Carbohydr Res, 1998 Dec 31, 314(3-4), 211 - 20 Structure of an extracellular polysaccharide produced by Lactobacillus rhamnosus strain C83; Vanhaverbeke C et al.; The extracellular polysaccharide produced by Lactobacillus rhamnosus strain C83 was found to be composed of D-glucose and D-galactose in a molar ratio of 2:3 . The primary structure of the polysaccharide was shown by sugar analysis, methylation analysis, FABMS, partial acid hydrolysis and nuclear magnetic resonance (NMR) spectroscopy to consist of a pentasaccharide repeating unit having the following structure: -->3)-alpha-D-Glcp-(1-->2)-beta-D-Galf-(1-->6)-alpha-D-Galp-(1-->6 )-alpha-D -Glcp-(1-->3)-beta-D-Galf-(1--> Eur J Clin Nutr, 1999 Apr, 53(4), 277 - 80 Yoghurt enriched with Lactobacillus acidophilus does not lower blood lipids in healthy men and women with normal to borderline high serum cholesterol levels; de Roos NM et al.; OBJECTIVE: To investigate whether intake of Lactobacillus acidophilus strain L-1 lowers serum cholesterol in healthy men and women . DESIGN: Randomised, placebo-controlled parallel trial . SETTING: Subjects were free-living . Blood sampling and distribution of yoghurts were administered at a local hospital . SUBJECTS: Seventy-eight adult men and women with cholesterol levels of 3.9-7.8 mmol/L (mean +/- s.d., 5.4+/-0.7) . INTERVENTIONS: Subjects consumed 500 mL of control yoghurt daily for two weeks . They were then randomly allocated to receive 500 mL per day of control yoghurt or of yoghurt enriched with Lactobacillus acidophilus L-1 for another six weeks . The yoghurts were spiked with a trace of lithium; compliance as assessed by plasma lithium was excellent . RESULTS: Energy and nutrient intake was constant, and identical for the two groups . Mean body weight was stable . Baseline blood lipid concentrations in the control and treatment groups were highly similar . The effect of consumption of Lactobacillus acidophilus L-1 vs . control on total cholesterol was -0.02 mmol/L (95% CI, -0.18-0.15) after three weeks and 0.04 mmol/L (95% CI, -0.12-0.20) after six weeks . Serum LDL and HDL-cholesterol and triacylglycerol levels were also unaffected . CONCLUSIONS: Yoghurt enriched with Lactobacillus acidophilus L-1 does not lower serum cholesterol in men and women with normal to borderline high cholesterol levels. Quintessence Int, 1997 Jan, 28(1), 27 - 9 Monitoring the caries risk of orthodontic patients; Ulukapi H et al.; Several caries activity tests were used to investigate the caries risk of patients with fixed orthodontic appliances . Twenty young individuals, with a mean age of 11 years, participated in this study . Ten subjects were free of any orthodontic appliances and formed the control group . The other 10 individuals had been wearing fixed orthodontic appliances for at least 4 months . Stimulated saliva samples were obtained from all patients . Saliva samples were used to investigate the salivary flow rate, the salivary buffer capacity, and mutans streptococci and lactobacilli counts . The orthodontic patients exhibited a significant increase in salivary flow rate . Results of statistical analyses were nonsignificant in all other tests, suggesting that fixed orthodontic appliances are not the sole factor increasing the patient's caries risk during orthodontic treatment. Am J Obstet Gynecol, 1999 May, 180(5), 1080 - 7 Effects of multiple applications of benzalkonium chloride and nonoxynol 9 on the vaginal epithelium in the pigtailed macaque (Macaca nemestrina); Patton DL et al.; OBJECTIVE: Safe and effective vaginally applied microbicides could help to control the continuing spread of sexually transmitted diseases . STUDY DESIGN: This study used nonhuman primates to test the effects of multiple applications of nonoxynol 9, benzalkonium chloride, or a combination on vaginal flora and lower reproductive tract tissues . Fourteen monkeys (Macaca nemestrina) received daily vaginal applications of nonoxynol 9, benzalkonium chloride, or both for 3 to 4 days . Vaginal microflora and colposcopic observations were made at baseline and during and after completion of treatments . Cervical biopsy specimens were collected from a subset of animals . RESULTS: Cervical erythema and vaginal erythema were observed in all 3 treatment groups . Cervical papillae and epithelial disruption were present in both the nonoxynol 9 and the nonoxynol 9 plus benzalkonium chloride groups . Vaginal epithelial disruption was noted in both the benzalkonium chloride and the nonoxynol 9 plus benzalkonium chloride groups . Cervical biopsy specimens from each group revealed acute inflammatory infiltrates with occasional plasma cells and lymphoid follicles . Detection of most microorganisms, including viridans streptococci, decreased in the benzalkonium chloride and the nonoxynol 9 plus benzalkonium chloride groups . Detection of Lactobacillus species decreased in the benzalkonium chloride group . All microflora levels recovered after several days without microbicide use . CONCLUSIONS: Although nonoxynol 9 is currently the only microbicide approved for use as a spermicide in the United States, its repeated use may be detrimental to the epithelial tissues of the female reproductive tract . Benzalkonium chloride, currently approved for use in other countries, not only may damage epithelial tissues but also appears to reduce the population of potentially protective Lactobacillus species in the vagina. Mol Genet Metab, 1999 May, 67(1), 23 - 35 Folate-homocysteine interrelations: potential new markers of folate status; Lucock MD et al.; We report a transient drop in plasma Hcy and Cys following a single oral dose of PteGlu . The thiol change was concomitant with both the peak plasma 5CH3H4PteGlu1 level (by HPLC) and the maximum plasma Lactobacillus casei activity which reflects absorption of unmodified PteGlu . The significant reciprocal association of Hcy with radioassay RBC folate (r = -0.28, 99% CI -0.48, -0.05, P = 0.0016), serum folate (r = -0.37, 99% CI -0.56, -16, P = 0.0001), and vitamin B12 (r = -0.42, 99% CI -0.59, -21, P = 0.0001) is shown and reflects the long-term nutritional effect of B vitamins on this important, potentially atherogenic thiol . These are now well-established associations . We extend the potential for investigation of folate metabolism in health and disease by evaluating a range of new folate indices which are based on erythrocyte coenzymes . These have been looked at independently and in association with established parameters . Erythrocyte methylfolates (mono- to hexaglutamate-5CH3H4PteGlu1-6), formylfolates (tri- to pentaglutamate-5CHOH4PteGlu3-5),formiminotetrahydrofolate (formiminoH4PteGlu1), unsubstituted tetrahydrofolate (H4PteGlu1), andpara-aminobenzoylglutamate (P-ABG) have been measured by HPLC with fluorescence detection . A positive linear association exists between (i) H4PteGlu1 and radioassay RBC folate (r = 0.50, 99% CI 0 . 07, 0.77, P = 0.0036), and (ii) H4PteGlu1 and tetraglutamates of both formyl- and methylfolate (r = 0.52, 99% CI 0.10, 0.78, P = 0 . 0022, and r = 0.56, 99% CI 0.15, 0.80, P = 0.0009, respectively) . Since, in addition, a reciprocal linear association exists between Hcy and tetraglutamyl formylfolate (r = -0.41, 99% CI -0.73, 0.05, P = 0.0206), erythrocyte tetraglutamates may be a good reflection of the bodies' active coenzyme pools . Pentaglutamyl formylfolate, the longest oligo-gamma-glutamyl chain form of this coenzyme may be a good indicator of folate depletion . The abundance of this coenzyme both increases with increasing Hcy (r = 0.55, 99% CI 0.13, 0.80, P = 0.0014) and increases as H4PteGlu1, the principle folate congener, decreases (r = -0.59, 99% CI -0.82, -0.20, P = 0.0004) . Furthermore, the apparent equilibrium between substrate (5CH3H4PteGlu1) and product (H4PteGlu1) of methionine synthase is significantly associated with the abundance of 5CHOH4PteGlu5 (r = -0.53, 99% CI -0 . 