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PubMed1991

Inhibition of the growth of Streptococcus mutans, Streptococcus sobrinus and Lactobacillus casei by oral peroxidase systems in human saliva.

Streptococcus mutans, Strep. sobrinus and Lactobacillus casei were grown in glucose-supplemented, sterilized, human whole saliva, adjusted to pH 5, 6 or 7. Components of the antibacterial peroxidase system--hypothiocyanous acid (HOSCN) and hypothiocyanite ions (OSCN-)--were generated by adding exogenous H2O2 to sterilized saliva containing endogenous peroxidases and thiocyanate (SCN-) ions. HOSCN/OSCN- generation was proportional to the amount of H2O2 added, and more HOSCN/OSCN- was detected in saliva at pH 7 than at pH 5. However, the growth of mutans streptococci and L. casei was inhibited at pH 5 by HOSCN/OSCN-, whereas no inhibition was found at pH 7. The findings show that (a) sufficient amount of HOSCN/OSCN- will inhibit the growth of cariogenic bacteria in human saliva at pH 5; (b) this amount of HOSCN/OSCN- can be generated in saliva by exogenously added H2O2; and (c) peroxidase systems have stronger antistreptococcal effects in human whole saliva than in phosphate buffer.

Anti-Bacterial Agents↗

PubMed1981

The inactivation of thymidylate synthase by diethyl pyrocarbonate.

Thymidylate synthase (5,10-methylenetetrahydrofolate: dUMP C-methyltransferase, EC 2.1.1.45) from Lactobacillus casei was inactivated by treatment with diethyl pyrocarbonate. The inactivation was apparently due to the modification of a large proportion of the enzyme's histidine residues. Neither the substrate dUMP nor the product dTMP prevented inactivation by diethyl pyrocarbonate. The inactivated enzyme was not reactivated by treatment with hydroxylamine. These results indicate that histidine residues are involved in the maintenance of enzyme structure.

Chemical Phenomena↗

PubMed2005

[Probiotics as therapeutic agents in irritable bowel syndrome].

Probiotics are defined as living micro-organisms which, when administered in large amounts, confer a health benefit on the host. The use of probiotics in the therapy of infectious bowel diseases as well as maintaining remission of ulcerative colitis and in pouchitis is evidence-based. Also, in several studies proof could be supplied that specific probiotics relieve the symptoms and the course of irritable bowel syndrome. Some trials showed a significant improvement of irritable bowel syndrome-related constipation via Lactobacillus casei Shirota and E. coli Nissle 1917. Lactobacillus plantarum has been proven effective in reducing pain and abdominal bloating. However, in most of the studies rather small numbers of patients were examined. Furthermore, these studies do not always closely follow scientific standards (randomised, double-blind, placebo-controlled). Therefore, confirmatory studies are necessary to examine the effect of probiotics in irritable bowel syndrome.

Follow-Up Studies↗

PubMed1999

Disulfide engineering at the dimer interface of Lactobacillus casei thymidylate synthase: crystal structure of the T155C/E188C/C244T mutant.

The crystal structure of a covalently cross-linked Lactobacillus casei thymidylate synthase has been determined at 2.8 A resolution. The sites for mutation to achieve the bis-disulfide linked dimer were identified using the disulfide modeling program MODIP. The mutant so obtained was found to be remarkably thermostable. This increase in stability has been reasoned to be entirely a consequence of the covalent gluing between the two subunits.

Crystallography, X-Ray↗

PubMed1989

Competition between Streptococcus mutans and Lactobacillus casei in mixed continuous culture.

