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PubMed1987

Purification and characterization of ribitol-5-phosphate and xylitol-5-phosphate dehydrogenases from strains of Lactobacillus casei.

A simple three-step procedure is described which yields electrophoretically homogeneous preparations of ribitol-5-phosphate dehydrogenase and xylitol-5-phosphate dehydrogenase. The former enzyme is a 115,000-molecular-weight protein composed of two subunits of identical size and is specific for its substrate, ribitol. The xylitol-5-phosphate dehydrogenase exists as a tetrameric protein with a molecular weight of 180,000; this enzyme oxidizes the phosphate esters of both xylitol and D-arabitol. Characterization of the physical, kinetic, and immunological properties of the two enzymes suggests that the functionally similar enzymes may not be structurally related.

D-Xylulose Reductase↗
PubMed1998

Determination of folate patterns in mouse plasma, erythrocytes, and embryos by HPLC coupled with a microbiological assay.

Folates are important cofactors in one-carbon metabolism. Disturbances in folate homeostasis and metabolism may be related to an increased risk of cardiovascular disease and carcinogenesis and may lead to congenital malformations, namely neural tube defects. Determination of these compounds in biological samples is often a problem due to the existence of numerous folate metabolites, their relative instability, and the low contents in serum and most tissues. As existing methods have distinct limitations, we developed a method, which facilitates the separation as well as the sensitive detection of eight folates by coupling HPLC with a microbiological assay. After a simple sample preparation, including deproteinization and enzymatic hydrolysis of folate polyglutamates, extracts were chromatographed, fractions were collected on microtiter plates, and folates were quantitated using the Lactobacillus casei assay. The raw data were processed using a computing system after reconstructing the HPLC chromatogram with the bacterial growth data. Using the described method, the eight physiologically occurring folate monoglutamates could be simultaneously determined. The detection limits were 2-20 fmol per injection. The application of the method was demonstrated with the analysis of the folate pattern in milligram or sub-milligram quantities of plasma, erythrocyte, and embryos of pregnant mice during organogenesis.

Animals↗
PubMed1992

Purification and crystallization of Lactobacillus casei folylpolyglutamate synthetase expressed in Escherichia coli.

Folylpolyglutamate synthetase (FPGS) from Lactobacillus casei has been crystallized with polyethylene glycol and acetate buffer at pH 5.0. The enzyme was obtained from Escherichia coli strain SF4 harboring the L. casei FPGS chromosomal gene on a pEMBL vector (pGT3-8.1). Crystals of the enzyme were obtained which diffract to 2.6 A resolution. The crystals are monoclinic, space group P2(1), with unit cell dimensions of a = 54.07 A, b = 45.83 A, c = 84.37 A and beta = 107.92 degrees. A unit cell contains one molecule of the 43,000 Da enzyme per asymmetric unit. A complete X-ray data set on the native crystals has been collected.

Escherichia coli↗
PubMed1991

Immunoadjuvant activity of oral Lactobacillus casei: influence of dose on the secretory immune response and protective capacity in intestinal infections.

Lactobacilli, often used as effectors of host functions, could play an important role in maintaining human health by controlling other intestinal microorganisms capable of producing harmful effects. Using an experimental model, we studied the effect of different oral doses of Lactobacillus casei on the secretory IgA response and the protective capacity of the microorganism in preventing intestinal infections. The optimization of the protective dose of Lb. casei by previous feeding and the use of the lactobacillus as an immunological way to control enteric infections were investigated. We found that conventional mice were protected against infection with Salmonella typhimurium and Escherichia coli by previous feeding for 2 consecutive days with a daily Lb. casei dose of 1.2 x 10(9) cfu/mouse. Previous feeding for 7 d proved less effective, and feeding for 5 d afforded no protection at all. We were also able to demonstrate that the protective effect of Lb. casei against Sal. typhimurium and Esch. coli was connected mainly with the high level of IgA antipathogen antibodies present in intestinal secretions. beta-Glucuronidase (EC 3.2.1.31) and beta-galactosidase (EC 3.2.1.23) activities, measured both in the intestinal fluid and histological samples, showed a marked increase in intestinal inflammatory response on day 5 of feeding. These results show that Lb. casei plays an important role in the prevention of enteric infections, a low dose being enough for protection against intestinal infections by increasing IgA secretion into the intestinal lumen, thus providing adequate defences for the mucosal surface. A previously administered dose of this magnitude could therefore be used as an oral adjuvant in preventing enteric infections.

Adjuvants, Immunologic↗
PubMed1983

5-p-benzoquinonyl-2'-deoxyuridine 5'-phosphate: a possible mechanism-based inhibitor of thymidylate synthetase.