79, -0.11, P = 0.0018) . This suggests that low methionine synthase activity for whatever reason (metabolic or dietary) may lead to an increase in the relative abundance of 5CHOH4PteGlu5 . Like 5CHOH4PteGlu5, evidence is given that 5CH3H4PteGlu6, the longest oligo-gamma-glutamyl chain form of this particular coenzyme pool, may also be a good indicator of folate depletion . This is shown by a change in the relative proportion of erythrocyte methylfolate polyglutamates following supplementation with 400 microg/day PteGlu . Short-chain polyglutamates of methylfolate (5CH3H4PteGlu1--> 5CH3H4PteGlu4) increase in proportion to the total methylfolate pool, while long-chain polyglutamates of methylfolate (5CH3H4PteGlu5 and particularly 5CH3H4PteGlu6) decrease in their relative abundance . Chem Biol, 1999 May, 6(5), 319 - 31 Structure-based discovery and in-parallel optimization of novel competitive inhibitors of thymidylate synthase; Tondi D et al.; BACKGROUND: The substrate sites of enzymes are attractive targets for structure-based inhibitor design . Two difficulties hinder efforts to discover and elaborate new (nonsubstrate-like) inhibitors for these sites . First, novel inhibitors often bind at nonsubstrate sites . Second, a novel scaffold introduces chemistry that is frequently unfamiliar, making synthetic elaboration challenging . RESULTS: In an effort to discover and elaborate a novel scaffold for a substrate site, we combined structure-based screening with in-parallel synthetic elaboration . These techniques were used to find new inhibitors that bound to the folate site of Lactobacillus casei thymidylate synthase (LcTS), an enzyme that is a potential target for proliferative diseases, and is highly studied . The available chemicals directory was screened, using a molecular-docking computer program, for molecules that complemented the three-dimensional structure of this site . Five high-ranking compounds were selected for testing . Activity and docking studies led to a derivative of one of these, dansyltyrosine (Ki 65 microM) . Using solid-phase in-parallel techniques 33 derivatives of this lead were synthesized and tested . These analogs are dissimilar to the substrate but bind competitively with it . The most active analog had a Ki of 1.3 microM . The tighter binding inhibitors were also the most specific for LcTS versus related enzymes . CONCLUSIONS: TS can recognize inhibitors that are dissimilar to, but that bind competitively with, the folate substrate . Combining structure-based discovery with in-parallel synthetic techniques allowed the rapid elaboration of this series of compounds . More automated versions of this approach can be envisaged. FEMS Microbiol Rev, 1999 Apr, 23(2), 131 - 51 Glucansucrases: mechanism of action and structure-function relationships; Monchois V et al.; Glucansucrases are produced principally by Leuconostoc mesenteroides and oral Streptococcus species, but also by the lactic acid bacteria (Lactococci, Lactobacilli) . They catalyse the synthesis of high molecular weight D-glucose polymers, named glucans, from sucrose . In the presence of efficient acceptors, they catalyse the synthesis of low molecular weight oligosaccharides . Glucosidic bond synthesis occurs without the mediation of nucleotide activated sugars and cofactors are not necessary . Glucansucrases have an industrial value because of the production of dextrans and oligosaccharides and a biological importance by their key role in the cariogenic process . They were identified more than 50 years ago . The first glucansucrase encoding gene was cloned more than 10 years ago . But the mechanism of their action remains incompletely understood . However, in order to synthesise oligosaccharides of biological interest or to develop vaccines against dental caries, elucidation of the factors determining the regiospecificity and the regioselectivity of glucansucrases is necessary . The cloning of glucansucrase encoding genes in addition to structure-function relationship studies have allowed the identification of important amino acid residues and have shown that glucansucrases are composed of two functional domains: a core region (ca . 1000 amino acids) involved in sucrose binding and splitting and a C-terminal domain (ca . 500 amino acids) composed of a series of tandem repeats involved in glucan binding . Enzymology studies have enabled different models for their action mechanism to be proposed . The use of secondary structure prediction has led to a clearer knowledge of structure-function relationships of glucansucrases . However, mainly due to the large size of these enzymes, data on the three-dimensional structure of glucansucrases (given by crystallography and modelling) remain necessary to clearly identify those features which determine function. Lancet, 1999 May 1, 353(9163), 1485 - 8 Atopy in children of families with an anthroposophic lifestyle; Alm JS et al.; BACKGROUND: Increased prevalence of atopic disorders in children may be associated with changes in types of childhood infections, vaccination programmes, and intestinal microflora . People who follow an anthroposophic way of life use antibiotics restrictively, have few vaccinations, and their diet usually contains live lactobacilli, which may affect the intestinal microflora . We aimed to study the prevalence of atopy in children from anthroposophic families and the influence of an anthroposophic lifestyle on atopy prevalence . METHODS: In a cross-sectional study, 295 children aged 5-13 years at two anthroposophic (Steiner) schools near Stockholm, Sweden, were compared with 380 children of the same age at two neighbouring schools in terms of history of atopic and infectious diseases, use of antibiotics and vaccinations, and social and environmental variables . Skin-prick tests were done for 13 common allergens, and we took blood samples from children and their parents for analysis of allergen-specific serum IgE-antibodies . FINDINGS: At the Steiner schools, 52% of the children had had antibiotics in the past, compared with 90% in the control schools . 18% and 93% of children, respectively, had had combined immunisation against measles, mumps, and rubella, and 61% of the children at the Steiner schools had had measles . Fermented vegetables, containing live lactobacilli, were consumed by 63% of the children at Steiner schools, compared with 4.5% at the control schools . Skin-prick tests and blood tests showed that the children from Steiner schools had lower prevalence of atopy than controls (odds ratio 0.62 {95% CI 0.43-0.91}) . There was an inverse relation between the number of characteristic features of an anthroposophic lifestyle and risk of atopy (p for trend=0.01) . INTERPRETATION: Prevalence of atopy is lower in children from anthroposophic families than in children from other families . Lifestyle factors associated with anthroposophy may lessen the risk of atopy in childhood. Mol Microbiol, 1999 Apr, 32(2), 427 - 36 Autoaggregation of Lactobacillus reuteri is mediated by a putative DEAD-box helicase; Roos S et al.; We have cloned and sequenced a gene from Lactobacillus reuteri that encodes a 56 kDa protein, which mediates autoaggregation of the bacteria . Using an antiserum raised against extracellular proteins from the pig intestinal isolate L . reuteri 1063, we screened a genomic lambda library derived from the same strain . Affinity purification of recombinant protein from the isolated lambda clones showed that one type of clone expressed a protein that efficiently aggregated the parental strain when added to the bacteria . Subcloning and introduction of the corresponding gene, here denoted aggHinto the L . reuteri type strain markedly enhanced aggregation . Furthermore, insertional inactivation of aggH in strain 1063 resulted in an autoaggregation-deficient phenotype . Finally, an affinity-purified and cleaved fusion of AggH protein and the maltose-binding protein, MBP, strongly promoted aggregation of L . reuteri 1063, whereas the uncleaved fusion protein was inactive . Sequencing of aggH revealed that the corresponding protein has extensive sequence homology to the large family of ATP-dependent DEAD-box helicases . These results are intriguing in view of earlier data on the promotion of genetic exchange in Lactobacillus by aggregation. Int J Immunopharmacol, 1999 Feb, 21(2), 121 - 31 Lactic acid bacterium potently induces the production of interleukin-12 and interferon-gamma by mouse splenocytes; Kato I et al.; We previously have reported that the lactic acid bacterium, Lactobacillus casei strain Shirota (LcS), shows marked antitumor activities and an ability to modify immune responses . In this study, we examined whether LcS can induce the production of interleukin (IL)-12 and interferon-gamma (IFN-gamma), which are important cytokines for antitumor and antimicrobial immunity, from murine