In natural oral ecosystems, succession of Streptococcus mutans and Lactobacillus species occurs in bacterial communities which can be associated with the development of carious lesions. The objective of this study was to explore the interaction between 2 strains of S. mutans (SF2452) and Lactobacillus casei (RB1014) when grown together in continuous culture under conditions of varying environmental pH and in the presence of fluoride. When the strains, isolated from approximal carious lesions, were grown initially at a dilution rate of 0.1 h-1 under a glucose limitation at pH 7.0, a stable mixed community developed (Days 5-10) with S. mutans as the dominant population. When the pH control was removed (Day 10), the binary culture lowered the pH to 5.1 causing a small reduction in the numbers of S. mutans and allowing L. casei to predominate. A more dramatic effect was seen when the environmental pH was rapidly lowered to 4.8 by the addition of acid. In this case, the S. mutans population was significantly reduced and the L. casei population rose to the level previously held by S. mutans. Re-adjustment of the pH to 7.0 resulted in a re-establishment of the population ratios prior to the addition of acid. The possibility that S. mutans was capable of aciduric adaptation during a biologically-generated pH reduction was examined by mixing cultures of both organisms after each had been grown to steady state at pH 5.5 in separate chemostats.

Fluorides↗

PubMed2006

Calcium modulates interactions between bacteria and hydroxyapatite.

Bacterial adhesion onto hydroxyapatite is known to depend on the surface properties of both the biomaterial and the bacterial strain, but less is known about the influence of the composition of the aqueous medium. Here, the adhesion of Streptococcus mutans and 3 different Lactobacilli on powdered hydroxyapatite was shown to change with Ca2+ concentration. The effect depends on the surface properties of each strain. Adhesion of Lactobacillus fermentum and salivarius (and of Streptococcus mutans at low Ca2+) was enhanced with increasing Ca2+ concentration. Lactobacillus casei was efficiently removed by adhesion on hydroxyapatite, even without Ca2+ addition, and the effect of this ion was only marginal. The results are interpreted in terms of Ca2+-mediated adhesion, and relative to the hydrophobic properties of each strain and the electrical properties of the bacterial and solid surfaces (electrophoretic mobility).

Adsorption↗

PubMed1986

Crystallization and crystallographic data for new forms of thymidylate synthase from Lactobacillus casei.

Several new crystal forms of thymidylate synthase (5,10-methlenetetrahydrofolate:dUMP C-methyltransferase; EC 2.1.1.45) were obtained by controlled pH change. In the crystals the dimeric molecule has a 2-fold symmetry axis coinciding with crystallographic symmetry. The crystals scatter to at least 2.7 A resolution in the synchrotron X-ray beam and appear to be suitable for high-resolution X-ray diffraction analysis. The crystals were successfully derivatized and preliminary results are reported for the covalent inhibitory ternary complex of thymidylate synthase, 5-fluoro-2'-deoxyuridylate and 5,10-methylenetetrahydrofolate.

Crystallography↗

PubMed1993

Stereospecific assignments of the leucine methyl resonances in the 1H NMR spectrum of Lactobacillus casei dihydrofolate reductase.

A general method is described for the stereospecific assignment of methyl resonances in protein NMR spectra based on selective deuteration procedures. A selectively deuterated dihydrofolate reductase from L. casei was prepared by incorporating stereoselectively deuterated L-leucine, (2S,4R)[5,5,5-2H3]leucine. By comparing the COSY spectra of the dihydrofolate reductase-methotrexate complexes formed using deuterated and non-deuterated enzyme the stereospecific assignments for resonances of all 13 leucine residues were obtained by noting the absence of cross-peaks in spectra from the deuterated proteins.

Bacterial Proteins↗

PubMed1991

Affinity labeling of folate transport proteins with the N-hydroxysuccinimide ester of the gamma-isomer of fluorescein-methotrexate.

Fluorescein-methotrexate, a derivative in which the fluorophore is linked via a diaminopentane spacer to either the alpha- or gamma-carboxyl group of the glutamate moiety in the drug [Gapski et al. (1975) J. Med. Chem. 18, 526-528], has been synthesized by an improved procedure and separated by DEAE-Trisacryl chromatography into the alpha- and gamma-isomers (alpha-F-MTX and gamma-F-MTX). Each isomer was characterized by mass spectrometry, elemental analysis, absorbance spectrum, TLC, and reversed-phase HPLC. Identity of the isomers was established by the following enzymatic criteria: (a) gamma-F-MTX (but not the alpha-isomer) was hydrolyzed at the pteroate-glutamate bond by carboxypeptidase G2 to yield 4-amino-4-deoxy-10-methylpteroate and gamma-glutamyldiaminopentane-fluorescein; and (b) gamma-F-MTX was a much better inhibitor of human dihydrofolate reductase than the alpha-isomer (Ki values of 0.079 and 4.6 nM). alpha- and gamma-F-MTX were comparable as inhibitors (Ki values of 1.6 and 0.6 microM) of the transport system for reduced folates and MTX in L1210 cells, but the transporter in Lactobacillus casei was inhibited only by the gamma-isomer (Ki = 4.3 microM). The gamma-isomer, therefore, was selected for covalent labeling of proteins. When L. casei folate transport protein (18 kDa) was treated with gamma-F-MTX that had been activated with N-hydroxysuccinimide (NHS), the protein was readily visualized as a fluorescent band on SDS-PAGE electrophoretograms. The probe was also able to detect the transporter in membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗

