductase activity, observed when cells are exposed to amethopterin, is due largely to a decreased susceptibility of the enzyme-inhibitor complex to degradation. Bound TPNH also increases the half-life of dihydrofolate reductase as shown by the fact that forms (I) and (II) of the L. casei enzyme had tx12 values of approximately 3 and 9 hr.

Blood and urinary vitamin Be and urinary 4-pyridoxic acid values are reported for 3 rhesus monkeys before and after a 5-day period of pyridoxol-HCl sup plementation. Blood and liver vitamin Be values are given for a group of free-ranging howler monkeys. Microbiological procedures employing Saccharomyces carlsbergensis ATCC 9080, Streptococcus faecium 0 51 NCDO 1229 and Lactobacillus casei ATCC 7469 were used for the vitamin B8 analyses and a microprocedure of the lactone method was used for 4-pyridoxic acid determinations. Large increments of vitamin Be in blood and urine and increased excretion of 4-pyridoxic acid were observed in the rhesus monkeys following supplementation. Of significance was the prolonged elevation of values several days after supplementation had ceased. Blood values for the howler monkeys were higher than those for the rhesus. Differential assay of liver hydrolysates from howler monkeys revealed that most of the vitamin was present in the pyridoxamine form. A lesser amount was in the pyridoxal form and the amount of pyridoxol was negligible.

SUMMARY The influence of 11 anthocyanin pigments on glucose oxidation by resting cells of Escherichia coli K-12, Staphylococcus aureus, Lactobacillus casei 7469, Salmonella enteritidis, S. typhosa, Proteus vulgaris, and Aerobacter aerogenes was determined with a manometric technique. Malvidin, delphinidin, and the monoglucosides of malvidin, pelargonidin, petunidin, and delphinidin generally inhibited glucose oxidation by the test organisms, but in the absence of glucose the organisms oxidized the pigments. The action of the anthocyanins on respiration varied with PH. Apigeninidin chloride, 5.desoxy-apigeninidinchloride- 4′-methyl-ether, and 5-desoxy-3′-methoxy-apigeninidin-chloride-4′-methyl-ether inhibited glucose oxidation by S. aureus. The latter two compounds and 3′-methoxy-apigeninidin-chloride-4′-methyl-ether had a similar effect on E. coli. The depressing effect of glycosidic anthocyanins on the respiration of S. aureus was reversed by the addition of MgCl2.

Summary Lactobacillus casei types were isolated from several samples of Cheddar cheese. Types capable of producing H 2 S in the growth medium were found only in cheese with a flavor score of 39.0 or better. The ability of the isolates to deaminate serine varied, but the strains showing the highest activity were isolated from the better samples of cheese. Some experimental cheese was examined for –SH groups and "free" H 2 S. Both of these values increased as the cheese matured. At 6 months the cheese containing the highest relative concentration of "free" H 2 S received the highest Cheddar flavor intensity score.

1. The composition of guinea-pig liver folates and the biochemical route of formation of liver folates from injected tritium-labelled pteroylglutamic acid (folic acid) have been studied. 2. Endogenous folate was measured by microbiological assay with Lactobacillus casei and Streptococcus faecalis, with and without deconjugation of whole liver pteroylpolyglutamates (conjugated folates). Individual folate compounds were identified by microbiological assay after fractionation of liver folates by DEAE cellulose ion-exchange column chromatography. 3. Liver folate in the guinea-pig consists of about 84–87% reduced pteroylpolyglutamates with more than three glutamate moieties/molecule, about 12–15% reduced pteroyltriglutamates, about 1% reduced pteroyldiglutamates and only traces of reduced pteroylmonoglutamates. 4. About 53% of the liver folate consists of methylated derivatives. 5. Injected pteroylglutamic acid was first rapidly reduced and formylated or methylated. Glutamate moieties were then added, probably singly, to form di-, tri- and poly-glutamates. This was a relatively slow process with a hold-up at the triglutamate stage. 6. The proportion of the labelled pteroylglutamic acid in the polyglutamate form approximated to the proportion of endogenous folates in this form after 3–4 days. 7. The amount of radioactive folate in the liver increased progressively from 1 to 84 h after injection of a standard amount of radioactive pteroylglutamic acid.