splenocytes in vitro in order to clarify the mechanisms of its immune modification . Stimulation by LcS induced a marked production of IL-12 by X-ray-irradiated splenocytes (X-irr-Spl) . The production of IL-12 by X-irr-Spl was independent of the presence of nylon wool column-passed splenocytes (NW-Spl) . IFN-gamma was produced by splenocytes by the stimulation with concanavalin A (Con A) . LcS showed a synergistic stimulatory effect on the ConA-induced production of IFN-gamma . In addition, X-irr-Spl were required for the IFN-gamma; production by NW-Spl treated with LcS . The IFN-gamma production was reduced by anti-IL-12 antibody treatment . NW-Spl produced IFN-gamma following treatment with recombinant IL-12 . Thus, we confirmed that IFN-gamma production by splenocytes was the result of the production of IL-12 from X-irr-Spl stimulated by LcS . Furthermore, in BALB/c mice, the oral administration of viable LcS augmented the production of IFN-gamma but not that of IL-4 or IL-5 by splenocytes . Thus, we suggested that LcS primarily activated X-irr-Spl, probably macrophages, and these cells secreted IL-12 . The IL-12 induced by LcS stimulated the production of IFN-gamma. Biosci Biotechnol Biochem, 1999 Mar, 63(3), 581 - 4 Antioxidant activity of a medicine based on Aspergillus oryzae NK koji measured by a modified t-butyl peroxyl radical scavenging assay; Kawasumi T et al.; A koji-based medicine composed of powder of Aspergillus oryzae NK koji, dried yeast, and lactobacilli koji had high antioxidant activity measured by a modified t-butyl peroxyl radical scavenging assay . This activity was mainly derived from A . oryzae NK koji . Digestion of koji-making grain germ medium with several commercial enzymes also increased antioxidant activity . By two weeks of oral administration of A . oryzae NK koji, the serum lipid peroxide levels elevated in STZ-induced diabetic rats could be decreased significantly. Infect Immun, 1999 May, 67(5), 2653 - 5 In vitro adhesion and platelet aggregation properties of bacteremia-associated lactobacilli; Kirjavainen PV et al.; Eight bacteremia-associated Lactobacillus strains were evaluated in vitro for the ability to adhere to human intestinal mucosa and to aggregate platelets . Adherence varied significantly among the strains, and platelet aggregation was induced by three strains . In conclusion, strong binding ability does not appear to be a prerequisite for the involvement of lactobacilli in bacteremia or to their ability to aggregate platelets. Infect Immun, 1999 May, 67(5), 2464 - 74 Insertional inactivation of genes responsible for the D-alanylation of lipoteichoic acid in Streptococcus gordonii DL1 (Challis) affects intrageneric coaggregations; Clemans DL et al.; Most human oral viridans streptococci participate in intrageneric coaggregations, the cell-to-cell adherence among genetically distinct streptococci . Two genes relevant to these intrageneric coaggregations were identified by transposon Tn916 mutagenesis of Streptococcus gordonii DL1 (Challis) . A 626-bp sequence flanking the left end of the transposon was homologous to dltA and dltB of Lactobacillus rhamnosus ATCC 7469 (formerly called Lactobacillus casei) . A 60-kb probe based on this flanking sequence was used to identify the homologous DNA in a fosmid library of S . gordonii DL1 . This DNA encoded D-alanine-D-alanyl carrier protein ligase that was expressed in Escherichia coli from the fosmid clone . The cloned streptococcal dltA was disrupted by inserting an ermAM cassette, and then it was linearized and transformed into S . gordonii DL1 for allelic replacement . Erythromycin-resistant transformants containing a single insertion in dltA exhibited a loss of D-alanyl esters in lipoteichoic acid (LTA) and a loss of intrageneric coaggregation . This phenotype was correlated with the loss of a 100-kDa surface protein reported previously to be involved in mediating intrageneric coaggregation (C . J . Whittaker, D . L . Clemans, and P . E . Kolenbrander, Infect . Immun . 64:4137-4142, 1996) . The mutants retained the parental ability to participate in intergeneric coaggregation with human oral actinomyces, indicating the specificity of the mutation in altering intrageneric coaggregations . The mutants were altered morphologically and exhibited aberrant cell septa in a variety of pleomorphs . The natural DNA transformation frequency was reduced 10-fold in these mutants . Southern analysis of chromosomal DNAs from various streptococcal species with the dltA probe revealed the presence of this gene in most viridans streptococci . Thus, it is hypothesized that D-alanyl LTA may provide binding sites for the putative 100-kDa adhesin and scaffolding for the proper presentation of this adhesin to mediate intrageneric coaggregation. Appl Environ Microbiol, 1999 May, 65(5), 1910 - 4 Potential of conjugal transfer as a strategy for the introduction of recombinant genetic material into strains of lactobacillus helveticus Thompson JK, McConville KJ, McReynolds C, Collins MA. Cointegrates generated between a plasmid pIP501 deletion derivative (pVA797) and nonconjugative shuttle vector pSA3 were confirmed as capable of exconjugation from lactococci into a range of strains of Lactobacillus helveticus with the concomitant expression of a recombinant gene . The plasmid cointegrate that was formed appeared to be segregationally stable at 37 degrees C in some host strains . In all strains, however, the plasmid became increasingly unstable as the incubation temperature was raised . The technique offers not only a generalized method for the introduction of novel genetic material into this important industrial microbe but also the possibility of exploiting the thermal sensitivity of the plasmid to enable it to act as a delivery system for the integration of cloned genes into the bacterial chromosome, at restrictive temperatures, by recombination at regions of homology. Appl Environ Microbiol, 1999 May, 65(5), 2035 - 40 Cell-wall-bound proteinase of Lactobacillus delbrueckii subsp . lactis ACA-DC 178: characterization and specificity for beta-casein; Tsakalidou E et al.; Lactobacillus delbrueckii subsp . lactis ACA-DC 178, which was isolated from Greek Kasseri cheese, produces a cell-wall-bound proteinase . The proteinase was removed from the cell envelope by washing the cells with a Ca2+-free buffer . The crude proteinase extract shows its highest activity at pH 6.0 and 40 degrees C . It is inhibited by phenylmethylsulfonyl fluoride, showing that the enzyme is a serine-type proteinase . Considering the substrate specificity, the enzyme is similar to the lactococcal PI-type proteinases, since it hydrolyzes beta-casein mainly and alpha- and kappa-caseins to a much lesser extent . The cell-wall-bound proteinase from L . delbrueckii subsp . lactis ACA-DC 178 liberates four main peptides from beta-casein, which have been identified. Appl Environ Microbiol, 1999 May, 65(5), 1919 - 23 Feed deprivation affects crop environment and modulates Salmonella enteritidis colonization and invasion of leghorn hens; Durant JA et al.; Leghorn hens over 50 weeks of age were assigned to two treatment groups designated as either unmolted controls or molted . A forced molt was induced by a 9-day feed withdrawal, and each hen was challenged orally with 10(5) Salmonella enteritidis organisms on day 4 of feed withdrawal . On days 4 and 9 of molt, the numbers of lactobacilli and the concentrations of lactate, acetate, propionate, and butyrate, and total volatile fatty acids in the crops decreased while crop pH increased significantly (P < 0.05) in the molted hens compared to the controls . S . enteritidis crop and cecal colonization, in addition to spleen and liver invasion, increased significantly (P < 0.05) in the molted hens compared to the controls . The invasive phenotype of Salmonella spp . is complex and requires several virulence genes which are regulated by the transcriptional activator HilA . Samples of the crop contents from the molted and unmolted birds were pooled separately, centrifuged, and filter sterilized . The sterile crop contents were then used to measure the expression of hilA . By using a lacZY transcriptional fusion to the hilA gene in S . enteritidis, we found that hilA expression was 1.6- to 2.1-fold higher in the crop contents from molted birds than in those from control birds in vitro . The results of the study