PubMed1984

Crystallography, quantitative structure-activity relationships, and molecular graphics in a comparative analysis of the inhibition of dihydrofolate reductase from chicken liver and Lactobacillus casei by 4,6-diamino-1,2-dihydro-2,2-dimethyl-1-(substituted-phenyl)-s-triazine s.

The inhibition of dihydrofolate reductase from chicken liver and from Lactobacillus casei has been studied with 4,6-diamino-1,2-dihydro-2,2-dimethyl-1-(substituted-phenyl)-s-triazines. It was found that for the chicken enzyme, inhibitor potency for 101 triazines was correlated by the following equation: log 1/Kiapp = 0.85 sigma tau' - 1.04 log (beta X 10 sigma tau' + 1) + 0.57 sigma + 6.36. The parameter tau' indicates that for certain substituents, tau = 0. In the case of the L. casei DHFR results, meta and para derivatives could not be included in the same equation. For 38 meta-substituted compounds, it was found that log 1/Kiapp = 0.38 tau'3-0.91 log (beta X 10 tau'3 + 1) + 0.71I + 4.60 and for 32 para-substituted phenyltriazines log 1/Kiapp = 0.44 tau'4-0.65 log (beta tau'4 + 1') - 0.90 upsilon + 0.69I + 4.67. In the L. casei equation, I is an indicator variable for substituents of the type CH2ZC6H4-Y and ZCH2C6H4-Y, where Z = O, NH, S, or Se. The parameter upsilon is Charton's steric parameter, which is similar to Taft's Es. The mathematical models obtained from correlation analysis are compared with stereo color graphics models.

Animals↗

PubMed1999

Antimicrobial effect of acidified nitrite on cariogenic bacteria.

The antimicrobial agent nitric oxide (NO) is formed in the mouth and its concentration is directly related to salivary nitrite, which in turn is related to dietary nitrate intake. The aim of this study was to determine whether nitrite under acidic conditions will have an inhibitory effect, possibly occurring through NO production, on Streptococcus mutans, Lactobacillus casei and Actinomyces naeslundii. Whereas the growth of S. mutans was inhibited by a more acid pH, the addition of nitrite caused a marked, further dose-dependent reduction in bacterial numbers after 24 h of exposure. Similar effects were observed with A. naeslundii and L. casei. The ability of these bacteria to recover from nitrite exposure was also markedly affected by nitrite concentration. At acidity levels below pH 7, low concentrations of nitrite (0.2 mM) caused effective complete killing of S. mutans, with similar effects on the other organisms tested. These results demonstrate the possibility that nitrite in saliva has an effect on the growth and survival of cariogenic bacteria.

Actinomyces↗

PubMed2003

An immunohistological study of the localization of bacteria invading root pulpal walls of teeth with periapical lesions.

We immunohistologically examined the prevalence and localization of bacteria invading dentinal tubules of the roots of teeth with infected canals. Forty extracted teeth with apical lesions were selected and divided into two groups: a group of untreated teeth and a group of canal-enlarged teeth. The bacteria in the specimens were detected by Brown-Brenn stain and the labeled-streptavidin-biotin method with specific antisera for 16-bacteria. Seventy percent of the examined teeth showed bacteria invading the dentinal tubules of the roots. Fusobacterium nucleatum, Eubacterium alactolyticum, E. nodatum, Lactobacillus casei, and Peptostreptococcus micros were abundant. Even in the canal-enlarged group, invasion of bacteria was observed in 65% of teeth. This study revealed the actual condition of bacteria in infected root dentin and suggested that the canal-enlargement procedure could not completely remove all the bacteria in the infected dentinal tubules of the root.