The title compound (1), designed as a suicide inhibitor of thymidylate synthetase, can be prepared by silver(II) oxide oxidative demethylation of the corresponding dimethoxyphenyl derivative. Compound 1 shows time-dependent inactivation of thymidylate synthetase (methotrexate-resistant Lactobacillus casei) and saturation kinetics, and the inactivation is responsive to substrate protection. The inactivation is not reversible on prolonged dialysis in attempts to remove the inhibitor. The rate constant for inactivation is 0.065 s-1; the dissociation constant (Ki) was estimated to be 2 microM. The kinetics of this inactivation are compared to inactivation caused by model thiol reagents that do not have affinity for the active site of thymidylate synthetase.

Deoxyuracil Nucleotides↗
PubMed1998

Quantitative structure-activity relationships of 2, 4-diamino-5-(2-X-benzyl)pyrimidines versus bacterial and avian dihydrofolate reductase.

Quantitative structure-activity relationships (QSAR) have been formulated for a set of 15 2,4-diamino-5-(2-X-benzyl)pyrimidines versus dihydrofolate reductase from Lactobacillus casei and chicken liver. QSARs were also developed for comprehensive data sets containing mono-, di-, and trisubstituted benzyl derivatives. Particular emphasis was placed on the role played by ortho substituents in the overall binding process and subsequent inhibition of the catalytic process in both the prokaryotic and eucaryotic DHFRs. Comparisons between the two QSARs reveal subtle differences at specific positions which can be optimized to design more selective antibacterial agents.

Animals↗
PubMed1994

Mixed lactic acid-alcoholic fermentation by Saccharomyces cerevisiae expressing the Lactobacillus casei L(+)-LDH.

We describe the construction of a Saccharomyces cerevisiae strain expressing the gene encoding the L(+)-lactate dehydrogenase [L(+)-LDH)] from Lactobacillus casei. The recombinant strain is able to perform a mixed lactic acid-alcoholic fermentation. Yeast cells expressing the L(+)-LDH gene from the yeast alcohol dehydrogenase (ADH1) promoter on a multicopy plasmid simultaneously convert glucose to both ethanol and lactate, with up to 20% of the glucose transformed into L(+)-lactate. Such strains may be used in every field where both biological acidification and alcoholic fermentation are required.

Alcohol Dehydrogenase↗
PubMed1983

Increased urinary excretion and prolonged turnover time of folic acid during ethanol ingestion.

The effect of ethanol on the kinetics of folic acid urinary excretion was studied in five chronic alcoholic patients who were maintained in a folate replete state. For 17 days, 95% ethanol was administered orally at 2-h intervals (X9). The folate pool(s) were labeled with the tritium labeled pteroyl glutamic acid at the start of the period of ethanol ingestion and at the start of a control abstinent period. The 24-h urine volume did not change with consumption of ethanol. Total urinary folate excretion was significantly greater by 20 to 40% in four of five patients during the ethanol administration period (p less than 0.05). The fall-off in urinary radioactivity corrected for total folate excretion (dpm/microgram Lactobacillus casei folate) was resolved into a biexponential decay with two distinct slopes. Ethanol caused the slope of the second component to flatten significantly with mean t1/2 of 63.7 days on ethanol versus 9.6 days off (p less than 0.05). These kinetic results are consistent with the known interference by ethanol of folate utilization in a functional tissue pool.

Adult↗
PubMed2006

Lactobacillus casei metabolic potential to utilize citrate as an energy source in ripening cheese: a bioinformatics approach.

AIMS: To identify potential pathways for citrate catabolism by Lactobacillus casei under conditions similar to ripening cheese. METHODS AND RESULTS: A putative citric acid cycle (PCAC) for Lact. casei was generated utilizing the genome sequence, and metabolic flux analyses. Although it was possible to construct a unique PCAC for Lact. casei, its full functionality was unknown. Therefore, the Lact. casei PCAC was evaluated utilizing end-product analyses of citric acid catabolism during growth in modified chemically defined media (mCDM), and Cheddar cheese extract (CCE). Results suggest that under energy source excess and limitation in mCDM this micro-organism produces mainly L-lactic acid and acetic acid, respectively. Both organic acids were produced in CCE. Additional end products include D-lactic acid, acetoin, formic acid, ethanol, and diacetyl. Production of succinic acid, malic acid, and butanendiol was not observed. CONCLUSIONS: Under conditions similar to those present in ripening cheese, citric acid is converted to acetic acid, L/D-lactic acid, acetoin, diacetyl, ethanol, and formic acid. The PCAC suggests that conversion of the citric acid-derived pyruvic acid into acetic acid, instead of lactic acid, may yield two ATPs per molecule of citric acid. Functionality of the PCAC reductive route was not observed. SIGNIFICANCE AND IMPACT OF THE STUDY: This research describes a unique PCAC for Lact. casei. Additionally, it describes the citric acid catabolism end product by this nonstarter lactic acid bacteria during growth, and under conditions similar to those present in ripening cheese. It provides insights on pathways preferably utilized to derive energy in the presence of limiting carbohydrates by this micro-organism.