suggest that the changes in the microenvironment of the crop caused by feed deprivation are important regulators of S . enteritidis survival and influence the susceptibility of molted hens to S . enteritidis infections . Furthermore, our in vitro results on the expression of hilA suggest that the change in crop environment during feed withdrawal has the potential to significantly affect virulence by increasing the expression of genes necessary for intestinal invasion. Appl Microbiol Biotechnol, 1999 Mar, 51(3), 358 - 63 Lactobacillus sanfranciscensis CB1: manganese, oxygen, superoxide dismutase and metabolism; De Angelis M et al.; The latency phase, growth rate, cell yield and end-products of Lactobacillus sanfranciscensis CB1 were affected by oxygen and the supply of 225 microM Mn2+ . Mn2+ was especially related to the highest substrate consumption . Aerobiosis and Mn2+ supply were responsible for the highest superoxide dismutase activity . An auto-inhibitory accumulation of H2O2 meant that the survival of air-grown cells supplied with Mn2+ rapidly decreased during the stationary phase . As shown by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, Mn2+ supply influenced protein expression . As shown by non-denaturating zymograms, Lb . sanfranciscensis CB1 expressed an approximately 12.5-kDa superoxide dismutase, which is probably Mn-dependent . The enzyme was insensitive to H2O2 treatment, was not induced by the presence of paraquat under aerobic conditions and was relatively stable at pH 4.0 . Sourdoughs that contained high levels of oxygen enhanced cell growth, acidification and acetic acid production by Lb . sanfranciscensis CB1. Appl Microbiol Biotechnol, 1999 Mar, 51(3), 316 - 24 Continuous production of L(+)-lactic acid by Lactobacillus casei in two-stage systems; Bruno-Barcena JM et al.; A two-stage two-stream chemostat system and a two-stage two-stream immobilized upflow packed-bed reactor system were used for the study of lactic acid production by Lactobacillus casei subsp casei . A mixing ratio of D12/D2 = 0.5 (D = dilution rate) resulted in optimum production, making it possible to generate continuously a broth with high lactic acid concentration (48 g l-1) and with a lowered overall content of initial yeast extract (5 g l-1), half the concentration supplied in the one-step process . In the two-stage chemostat system, with the first stage at pH 5.5 and 37 degrees C and a second stage at pH 6.0, a temperature change from 40 degrees C to 45 degrees C in the second stage resulted in a 100% substrate consumption at an overall dilution rate of 0.05 h-1 . To increase the cell mass in the system, an adhesive strain of L . casei was used to inoculate two packed-bed reactors, which operated with two mixed feedstock streams at the optimal conditions found above . Lactic acid fermentation started after a lag period of cell growth over foam glass particles . No significant amount of free cells, compared with those adhering to the glass foam, was observed during continuous lactic acid production . The extreme values, 57.5 g l-1 for lactic acid concentration and 9.72 g l-1 h-1 for the volumetric productivity, in upflow packed-bed reactors were higher than those obtained for free cells (48 g l-1 and 2.42 g l-1 h-1) respectively and the highest overall L(+)-lactic acid purity (96.8%) was obtained in the two-chemostat system as compared with the immobilized-cell reactors (93%). Int J Antimicrob Agents, 1999 Feb, 11(2), 151 - 7 Three kinds of antibacterial substances from Lentinus edodes (Berk.) Sing . (Shiitake, an edible mushroom); Hirasawa M et al.; Three kinds of antibacterial substances were extracted by chloroform, ethylacetate or water from dried Shiitake mushrooms (Lentinus edodes) . These substances possess efficient antibacterial activities against Streptococcus spp., Actinomyces spp., Lactobacillus spp., Prevotella spp., and Porphyromonas spp . of oral origin . In contrast, other general bacteria, such as Enterococcus spp., Staphylococcus spp., Escherichia spp., Bacillus spp., and Candida spp . were relatively resistant to these substances . Chloroform extracts had bactericidal activity against both growing and resting bacterial cells of S . mutans and P . intermedia, whereas the other two extracts showed bacteriostatic activity against both growing and resting bacterial cells of S . mutans and resting bacterial cell of P . intermedia . The antibacterial activity of chloroform extracts and ethylacetate extracts were relatively heat-stable . The water extract was heat-labile. Gastroenterology, 1999 May, 116(5), 1107 - 14 Lactobacillus species prevents colitis in interleukin 10 gene-deficient mice; Madsen KL et al.; BACKGROUND & AIMS: Intestinal luminal microflora, or their products, are likely an important initiating factor in the pathogenesis of inflammatory bowel disease . The aim of this study was to determine the role of colonic aerobic luminal bacteria and Lactobacillus species in the development of colitis in interleukin (IL)-10 gene-deficient mice . METHODS: Intestine from 2-16-week-old mice was scored histologically and cultured for bacteria . Lactobacillus sp . repopulation of the colonic lumen was achieved via daily rectal delivery of Lactobacillus reuteri or oral lactulose therapy . RESULTS: At 2 weeks of age, IL-10 gene-deficient mice showed no colonic injury but did display abnormal colonic bacterial colonization with increased colonic mucosal aerobic adherent and translocated bacteria in conjunction with reduced Lactobacillus sp . levels . In association with the abnormal colonic bacterial colonization, colitis developed by 4 weeks of age . Restoring Lactobacillus sp . to normal levels reduced levels of colonic mucosal adherent and translocated bacteria and attenuated the development of the colitis . CONCLUSIONS: In the neonatal period, IL-10 gene-deficient mice have decreased levels of colonic Lactobacillus sp . and an increase in colonic mucosal adherent and translocated bacteria . Normalizing Lactobacillus sp . levels reduced colonic mucosal adherent and translocated bacteria and prevented colitis. Nucleic Acids Res, 1999 May 15, 27(10), 2145 - 55 The complete genome sequence of the Streptomyces temperate phage straight phiC31: evolutionary relationships to other viruses; Smith MC et al.; The completed genome sequence of the temperate Streptomyces phage straight phiC31 is reported . straight phiC31 contains genes that are related by sequence similarities to several other dsDNA phages infecting many diverse bacterial hosts, including Escherichia, Arthrobacter, Mycobacterium, Rhodobacter, Staphylococcus, Bacillus, Streptococcus, Lactobacillus and Lactococcus . These observations provide further evidence that dsDNA phages from diverse bacterial hosts are related and have had access to a common genetic pool . Analysis of the late genes was particularly informative . The sequences of the head assembly proteins (portal, head protease and major capsid) were conserved between straight phiC31, coliphage HK97, staphylococcal phage straight phiPVL, two Rhodobacter capsulatus prophages and two Mycobacterium tuberculosis prophages . These phages and prophages (where non-defective) from evolutionarily diverse hosts are, therefore, likely to share a common head assembly mechanism i.e . that of HK97 . The organisation of the tail genes in straight phiC31 is highly reminiscent of tail regions from other phage genomes . The unusual organisation of the putative lysis genes in straight phiC31 is discussed, and speculations are made as to the roles of some inessential early gene products . Similarities between certain phage gene products and eukaryotic dsDNA virus proteins were noted, in particular, the primase/helicases and the terminases (large subunits) . Furthermore, the complete sequence clarifies the overall transcription map of the phage during lytic growth and the positions of elements involved in the maintenance of lysogeny. J Appl Microbiol, 1999 Apr, 86(4), 682 - 8 Partial characterization and plasmid linkage of a non-proteinaceous antimicrobial compound in a Lactobacillus casei strain of vegetable origin; Scolari G et al.; Lactobacillus casei IMPC LC34 of vegetable origin produces a non-proteinaceous inhibitory compound with a broad spectrum of activity towards Gram-positive and Gram-negative bacteria, including pathogens . The active substance, mainly produced in