Adult↗

PubMed1996

The establishment of a community of oral bacteria that controls the growth of Candida albicans in a chemostat.

The purpose of this study was to establish and identify a community of oral bacteria that controls the growth of Candida albicans in the chemostat. The chemostat was operated under glucose-limiting conditions at a dilution rate of 0.05 h-1 and inoculated with a yeast-free suspension of a tongue scraping. After a steady state had been reached, it was inoculated with C. albicans to establish the yeast and determine whether its growth could be contained. The steady-state community comprised the species Streptococcus sanguis, Streptococcus sobrinus, Streptococcus mitis, Lactobacillus casei, Eubacterium saburreum, Veillonella dispar and Fusobacterium nucleatum. Bacteroides gracilus and Haemophilus segnis were also detected but infrequently. Yeast growth was suppressed and yeast cells were lost at the same rate as the theoretical washout rate. It is concluded that this mixed community of oral bacteria can be used to identify the parameters that maintain the equilibrium between oral bacteria and C. albicans in the oral cavity.

Antibiosis↗

PubMed1987

Atomic structure of thymidylate synthase: target for rational drug design.

The atomic structure of thymidylate synthase from Lactobacillus casei was determined at 3 angstrom resolution. The native enzyme is a dimer of identical subunits. The dimer interface is formed by an unusual association between five-stranded beta sheets present in each monomer. Comparison of known sequences with the Lactobacillus casei structure suggests that they all have a common core structure around which loops are inserted or deleted in different sequences. Residues from both subunits contribute to each active site. Two arginine side chains can contribute to binding phosphate on the substrate. The side chains of several conserved amino acids can account for other determinants of substrate binding.

Amino Acid Sequence↗

PubMed2000

Quantitative approach in the study of adhesion of lactic acid bacteria to intestinal cells and their competition with enterobacteria.

To describe the phenomena of bacterial adhesion to intestinal cells and the competition for adhesion between bacteria, mathematical equations based on a simple dissociation process involving a finite number of bacterial receptors on intestinal cell surface were developed. The equations allow the estimation of the maximum number of Lactobacillus sp. and Escherichia coli cells that can adhere to Caco-2 cells and intestinal mucus; they also characterize the affinity of the bacteria to Caco-2 cells and intestinal and fecal mucus and the theoretical adhesion ratio of two bacteria present in a mixed suspension. The competition for adhesion between Lactobacillus rhamnosus GG and E. coli TG1 appeared to follow the proposed kinetics, whereas the competition between Lactobacillus casei Shirota and E. coli TG1 may involve multiple adhesion sites or a soluble factor in the culture medium of the former. The displacement of the adhered Lactobacillus by E. coli TG1 seemed to be a rapid process, whereas the displacement of E. coli TG1 by the Lactobacillus took more than an hour.

Bacterial Adhesion↗

PubMed1998

Study of the morphology of the cell walls of some strains of lactic acid bacteria and related species.

The objective of the present study was to find an explanation for the biological effect of the bacteria present in a biotherapeutic milk (Lactobacillus casei CRL 431 and Lactobacillus acidophilus CRL 730). The ability of bacterial cell walls to induce an immune response when introduced into an organism is well known. In this paper we specifically analyzed the morphology of these cell walls. Besides the two bacterial strains used in the fermented milk, two other lactic acid bacteria, belonging to another genus and unable to induce an immune system response, as well as a strain of Propionibacterium, of which the immune modulating capacity is known, were used in this work. We found a structural particularly in strains with immunostimulating capacity (L. casei CRL 431 and P. acidopropionici CRL 1198): molecules which protrude from the cell surface. In L. casei CRL 431 these molecules were identified as lectins because they are able to agglutinate yeast cells treated with glutaraldehyde and glycine. The structures protruding from P. acidipropionici CRL 1198 cells were teichoic acids. Teichoic acid and lectin-like structures can participate in adhesion to intestinal cells. The immunostimulation observed can be induced by the adhesion phenomenon.

