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PubMed1978

Dihydrofolate reductase from amethopterin-resistant Lactobacillus casei. Sequences of the cyanogen bromide peptides and complete sequences of the enzyme.

The complete amino acid sequence of dihydrofolate reductase from an amethopterin-resistant strain of Lactobacillus casei has been determined by sequence analysis of peptides produced by cleavage with cyanogen bromide, trypsin, staphylococcal protease, and myxobacter protease. Comparison of this sequence with those of reductases from other bacterial sources shows that the enzymes are homologous. The Lactobacillus casei reductase sequences shows a 29% sequence identity with that of the Escherichia coli enzyme and a 34% identity with the sequence of the enzyme from Streptococcus faecium. The NH2-terminal 68 residues of the L. casei reductase show a 54% sequence identity with that of the enzyme from S. faecium.

Amino Acid Sequence↗
PubMed2003

Use of group-specific and RAPD-PCR analyses for rapid differentiation of Lactobacillus strains from probiotic yogurts.

The increasing interest in probiotic lactobacilli implicates the requirement of techniques that allow a rapid and reliable identification of these organisms. In this study, group-specific PCR and RAPD-PCR analyses were used to identify strains of the Lactobacillus casei and Lactobacillus acidophilus groups most commonly used in probiotic yogurts. Group-specific PCR with primers for the L. casei and L. acidophilus groups, as well as L. gasseri/johnsonii, could differentiate between 20 Lactobacillus strains isolated from probiotic yogurts and assign these into the corresponding groups. For identification of these strains to species or strain level, RAPD profiles of the 20 Lactobacillus strains were compared with 11 reference strains of the L. acidophilus and L. casei group. All except one strain could be attributed unambigously to the species L. acidophilus, L. johnsonii, L. crispatus, L. casei, and L. paracasei. DNA reassociation analysis confirmed the classification resulting from the RAPD-PCR.

DNA, Bacterial↗
PubMed1986

Folate analogues. 26. Syntheses and antifolate activity of 10-substituted derivatives of 5,8-dideazafolic acid and of the poly-gamma-glutamyl metabolites of N10-propargyl-5,8-dideazafolic acid (PDDF).

The poly-gamma-glutamyl derivatives of n10-propargyl-5,8-dideazafolic acid (PDDF) with a chain length of up to five glutamate residues were synthesized from N10-propargyl-5,8-dideazapteroic acid by the solid-phase procedure. These compounds were evaluated for their antifolate activity using folate-requiring microorganisms and intact and permeabilized L1210 cells and as inhibitors of dihydrofolate reductase and thymidylate synthase derived from L. casei. The polyglutamylated derivatives of PDDF (1) were more active than the parent compound in inhibiting the growth of L. casei, thymidylate synthesis in permeabilized L1210 cells, and L. casei thymidylate synthase. Two analogues of 5,8-dideazafolic acid (2 and 3), one with a 2-butyne and another with a cyclopropylmethyl substituent at N10, were also synthesized and evaluated for their antifolate activities using the above-mentioned test systems. They were considerably less active than PDDF or its polyglutamylated derivatives. N10-Propargyl-5,8-dideazapteroyl tri-, tetra-, and pentaglutamates were equipotent with 5-fluorodeoxyuridylate as inhibitors of thymidylate synthesis in permeabilized L1210 cells. The polyglutamyl metabolites of PDDF were shown to be the most potent antifolate inhibitors of L. casei and L1210 thymidylate synthases yet described.

Animals↗
PubMed1983

Folate analogues. 22. Synthesis and biological evaluation of two analogues of dihydrofolic acid possessing a 7,8-dihydro-8-oxapterin ring system.