the stationary phase of growth, is insensitive to proteolytic enzymes, lipase and catalase, and is stable at 121 degrees C for 30 min . The inhibitory activity was detected either at 8 degrees C or at 37 degrees C . The active compound does not contain glucidic groups, is inactivated by Na-metaperiodate, and its molecular mass is between 2000 and 5000 Da . Plasmid curing experiments showed that both antimicrobial compound immunity and production determinants were encoded by an 8.8 kbp plasmid . The effectiveness of the active agent was verified on ready-to-use vegetables, using either the Lact . casei strain or its culture supernatant fluid as inoculant, compared with cured clone . The application potential of the Lact . casei strain or its culture supernatant fluid for assuring the microbiological safety of ready-to-use vegetables is discussed. Protein Sci, 1999 Apr, 8(4), 930 - 3 Disulfide engineering at the dimer interface of Lactobacillus casei thymidylate synthase: crystal structure of the T155C/E188C/C244T mutant; Velanker SS et al.; 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. Caries Res, 1999 May-Jun, 33(3), 191 - 5 Associations between microbiological and salivary caries activity tests and caries experience in Hungarian adolescents; Gabris K et al.; The aim of the study was to assess caries prevalence in connection with salivary caries-related findings in 349 14- to 16-year-old Hungarian adolescents living in two different cities . DMFT, DMFS means, stimulated salivary flow, buffer capacity, mutans streptococci, lactobacilli and candida counts in saliva were determined . The ratio of caries-free adolescents was 4.6% in the total population sample, DMFT mean values were 7.24+/-4.86, DMFS means 10.50+/-8.35 . Mean secretion rate of stimulated saliva was 0 . 84+/-0.50; a low buffer capacity was found in 6.3% of the examined children . The ratio of carriers of mutans streptococci, lactobacilli and yeasts in saliva was 89.7, 73.9 and 47.7%, respectively . DMFT and DMFS values as well as mutans streptococci and candida counts were lower in the capital than in the other city . Statistically significant correlations were found between DMFT, DMFS mean values, and salivary microbiological counts. Caries Res, 1999 May-Jun, 33(3), 178 - 84 A low concentration of carbonic anhydrase isoenzyme VI in whole saliva is associated with caries prevalence; Kivela J et al.; Carbonic anhydrases maintain pH homeostasis in various tissues of the human body by catalyzing the reversible reaction CO2 + H2O <=> HCO3- + H+ . Carbonic anhydrase isoenzyme VI (CA VI) is secreted into human saliva by the serous acinar cells of the parotid and submandibular glands . Although it represents about 3% of the total protein in stimulated parotid saliva, its exact physiological significance in the saliva has not been established . In the present study, saliva samples were collected under strictly controlled conditions from young, healthy men and assayed for CA VI concentrations using a specific time-resolved immunofluorometric assay . Salivary secretion rate, pH, buffering capacity, alpha-amylase activity levels, lactobacillus and Streptococcus mutans counts were also determined, and the results were correlated with the dental status of the subjects . Salivary CA VI concentration, pH and buffering capacity values correlated negatively with the numbers of decayed, missing and filled teeth (DMFT index) . The correlations between salivary CA VI concentration and DMFT index were most significant in subjects with poor oral hygiene . No correlation was found between salivary CA VI concentration and lactobacillus or Streptococcus mutans counts . As predicted, salivary lactobacillus and Streptococcus mutans counts showed a close positive correlation with the DMFT index . In contrast, no significant correlation was seen between salivary secretion rate or amylase activity and the DMFT index . The present results indicate that low salivary CA VI concentrations are associated with increased caries prevalence, particularly in subjects with neglected oral hygiene. Oral Microbiol Immunol, 1999 Feb, 14(1), 43 - 8 Acid-induced acid tolerance and acidogenicity of non-mutans streptococci; Takahashi N et al.; Acid tolerance and acidogenicity of non-mutans streptococci and their capacity of acid adaptation were studied . The cells of non-mutans streptococci (Streptococcus sanguis {Streptococcus sanguinis}, Streptococcus gordonii, Streptococcus oralis and Streptococcus mitis) grown at pH 7.0 showed 0.0088% to 71% viability after acidification at pH 4.0 for 60 min, whereas the cells of mutans streptococci (Streptococcus mutans) were not killed by the acidification . Washed cells of non-mutans streptococci lowered pH to 4.04-4.33 in the presence of glucose, while mutans streptococci cells lowered pH to 3.70 . When the growth pH was shifted to 5.5 for 30-90 min, the viability of non-mutans streptococci after the acidification at pH 4.0 for 60 min increased (0.25% to 91%) and the minimum pH values of the cells in the presence of glucose decreased (3.90 4.19) . Along with the increase in acid tolerance and acidogenicity, non-mutans streptococci increased activities of H(+)-ATPase and arginine deiminase and amounts of stress proteins cross-reacting with 60 kDa and 70 kDa heat shock proteins (Hsp60 and Hsp70) . These results indicate that non-mutans streptococci were capable of increasing their acid tolerance and acidogenicity in response to environmental acidification . Furthermore, it is suggested that the acid adaptation observed in non-mutans streptococci cells could involve the induction of H(+)-ATPase, arginine deiminase and stress protein syntheses . The strains of non-mutans streptococci, which are pioneer bacteria for dental plaque formation and predominant in plaque microbial flora, may play a significant role in shifting the dental plaque environment toward acidic and consequently promoting the colonization of more acid-tolerant and acidogenic bacteria such as mutans streptococci and lactobacilli. Clin Exp Allergy, 1999 Mar, 29(3), 342 - 6 The intestinal microflora in allergic Estonian and Swedish 2-year-old children; Bjorksten B et al.; BACKGROUND: The prevalence of allergic diseases seems to have increased particularly over the past 35-40 years . Furthermore, allergic disease is less common among children in the formerly socialist countries of central and Eastern Europe as compared with Western Europe . It has been suggested that a reduced microbial stimulation during infancy and early childhood would result in a slower postnatal maturation of the immune system and development of an optimal balance between TH1- and TH2-like immunity . AIMS: To test the hypothesis that allergic disease among children may be associated with differences in their intestinal microflora in two countries with a low (Estonia) and a high (Sweden) prevalence of allergy . METHODS: From a prospective study of the development of allergy in relation to environmental factors, 29 Estonian and 33 Swedish 2-year-old children were selected . They were either nonallergic (n = 36) or had a confirmed diagnosis of allergy (n = 27) as verified by typical history and at least one positive skin prick test to egg or cow's milk . Weighed samples of faeces were serially diluted (10-2-10-9) and grown under anaerobic conditions . The counts of the various genera and species were calculated for each child . In addition, the relative amounts of the particular microbes were expressed as a proportion of the total count . RESULTS: The allergic children in Estonia and Sweden were less often colonized with lactobacilli (P < 0.01), as compared with the nonallergic children in the two countries . In contrast, the allergic children harboured higher counts of aerobic micro-organisms (P < 0 . 05), particularly coliforms (P < 0.01) and Staphylococcus aureus (P < 0.05) . The proportions of aerobic bacteria of the intestinal flora were also higher in the allergic children (P < 0.05), while the opposite was true for anaerobes (P < 0.05) . Similarly, in the allergic children the proportions of coliforms were higher (P < 0 . 