Acetic Acid↗
PubMed2002

Inducible promoter-repressor system from the Lactobacillus casei phage phiFSW.

With the aim to extend the presently available inducible gene expression systems for lactobacilli, we have isolated a thermoinducible promoter-repressor cassette from the temperate Lactobacillus casei phage phiFSW-TI in Escherichia coli. The phiFSW-TI promoter fragment was abutted to the plasmid-borne promoterless beta-glucuronidase (gusA) reporter gene and shown to direct its transcription in L. casei. In addition, the functionality of the promoter-repressor system was verified in the L. casei phiFSW-TI lysogen by showing that the gusA reporter gene, controlled by the isolated phiFSW-TI promoter, was repressed at 28 degrees C and expressed at 42 degrees C. Moreover, a homology search revealed that the C terminus of the isolated phiFSW repressor shows a high similarity to the small mutS-related domain of the MutS2 protein family that is unprecedented for phage-encoded repressor proteins.

Amino Acid Sequence↗
PubMed2004

Isolation and characterization of lactobacilli from some traditional fermented foods and evaluation of the bacteriocins.

Lactic acid bacteria (LAB) commonly used in food as starter cultures are known to produce antimicrobial substances such as bacteriocins and have great potential as food biopreservatives. LAB isolated from traditional fermented foods (appam batter and pickles) were screened for bacteriocin production. Two lactobacilli, LABB and LABP (one from each source) producing bacteriocins were characterized. Both the bacilli were homo-fermentative, catalase negative and micro-aerophilic in nature. LABB was found to be a thermobacterium growing at 45 degrees C while LABP was a streptobacterium growing at 15 degrees C. Both were able to grow at pH 4.5-8.6 but were intolerant to high salt concentration. They failed to produce gas from glucose as well as ammonia from arginine. Among the sugars examined they could not ferment arabinose, raffinose, rhamnose or xylose. Additionally, LABB could not ferment esculin, gluconate or mannose. LABB is identified as Lactobacillus acidophilus while LABP as Lb. casei. Their bacteriocins showed a broad inhibitory spectrum against the indicator organisms tested. They were active below pH 8.0 and after autoclaving as well. There was a complete loss of activity when treated with proteolytic enzymes such as trypsin indicating the proteinaceous nature of the active molecules. SDS-PAGE of partially purified bacteriocins indicated the molecular mass of the bacteriocin as 3.8 and 4.5 kDa for LABB and LABP respectively.

Bacteria↗
PubMed1993

Caseicin, a bacteriocin from Lactobacillus casei.

The intracellular bacteriocin caseicin 80 was purified from cell extracts of Lactobacillus casei strain B80. It is a thermolabile protein with an apparent molar mass of 42 kDa. As no plasmids were observed in the bacteriocinogenic strain it is assumed that caseicin is encoded by the bacterial chromosome. Using 14C-labelled precursors it was found that biosynthesis of DNA and proteins was influenced by caseicin but this inhibition is probably not the primary effect. The incorporation of fructose but not of glucose into cellular material was inhibited by caseicin.

Bacterial Proteins↗
PubMed1984

Riboflavin analogs utilized for metabolism by a Lactobacillus casei mutant.

Several new halogen-containing riboflavin analogs that are antagonists for the stock strain of Lactobacillus casei have been found to be utilized by a mutant strain of the microorganism for growth and acid production. Riboflavin analogs consisting of all combinations of a single aliphatic group (methyl or ethyl) and a single halogen group (chloro or bromo) in positions 7 and 8 of the isoalloxazine nucleus possess this property. The analog devoid of substituents in positions 7 and 8 and the analog possessing a methyl group in position 7 and a cysteinyl group in position 8, were inert for both strains of the microorganism.

Acids↗
PubMed2003

Probiotics induce resistance to enteropathogens in a re-nourished mouse model.

Nutritional deficiency is commonly associated with impaired immune response and the relation between infection and malnutrition is synergic. Probiotics, especially lactic acid bacteria are immunomodulatory. The aim was to determine whether optimal doses of Lactobacillus casei and yogurt, used as adjuvants in a re-nutrition diet in a non-severe malnutrition experimental model, protect against Salmonella typhimurium and Escherichia coli. Groups of malnourished mice were used, which were re-nourished with milk for 7 or 14 d. After that, both groups of mice received the optimal doses of Lb. casei and yogurt supplements. We measured IgA+ and IgG+-B cells and phenotypic markers of T lymphocytes; CD3+, CD4+ and CD8+ cells. We also determined alphabeta and gammabeta T cell receptor (TCR). The ability to protect against Sal. typhimurium and Esch. coli infections and specific S-IgA were assessed. Probiotics complemented the effects of the re-nutrition diet, by stimulating recuperation of the activity of immune cells that improved protection against infections.