Two analogues of dihydrofolic acid possessing a 7,8-dihydro-8-oxapterin ring system have been synthesized and evaluated for their antifolate activities. These compounds, N-[(2-amino-4-hydroxy-7,8-dihydro-8-oxa-6-pteridinyl)benzoyl]-L-glutamic acid (3) and N-[[(2-amino-4-hydroxy-7,8-dihydro-8-oxa-6-pteridinyl) methyl]benzoyl]-L-glutamic acid (4), were synthesized by reacting the appropriately substituted alpha-halo ketones with 2,5-diamino-4,6-dihydroxypyrimidine (2). Elaboration of p-carbomethoxybenzaldehyde (5) to p-carbomethoxyphenacyl bromide (7) was accomplished by its oxidation with Jones reagent and the successive treatment of the oxidation product with SOCl2, CH2N2, and HBr. Commercially available p-vinylbenzoic acid (11) was converted to its glutamate conjugate 12 and was further converted to the bromo ketone, diethyl N-[p-(1-bromo-2-oxopropyl)benzoyl]-L-glutamate (17), by a series of reactions involving epoxidation, oxirane ring opening with HBr, Jones oxidation, Zn/HOAc reduction, and successive treatment of the reduction product 16 with SOCl2, CH2N2, and HBr. These bromo ketones, 7 and 17, upon reaction with pyrimidine 2, gave the diethyl esters of the target compounds, which were hydrolyzed to 3 and 4 with NaOH. Compound 4 underwent an interesting acid-catalyzed isomerization where the double bond of 4 was shifted from the 5,6-position to the 6,9-position to give the isomer 19. Both compounds 3 and 4 were inactive against Lactobacillus casei (ATCC 7469) and did not serve as synthetic substrates of L. casei dihydrofolate reductase. Compound 4 showed activity against Streptococcus faecium (ATCC 8043), but 3 was inactive against this organism.

Enterococcus faecalis↗
PubMed1995

Role of histidine 188 in fructose 1,6-bisphosphate- and divalent cation-regulated L-lactate dehydrogenase of Lactobacillus casei.

A fructose 1,6-bisphosphate [Fru(1,6)P2] and divalent cation-regulated allosteric L-lactate dehydrogenase (L-LDH) (EC 1.1.1.27) of Lactobacillus casei was highly produced in Escherichia coli cells, together with its mutant enzyme, in which His-188 was replaced by Asp. Under acidic conditions, the mutant enzyme showed positive allosteric regulations by the substrate pyruvate and its analogues, like the wild-type enzyme, but not by Fru(1,6)P2, which even inhibited the stimulative effects of the alternative activation factors. In addition, Mn2+ ions also showed greatly reduced inhibitory effects on the mutant enzyme. Under neutral conditions, on the other hand, the reaction of the mutant enzyme was slightly enhanced by Fru(1,6)P2, but not further stimulated by additional Mn2+ ions, unlike the case of the wild-type enzyme. These results indicate that His-188 is, though not essential for the regulation by the alternative factors, essential for the cooperative regulation by Fru(1,6)P2 and divalent cations in L. casei L-LDH.

Amino Acids↗
PubMed2006

Oral immunoadjuvant activity of Lactobacillus casei subsp. casei in dextran-fed layer chickens.

We recently reported that synbiotic Lactobacillus casei subsp. casei together with specific substrate dextran elicited an enhancement in humoral immune response against bovine serum albumin (BSA) as a model antigen in BALB/c mice. The present study was designed to evaluate the oral immunoadjuvant effects of the synbiotic in layer chickens. Using a PCR assay, L. casei subsp. casei was detected specifically in the intestinal chyme of chickens (10 d of age, Julia strain) fed ad libitum on a diet supplemented with 75 mg dextran/kg (dextran-supplemented diet, DSD) and administered orally with 10(7) colony-forming units (CFU) L. casei subsp. casei in 0.1 ml PBS with the aid of an intubation needle at 1, 2 and 3 d of age. Furthermore, oral administration of 10(7) CFU L. casei subsp. casei at 1-3 d of age significantly enhanced the production of anti-BSA antibody in DSD-fed chickens (60 d of age) administered orally with 1 mg BSA at 32 and 33 d of age and subcutaneously with 5 microg BSA at 33 d of age. In addition, among bacterial numbers tested, 10(6) CFU L. casei subsp. casei together with dextran induced an effective increase in humoral immune response to mixed inactivated vaccines against Newcastle disease and avian infectious bronchitis, and the treatment may be advantageous in protecting against these infectious diseases in chickens in actual application. These results suggest that dietary supplementation of L. casei subsp. casei with dextran leads to immunomodulation of humoral immune responses.