05) and bacteroides lower (P < 0.05) than in the nonallergic children . CONCLUSIONS: Differences in the indigenous intestinal flora might affect the development and priming of the immune system in early childhood, similar to what has been shown in rodents . The role of intestinal microflora in relation to the development of infant immunity and the possible consequences for allergic diseases later in life requires further study, particularly as it would be readily available for intervention as a means for primary prevention of allergy by the administration of probiotic bacteria. Int J Oncol, 1999 May, 14(5), 939 - 44 Prevention of colonic preneoplastic lesions by the probiotic Lactobacillus acidophilus NCFMTM in F344 rats; Rao CV et al.; The experiments described here were aimed at developing novel probiotic strains that may aid in the reduction of colon cancer risk . We assessed the potential anticancer properties of Lactobacillus acidophilus NCFMTM in male F344 rats using inhibition of the formation of azoxymethane (AOM)-induced aberrant crypt foci (ACF) in the colon as the measure of preventive efficacy . At 6 weeks of age, groups of rats were fed the experimental diets containing 0, 2% or 4% lyophilized cultures of L . acidophilus NCFMTM . At 7 weeks of age, all animals in each dietary group, except the vehicle-treated rats, were s.c . injected with AOM (15 mg/kg body weight) once weekly for two weeks . The vehicle-treated groups were given s.c . injections of normal saline . All rats were necropsied 10 weeks after the last AOM injection and ACF in formalin-fixed, methylene blue-stained colonic tissues were counted under the light microscope . The contents of the cecum were analyzed for bacterial beta-glucuronidase activity . Diet supplementation with the probiotic strain NCFMTM significantly suppressed AOM-induction of colonic ACF, in terms of total number, as well as crypt multiplicity and number of ACF/cm2 colon (P<0.01 - 0.001) . NCFMTM inhibited AOM-induced colonic ACF formation in a dose-dependent manner (P<0.01) . A significant dose-dependent reduction of cecal beta-glucuronidase activities was observed in the rats fed 2% (P<0.04) and 4% (P<0.0001) NCFMTM . These results suggest that Lactobacillus acidophilus NCFMTM may potentially prevent colon cancer development . Further studies are warranted to determine the full potential of this probiotic strain in preclinical efficacy studies. Am J Physiol, 1999 Apr, 276(4 Pt 1), G941 - 50 Probiotics inhibit enteropathogenic E . coli adherence in vitro by inducing intestinal mucin gene expression; Mack DR et al.; Probiotic agents, live microorganisms with beneficial effects for the host, may offer an alternative to conventional antimicrobials in the treatment and prevention of enteric infections . The probiotic agents Lactobacillus plantarum 299v and Lactobacillus rhamnosus GG quantitatively inhibited the adherence of an attaching and effacing pathogenic Escherichia coli to HT-29 intestinal epithelial cells but did not inhibit adherence to nonintestinal HEp-2 cells . HT-29 cells were grown under conditions that induced high levels of either MUC2 or MUC3 mRNA, but HEp-2 cells expressed only minimal levels of MUC2 and no MUC3 mRNA . Media enriched for MUC2 and MUC3 mucin were added exogenously to binding assays and were shown to be capable of inhibiting enteropathogen adherence to HEp-2 cells . Incubation of L . plantarum 299v with HT-29 cells increased MUC2 and MUC3 mRNA expression levels . From these in vitro studies, we propose the hypothesis that the ability of probiotic agents to inhibit adherence of attaching and effacing organisms to intestinal epithelial cells is mediated through their ability to increase expression of MUC2 and MUC3 intestinal mucins. J Bacteriol, 1999 Apr, 181(8), 2363 - 72 Regulated expression of the Streptococcus mutans dlt genes correlates with intracellular polysaccharide accumulation; Spatafora GA et al.; Intracellular polysaccharides (IPS) are glycogen-like storage polymers which contribute significantly to Streptococcus mutans-induced cariogenesis . We previously identified and cloned a locus from the S . mutans chromosome which is required for the accumulation of IPS . Sequencing of this locus revealed at least four contiguous open reading frames, all of which are preceded by a common promoter region and are transcribed in the same direction . Analysis of the amino acid sequence deduced from the first of these open reading frames (ORF1) revealed domains which are highly conserved among D-alanine-activating enzymes (DltA) in Lactobacillus rhamnosus (formerly Lactobacillus casei) and Bacillus subtilis . The deduced amino acid sequences derived from ORF2, -3, and -4 also exhibit extensive similarity to DltB, -C, and -D, respectively, in these microorganisms . However, Southern hybridization experiments indicate that this operon maps to a locus on the S . mutans chromosome which is separate from the glgP, glgA, and glgD genes, whose products are known mediators of bacterial IPS accumulation . We therefore assigned a new dlt designation to the locus which we had formerly called glg . We maintain that the dlt genes are involved in S . mutans IPS accumulation, however, since they complement a mutation in trans which otherwise renders S . mutans IPS deficient . In this study, we found that expression of the S . mutans dlt genes is growth phase dependent and is modulated by carbohydrates internalized via the phosphoenolpyruvate phosphotransferase system (PTS) . We demonstrated that the S . mutans dlt genes are expressed constitutively when non-PTS sugars are provided as the sole source of carbohydrate . Consistent with a role for the PTS in dlt expression is a similar constitutive expression of the dlt genes in an S . mutans PTS mutant grown in a chemically defined medium supplemented with glucose . In summary, these findings support a novel role for the dlt gene products in S . mutans IPS accumulation and suggest that dlt expression in this oral pathogen is subject to complex mechanisms of control imposed by growth phase, dietary carbohydrate, and other factors present in the plaque environment. Lett Appl Microbiol, 1999 Mar, 28(3), 159 - 63 Human ileostomy glycoproteins as a model for small intestinal mucus to investigate adhesion of probiotics; Tuomola EM et al.; Human ileostomy glycoproteins were used as a model for small intestinal mucus to investigate the adhesion of 12 Lactobacillus strains, one Lactococcus strain and one Propionibacterium strain, Both probiotic and dairy strains were tested . Adhesive and non-adhesive Escherichia coli strains were used as controls . All the strains were also tested for their adhesion to polystyrene . Adhesion to ileostomy glycoproteins and to polystyrene varied significantly among the strains tested . Lactobacillus rhamnosus (human isolate), Lactobacillus GG, Lact . acidophilus 1 and P . freudenreichii adhered to ileostomy glycoproteins . Adhesion was concentration-dependent and the most adhesive strains were able to saturate the substratum . These results indicate that human ileostomy glycoproteins can be used as a model system to select potential probiotic strains to complement the adhesion test with intestinal cell lines. J Virol, 1999 May, 73(5), 3920 - 9 Cooperative interaction of CI protein regulates lysogeny of Lactobacillus casei by bacteriophage A2; Garcia P et al.; The temperate bacteriophage A2 forms stable lysogens in Lactobacillus casei . The A2-encoded cI product (CI), which is responsible for maintaining the A2 prophage in the lysogenic state, has been purified . The CI protein, which is a monomer of 25.3 kDa in solution, specifically binds to a 153-bp DNA fragment that contains two divergent promoters, PL and PR . These promoters mediate transcription from cI and a putative cro, respectively . Three similar, although not identical, 20-bp inverted repeated DNA segments (operator sites O1, O2, and O3) were found in this segment . CI selectively interacts with O1, which is placed downstream from the transcription start point of the cro gene, and with O2 and O3, which overlap with the -35 region of the two promoters . Using a heterologous RNA polymerase, we have determined the transcription start points of PL and PR . CI exerts a negative effect on the in vitro transcription of PR by repositioning the RNA polymerase in a concentration-dependent manner . CI, when bound to O1 and O2, enhances the positioning of the RNA polymerase with the PL promoter . Our data indicate that the CI protein regulates the lytic and lysogenic pathways of the A2 phage. J Dairy Sci, 1999 Mar, 82(3), 520 - 6 Effect of applying lactic acid