Adjuvants, Immunologic↗

PubMed2002

Effects of antifolates on the binding of 5-fluoro-2'-deoxyuridine monophosphate to thymidylate synthase.

Folate based inhibitors of thymidylate synthase (TS) might facilitate binding of 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP) to TS similar to the natural reduced folate 5,10-methylenetetrahydrofolate (CH(2)-H(4)-folate). We studied the lipophilic, non-polyglutamatable antifolates Nolatrexed (NTX) and AG331 and antifolates, that can have a polyglutamate side chain like the natural folate CH(2)-H(4)-folate; GW1843U89, Raltitrexed (RTX) and Multi-targetted antifolate (MTA) and pentaglutamates (RTX-Glu(5) and MTA-Glu(5)). The capacity of these compounds to facilitate the binding of [(3)H]FdUMP to Lactobacillus casei TS and an ammoniumsulphate precipitate of human TS was investigated. Only NTX, RTX-Glu(5) and MTA-Glu(5) facilitated FdUMP binding to L. casei TS and their dissociation constant K(d) (0.2-0.7 microM) was low compared to CH(2)-H(4)-folate (2.0 microM). The small lipophilic molecule NTX was favorable to the larger AG331. Polyglutamylation, as indicated by the difference in effect of RTX vs. RTX-Glu(5) and MTA vs. MTA-Glu(5), seems to be important for a classical antifolate to facilitate binding of FdUMP to bacterial TS. Effects of antifolates on FdUMP binding to human TS were different. At a low concentration (0.05 microM) NTX, RTX-Glu(5) and MTA-Glu(5) facilitated 3-5 times higher binding of [(3)H]FdUMP to TS than CH(2)-H(4)-folate. At higher concentrations (0.3-5 microM) of NTX, RTX-Glu(5) and MTA-Glu(5) the FdUMP binding decreased. The complex remained stable in the absence of (anti)folate for at least 24hr. The K(d) values of the antifolates for human TS varied from 19 to 387 nM, while the K(d) of CH(2)-H(4)-folate for human TS was 351 nM. The Hill coefficients, which indicated the type of cooperativity of the antifolates in the binding of FdUMP to TS were positive (0.58-0.99) at low concentrations (<0.3 microM) and negative (-0.35 to -0.81) at concentrations >0.3 microM except for GW1843U89, which only showed negative cooperativity (-1.70). It was shown with [(14)C]NTX that when the binding of FdUMP decreased at high NTX concentrations, the binding of NTX to TS still increased. This also held for the natural substrate dUMP. The negative cooperativity of the antifolates was clearly concentration dependent. The difference between human and L. casei TS in the FdUMP binding assays with antifolates can possibly be explained by interaction of the two subunits of human TS, which was absent in L. casei TS. The binding of antifolates to one of the two subunits induced a conformational change of the other subunit. This change no longer allowed the binding of FdUMP or dUMP at the active site. In conclusion this study showed that antifolates enhanced the binding of FdUMP to TS, especially at low antifolate concentrations, that are also clinically achievable, e.g. in human plasma.

Binding, Competitive↗

PubMed1997

Isolation and characterization of promoters from the Lactobacillus casei temperate bacteriophage A2.

Random Sau3A1 DNA fragments from the temperate Lactobacillus bacteriophage A2 were cloned into the promoter-probe plasmid pGKV210. Seven DNA fragments with promoter activity were selected, after transformation of Escherichia coli and Lactococcus lactis, subsp, lactis, through the chloramphenicol resistance they conferred to the corresponding clones. The seven promoters were functional in Lactobacillus casei. Their strength was analysed by measuring the levels of chloramphenicol resistance and chloramphenicol acetyltransferase activity induced in each host. The nucleotide sequences of these fragments were determined and primer extension analysis was used to locate the initiation site of transcription from each promoter in E coli. The promoters contained -10 and -35 regions similar to the consensus sequences of E. coli and Lactobacillus promoters.

Bacteriophages↗