Adjuvants, Immunologic↗
PubMed2003

Superantigenic activity is responsible for induction of coronary arteritis in mice: an animal model of Kawasaki disease.

Kawasaki disease (KD) is a multisystem vasculitis and the leading cause of acquired heart disease in children of the developed world. Lactobacillus casei cell wall extract-induced coronary arteritis in mice mirrors KD in children. Here, we report that responses to L. casei cell wall extract possess all the hallmarks of a superantigen-mediated response: marked proliferation of naive T cells, non-classical major histocompatibility restriction with a hierarchy in the efficiency of different class II molecules to present this superantigen, a requirement for antigen presentation, but not processing, and stimulation of T cells in a non-clonal, TCR V(beta) chain-dependent fashion. This superantigenic activity directly correlates with the ability to induce coronary arteritis in mice. Taken together, our findings demonstrate that superantigenic activity in L. casei cell wall extract is responsible for induction of coronary artery disease.

Animals↗
PubMed1991

Shuttle plasmid vectors for Lactobacillus casei and Escherichia coli with a minus origin.

Recombinant plasmids which can be used as shuttle vectors between Escherichia coli and the industrially used strains of Lactobacillus casei were constructed. They have replication regions closely related to those of pUB110 and are likely to replicate by a rolling-circle mechanism via a plus-strand-specific DNA intermediate in L. casei. Both orientations of palA from the staphylococcal plasmid pC194 and those of the intergenic region from coliphage M13 are identified as active minus origins in L. casei, in contrast to the pAM alpha 1 delta 1-derived BA3 minus origin which does not function in L. casei. Stability of the plasmids increased in L. casei when one of these two active minus origins was inserted. All the DNA sequences of the constructed vectors were known.

Base Sequence↗
PubMed2000

Gut mucosa morphology and microflora changes in malnourished mice after renutrition with milk and administration of Lactobacillus casei.

Nutrition plays a key role in maintaining the balance of the intestinal microflora. Malnutrition disturbs the ecological barrier and induces histological damage. We evaluated modifications induced by renutrition with nonfat milk (NFM) and Lactobacillus casei administration (for 2 days) on the bacterial gut population and structural and ultrastructural gut modifications in malnourished mice. Balb/c mice suffering from a malnutrition process immediately after weaning (for 21 days) were divided into four groups and were given NFM for 0, 7, 14, and 21 days. Another group was treated in a similar way, but after different periods of NFM administration, mice in this group received L. casei for two consecutive days. All experimental animals were sacrificed by cervical dislocation, and both the microflora and the histological structure of the intestine were studied. In malnourished animals, a decrease in the numbers of Lactobacillus and anaerobic microorganisms was observed, whereas there was an increase in the number of Enterobacteriaceae. In animals treated with NFM and NFM plus L. casei, we could observe an important improvement in the microflora in the small and large intestines but no differences between both treatments. Structural and ultrastructural studies showed a slight improvement 7 days after treatment with NFM, and for 14 and 21 days after renutrition, the mice showed normal intestinal villi, whereas the additional feeding with L. casei for two consecutive days, after different periods of renutrition, yielded an earlier improvement (7 days).

Animals↗
PubMed2000

Multicentric study of the effect of milk fermented by Lactobacillus casei on the incidence of diarrhoea.