bacteria isolated from forage crops on fermentation characteristics and aerobic deterioration of silage; Cai Y et al.; Two selected strains, Lactobacillus casei FG 1 and Lactobacillus plantarum FG 10 that were isolated from forage crops were used as additives at 1.0 x 10(5) cfu/g of fresh matter to alfalfa, Italian ryegrass, and sorghum, and their effect on fermentation characteristics and aerobic deterioration of silage was studied . The three silages treated with strains FG 1 or FG 10 were well preserved; had significantly lower pH values, butyric acid, propionic acid, and ammonia N concentrations, gas production, and dry matter losses; and had significantly higher contents of residual water-soluble carbohydrates and lactic acid than did the respective control silages . Yeast counts were high in all treated silages and increased rapidly during aerobic exposure . As a result, treated silages spoiled faster upon aerobic exposure than did the respective control silages . Most yeasts isolated from deteriorated silages showed high tolerance to lactic acid but low tolerance to butyric acid, and they were able to grow at low pH conditions and assimilate lactic acid . The results confirmed that L . casei and L . plantarum improved fermentation quality but did not inhibit the growth of silage yeast or aerobic deterioration of the silage. J Dairy Sci, 1999 Mar, 82(3), 472 - 80 Bile salt hydrolase activity of three strains of Lactobacillus acidophilus; Corzo G et al.; Three strains of Lactobacillus acidophilus, two from human intestinal origin (016 and L1) and one from porcine intestinal origin (ATCC 43121), were tested for their bile salt deconjugation activity . The L . acidophilus ATCC 43121 had more deconjugating activity of both sodium glycocholate and sodium taurocholate at pH 6.5 than did either L . acidophilus 016 or L1 . The activity of intracellular bile salt hydrolase found in strain ATCC 43121 was 14-fold higher than that in either of the other two strains . The optimum pH for deconjugation of sodium glycocholate was between 4 and 5.5 for all three strains . For deconjugation of sodium taurocholate, the optimum pH was between 3.5 and 4.5 for strains L1 and ATCC 43121 and was between pH 5 and 6 for strain O16 . The molecular mass of the enzyme in all three strains of L . acidophilus was estimated to be 126 kDa by Sephadex G-200 gel filtration . All three strains exhibited more bile salt hydrolase activity towards sodium glycocholate than towards sodium taurocholate. J Dairy Sci, 1999 Mar, 82(3), 466 - 71 Measurement of bile salt hydrolase activity from Lactobacillus acidophilus based on disappearance of conjugated bile salts; Corzo G et al.; Bile salt hydrolase activity of Lactobacillus acidophilus was measured based on the disappearance of sodium glycocholate and sodium taurocholate from the reaction mixture using HPLC . The amount of sodium glycocholate and sodium taurocholate that disappeared was proportional to the amount of sodium cholate that appeared in the mixture as detected by HPLC . Sodium glycocholate did not precipitate at the enzyme reaction conditions (37 degrees C and pH 5.4) for determining bile salt hydrolase activity . The bile salt hydrolase assay was insensitive to low oxidation-reduction potential when measuring bile salt hydrolase from L . acidophilus, an intestinal microorganism . However, EDTA and freezing temperatures were necessary to maintain stability of the partially purified enzyme during storage. Biosci Biotechnol Biochem, 1999 Feb, 63(2), 373 - 8 Immunopotentiating activity of nigerooligosaccharides for the T helper 1-like immune response in mice; Murosaki S et al.; The immunopotentiating activity of nigerooligosaccharides (NOS), a mixture of nigerose, nigerosyl glucose and nigerosyl maltose, was studied in vitro and in vivo in mice . Mitogen-induced proliferation of splenocytes from normal mice was augmented in a dose-dependent manner by nigerose of NOS . NOS enhanced interleukin 12 (IL-12) and interferon-gamma (IFN-gamma) production by normal splenocytes in the presence of the potent IL-12 inducer, heat-killed Lactobacillus plantarum L-137, in vitro . Consistent with the in vitro finding, L . plantarum L-137-induced IL-12 production and IL-2-induced IFN-gamma production were augmented in mice fed with a 14.6% NOS diet for 2 weeks compared with mice fed with a control diet . Notably, mice fed with the NOS diet showed significantly longer survival time than the control mice after the induction of an endogenous infection by administering 5-fluorouracil in a lethal dose . Taken together, these results suggest that NOS may exert immunopotentiating activity through the activation of an IL-12-dependent T helper 1-like immune response. Histochem J, 1998 Feb, 30(2), 75 - 80 Visualization of vaginal flora in cervical smears using a modified microwave silver-staining method; Boon ME et al.; The advantage of studying the vaginal flora to determine the bacteria and fungi present in cervical smears (as opposed to cultivation of these micro-organisms) is that the micro-organisms can be observed in their natural habitat . However, they are only faintly stained by the conventional Papanicolaou method . Accordingly, contrast is weak and visualization poor . For this reason, we developed a modified microwave silver-staining method that can be performed retrospectively on stained smears . Bacteria and fungi stain distinctly black and can be studied in greater detail, and their inter-relationship can be visualized . Haematoxylin or Eosin counterstain allows us to visualize vaginal inhabitants in relation to epithelial cells . In the series presented here, we show that a modified microwave silver-staining method is well suited to studying the ecology of micro-organisms in smears taken from women presenting to their doctor with clinical symptoms . Using this staining method, we have shown that lactobacilli overgrowth is associated with symptoms. Appl Environ Microbiol, 1999 Apr, 65(4), 1450 - 4 Effectiveness of chemometric techniques in discrimination of lactobacillus helveticus biotypes from natural dairy starter cultures on the basis of phenotypic characteristics Gatti M, Contarini G, Neviani E. Lactobacillus helveticus is the dominant organism in natural starter cultures used for the production of typical Italian cheeses . In this study, 74 L . helveticus strains, isolated from grana and provolone cheese natural whey starters, were distinguished with respect to their origin by using both cell wall protein profiles and chemometric evaluation of some phenotypic parameters, such as the ability to acidify cultures and the presence of nonspecific proteolytic and peptidase activities . Cell wall protein patterns allowed L . helveticus strains to be distinguished with respect to their source of isolation . Among the different phenotypes studied, no single specific parameter permitted the two groups of strains to be separated . A good discrimination between the two groups of L . helveticus species was obtained by multivariate statistical techniques, which permitted the extraction of all of the discriminating information retained in the phenotypic activities . Associations between strain phenotype expression and dairy environmental ecosystem source are discussed. Appl Environ Microbiol, 1999 Apr, 65(4), 1384 - 9 Homofermentative production of D- or L-lactate in metabolically engineered Escherichia coli RR1; Chang DE et al.; We investigated metabolic engineering of fermentation pathways in Escherichia coli for production of optically pure D- or L-lactate . Several pta mutant strains were examined, and a pta mutant of E . coli RR1 which was deficient in the phosphotransacetylase of the Pta-AckA pathway was found to metabolize glucose to D-lactate and to produce a small amount of succinate by-product under anaerobic conditions . An additional mutation in ppc made the mutant produce D-lactate like a homofermentative lactic acid bacterium . When the pta ppc double mutant was grown to higher biomass concentrations under aerobic conditions before it shifted to the anaerobic phase of D-lactate production, more than 62.2 g of D-lactate per liter was produced in 60 h, and the volumetric productivity was 1.04 g/liter/h . To examine whether the blocked acetate flux could be reoriented to a nonindigenous L-lactate pathway, an L-lactate dehydrogenase gene from Lactobacillus casei was