Animals↗
PubMed1977

Methotrexate analogues. 9. Synthesis and biological properties of some 8-alkyl-7,8-dihydro analogues.

A series of 8-alkyl-7,8,-dihydromethotrexate analogues was prepared by direct alkylation of 7,8-dihydromethotrexate, after pilot studies were performed with simpler pteridines. These compounds are tested for in vitro inhibitory activity against Lactobacillus casei and as enzyme inhibitors against dihydrofolate reductase and thymidylate synthetase derived from this organism. All of the analogues were less inhibitory toward dihydrofolate reductase than was methotrexate but were more inhibitory toward thymidylate synthetase. The analogues were also evaluated for in vitro inhibitory activity against the CCRF-CEM human lymphoblastic leukemia cells. In vivo against the L-1210 leukemia in mice, several of the analogues exhibited some antileukemic activity.

Animals↗
PubMed1999

Synthesis of classical and a nonclassical 2-amino-4-oxo-6-methyl-5-substituted pyrrolo[2,3-d]pyrimidine antifolate inhibitors of thymidylate synthase.

Compounds 2-5 were designed as potential antifolate nonpolyglutamatable inhibitors of thymidylate synthase (TS). These analogues are structurally related to 2-amino-4-oxo-5-substituted quinazolines and 2-amino-4-oxo-5-substituted pyrrolo[2, 3-d]pyrimidines which have shown excellent inhibition of TS and, for the quinazoline, significant promise as clinically useful antitumor agents. Compounds 2-4 were synthesized by appropriate amine exchange reactions on pivaloyl-protected 5-dimethylaminomethyl-substituted 6-methyl pyrrolo[2,3-d]pyrimidine 7 which in turn was obtained from the Mannich reaction of pivaloylated-6-methyl pyrrolo[2, 3-d]pyrimidine 6. In instances where the amine exchange reaction was sluggish, the Mannich base was quaternized with methyl iodide which afforded much faster exchange reaction with improved yields. For compound 5, 4-mercaptopyridine was used as the nucleophile and reacted with 7. The analogues 2-4 inhibited Lactobacillus casei (lc) TS and recombinant human (h) TS with IC50 in the 10(-4) to 10(-5) M range. Compound 5 inhibited lcTS and hTS 20% at 26 and 25 microM, respectively. In addition, compound 5 inhibited the growth of Pneumocystis carinii and Toxoplasma gondii cells in culture by 76% at 32 x 10(-6) M and 50% at 831 x 10(-6) M, respectively.

Animals↗
PubMed1999

The effect of orally administered viable probiotic and dairy lactobacilli on mouse lymphocyte proliferation.

Four common Lactobacillus strains were screened for their effects on proliferation of mouse splenic lymphocytes. Mice received perorally 10(9) viable bacteria kg(-1) body weight for 7 days. Lactobacillus acidophilus treatment enhanced ex vivo basal proliferation (by 43%) and B-cell response at suboptimal and optimal concentrations of lipopolysaccharide (LPS) (by 27-28%). Conversely, Lactobacillus casei, Lactobacillus gasseri and Lactobacillus rhamnosus inhibited both basal proliferation (by 14-51%) and mitogen-stimulated lymphoproliferation, particularly at supra-optimal concentrations of concanavalin A (by 43-68%) and LPS (by 23-62%). Therefore, these Lactobacillus strains demonstrate strain-specific effects on B- and T-cells and may also alter the splenocyte sensitivity to the cytotoxic effects of mitogens.

Adjuvants, Immunologic↗
PubMed1999

Clones of Lactobacillus casei and Torulopsis glabrata associated with recurrent abdominal wall abscess.

Infectious disease caused by Lactobacillus sp has not been previously reported in Taiwan. We present a case of recurrent abdominal wall abscess in a chronically ill 36-year-old woman, and review the literature on Lactobacillus infection. Five isolates of L. casei were recovered from blood and pus samples, and two isolates of Torulopsis glabrata were isolated from two blood specimens 3 months apart. Two clones of L. casei and T. glabrata were identified by means of antibiotyping with the E test and molecular methods. The abscess was surgically removed because of poor response to 7 months of antimicrobial therapy for the second infectious episode. Recurrent Lactobacillus infection can occur in chronically ill or immunosuppressed patients. Treatment of these infections may require a longer duration of antibiotic therapy, or surgical intervention.

Abdominal Abscess↗