The aim of this study was to determine if supplementation of healthy children with milk fermented by yogurt cultures and Lactobacillus casei strain DN-114 001 could affect the incidence of acute diarrhoea when compared with traditional yogurt. The study was a multicentre, randomised, double-blind trial, conducted over four months, on 928 children aged, at inclusion, 6-24 months. The study consisted of two periods: supplementation and observation. Subjects were supplemented daily with 100 g of one of the two dairy products being tested: standard yogurt and milk fermented by yogurt cultures and Lactobacillus casei (10(8) cfu/ml). Frequency or duration of any diarrhoea episode was evaluated. As far as frequency was concerned there was a statistically significant difference between the groups, the incidence of diarrhoea being significantly reduced by supplementation with L. casei fermented milk (15.9%) compared with yogurt (22%) (p = 0.03). These results suggest an additional benefit of L. casei in acute diarrhoea in children compared with standard yogurt.

Acute Disease↗
PubMed1978

Measurement of folates in human plasma and erythrocytes by a radiometric microbiologic method.

A radiometric microbiologic assay for the determination of folic acid in human plasma and red blood cells is described. The assay is based upon the measurement of 14CO2 produced from the metabolism of [1-14C] gluconate by Lactobacillus casei. The 14CO2 evolved was shown to be proportional to the amount of added DL-N-5-methyltetrahydrofolage (DL-N-5-methyl FH4). A total of 26 normal plasma and 57 blood hemolysates were assayed in parallel by this radiometric and the standard (turbidimetric) microbiologic assay. The correlation coefficients for the two assays were r = 0.96 for plasma and r = 0.98 for red-cell folate. Lyophilization of L. casei was found to simplify this radiometric assay by eliminating routine maintenance and culture of this microorganism.

Biological Assay↗
PubMed2006

Experimental study of the effects of probiotics on Cryptosporidium parvum infection in neonatal rats.

To date, there is no efficient treatment for cryptosporidiosis and parasite eradication relies on innate and acquired immunity. In this study, we investigated the effect of administration of probiotic bacteria on the development and progression of the experimental infection in suckling rats. Rats were fed daily with 2.10(7) CFU of Lactobacillus casei-containing mixture, starting 2 days before the infection until the spontaneous clearance of the parasite. Effects on weight gain, parasite burden, mucosal histology and production of mucosal cytokines (IFNgamma, IL10 and TNFalpha) were studied. Although a trend to a more rapid clearance of parasites was noted in rats treated with probiotics, no significant effect of probiotics administration was observed in terms of weight gain, parasite burden, mucosal damage, or kinetics of mucosal cytokines during the course of infection. Overall, our results showed that the daily administration of L. casei-containing mixtures was unable to eradicate the parasite in our model.

Animals↗
PubMed1979

Folate analogues altered in the C9-N10 bridge region: 11-thiohomofolic acid.

The synthesis of 11-thiohomofolic acid (2) has been accomplished by an unambiguous procedure. Reaction of 1-chloro-4-[p-(carbomethoxy)thiophenoxy]-2-butanone (10) with hydroxylamine under carefully controlled conditions gave the corresponding oxime 33. Conversion of this oxime to 1-phthalimido-4-[p-(carbomethoxy)thiophenoxy]-2-butanone oxime (4) was carried out by its reaction with potassium phthalimide using crown 18 ether as a catalyst. Hydrazinolysis of compound 4 gave 1-amino-4-[p-(carbomethoxy)thiophenoxy]-2-butanone oxime (5), which was used for the construction of the title compound 2 by modification of the Boon and Leigh procedure. An alternate synthesis utilizing 1-hydroxy-4-[p-(carbomethoxy)thiophenoxy]-2-butanone (11) and 4-hydroxy-2,5,6-triaminopyrimidine has also been carried out. Compound 2 did not exhibit any antifolate activity against Lactobacillus casei or Streptococcus faecium. The dithionite reduction product, 7,8-dihydro-11-thiohomofolic acid, was able to function as a substrate of L. casei dihydrofolate reductase. The catalytic reduction product of 2, consisting of a mixture of diastereomers, exhibited powerful antifolate activity against both these organisms.

Folic Acid↗