introduced into a pta ldhA strain which lacked phosphotransacetylase and D-lactate dehydrogenase . This recombinant strain was able to metabolize glucose to L-lactate as the major fermentation product, and up to 45 g of L-lactate per liter was produced in 67 h . These results demonstrate that the central fermentation metabolism of E . coli can be reoriented to the production of D-lactate, an indigenous fermentation product, or to the production of L-lactate, a nonindigenous fermentation product. Int J Food Microbiol, 1999 Feb 18, 46(3), 219 - 29 Characterization of lactic acid bacteria isolated from a Thai low-salt fermented fish product and the role of garlic as substrate for fermentation; Paludan-Muller C et al.; Lactic acid bacteria (LAB) isolated from raw materials (fish, rice, garlic and banana leaves) and processed som-fak (a Thai low-salt fermented fish product) were characterized by API 50-CH and other phenotypic criteria . Lactococcus lactis subsp . lactis and Leuconostoc citreum were specifically associated with fish fillet and minced fish, Lactobacillus paracasei subsp . paracasei with boiled rice and Weisella confusa with garlic mix and banana leaves . In addition, Lactobacillus plantarum, Lactobacillus pentosus and Pediococcus pentosaceus were isolated from raw materials . A succession of aciduric, homofermentative lactobacillus species, dominated by Lb . plantarum/pentosus, was found during fermentation . In total, 9% of the strains fermented starch and 19% fermented garlic, the two main carbohydrate components in som-fak . The ability to ferment garlic was paralleled by a capacity to ferment inulin . An increased percentage of garlic fermenting strains was found during fermentation of som-fak, from 8% at day 1 to 40% at day 5 . No starch fermenting strains were isolated during fermentation . Three mixed LAB cultures, composed of either starch fermenting Lc . lactis subsp . lactis and Lb . paracasei subsp . paracasei, or garlic fermenting Lb . plantarum and Pd . pentosaceus, or a combination of these strains were inoculated into laboratory prepared som-fak with or without garlic . In som-fak without garlic, pH was above 4.8 after three days, irrespective of addition of mixed LAB cultures . The starch fermenting LAB were unable to ferment som-fak and sensory spoilage occurred after three days . Fermentation with the combined mix of starch and garlic fermenting strains led to production of 2.5% acid and a decrease in pH to 4.5 in two days . The fermentation was slightly slower with the garlic fermenting strains alone . This is the first report describing the role of garlic as carbohydrate source for LAB in fermented fish products. Int J Food Microbiol, 1999 Feb 18, 46(3), 207 - 17 Effect of ecological factors on the inhibitory spectrum and activity of bacteriocins; Ganzle MG et al.; The effect of food components and ecological factors on the activities of nisin, sakacin P and curvacin A was evaluated . Lactobacillus curvatus, Listeria innocua, Salmonella and Escherichia coli including E . coli O157:H7 were used as target organisms . Lecithin, casein, and divalent cations were antagonists of the bacteriocins at 0.1%, 0.1% and 10 mmol l(-1), respectively . A decrease in pH as well as the presence of EDTA, propyl-parabene or NaCl at concentrations of 0-1 mmol y(-1), 0-0.16 g l(-1), and 0-6% (w/w), respectively, increased the activity of all bacteriocins . These compounds as well as a pH < 5.5 rendered the Gram-negative target organisms sensitive against bacteriocins . Of practical importance is the respective effect of NaCl at concentrations > 5% which are achieved in fermentation and ripening processes, e.g . in production of fermented sausages . A characteristic response was observed for each of the bacteriocins . It is suggested that bacteriocins of lactic acid bacteria are effective against a wide range of microorganisms including E . coli O157:H7 if applied in combination with other preservative principles prevailing in foods. Antibiot Khimioter, 1999, 44(1), 23 - 5 {Effectiveness of acipole in prevention of enteric dysbacteriosis due to antibacterial therapy}; Oleinichenko EV et al.; Acipole (Lactobacillus acidophilus + Kefir greins) was used to manage antibiotic dysbacteriosis as an adverse reaction of antibacterial therapy . 120 patients treated with antibacterial drugs for acute pneumonia and exacerbations of chronic bronchitis were observed . 54 of them were treated under the routine regimen with antibiotics and 66 were additionally treated with the eubiotic acipole: 1 tablet (5 doses) 3 times a day 30 minutes before meal . Routine bacteriological examination of the feces was applied to all the patients . High frequency of bacteriologically revealed dysbacteriosis was stated . The therapy under the antibiotic + acipole regimen lowered the frequency of dysbacteriosis events and their severity . The fact that the use of acipole simultaneously with the routine antibacterial therapy prevented the development of dysbacteriosis clinical signs is of practical importance. J Bacteriol, 1999 Apr, 181(7), 2192 - 8 The bspA locus of Lactobacillus fermentum BR11 encodes an L-cystine uptake system; Turner MS et al.; BspA is a basic surface-exposed protein from Lactobacillus fermentum BR11 . Sequence comparisons have shown that it is a member of family III of the solute binding proteins . It is 89% identical to the collagen binding protein, Cnb, from Lactobacillus reuteri . Compared with the database of Escherichia coli proteins, BspA is most similar to the L-cystine binding protein FliY . To investigate the function of BspA, mutants depleted for BspA were generated by homologous recombination with a temperature-sensitive plasmid . These mutants were significantly impaired in their abilities to take up L-cystine . Uptake rates of L-glutamine, L-histidine, and L-lysine, which are substrates for other binding proteins with similarity to BspA, were unaffected . Evidence was obtained that BspA is necessary for maximal resistance to oxidative stress . Specifically, inactivation of BspA causes defective growth in the presence of oxygen and sensitivity to paraquat . Measurements of sulfhydryl levels showed that incubation of L . fermentum BR11 with L-cystine resulted in increased levels of sulfhydryl groups both inside and outside the cell; however, this was not the case with a BspA mutant . The role of BspA as an extracellular matrix protein adhesin was also addressed . L . fermentum BR11 does not bind to immobilized type I collagen or laminin above background levels but does bind immobilized fibronectin . Inactivation of BspA did not significantly affect fibronectin binding; therefore, we have not found evidence to support the notion that BspA is an extracellular matrix protein binding adhesin . As BspA is most probably not a lipoprotein, this report provides evidence that gram-positive bacterial solute binding proteins do not necessarily have to be anchored to the cytoplasmic membrane to function in solute uptake. Protein Sci, 1999 Mar, 8(3), 467 - 81 Structure and dynamics in solution of the complex of Lactobacillus casei dihydrofolate reductase with the new lipophilic antifolate drug trimetrexate; Polshakov VI et al.; We have determined the three-dimensional solution structure of the complex of Lactobacillus casei dihydrofolate reductase and the anticancer drug trimetrexate . Two thousand seventy distance, 345 dihedral angle, and 144 hydrogen bond restraints were obtained from analysis of multidimensional NMR spectra recorded for complexes containing 15N-labeled protein . Simulated annealing calculations produced a family of 22 structures fully consistent with the constraints . Several intermolecular protein-ligand NOEs were obtained by using a novel approach monitoring temperature effects of NOE signals resulting from dynamic processes in the bound ligand . At low temperature (5 degrees C) the trimethoxy ring of bound trimetrexate is flipping sufficiently slowly to give narrow signals in slow exchange, which give good NOE cross peaks . At higher temperature these broaden and their NOE cross peaks disappear thus allowing the signals in the lower-temperature spectrum to be identified as NOEs involving ligand protons . The binding site for trimetrexate is well defined and this was compared with the binding sites in related complexes formed with methotrexate and trimethoprim . No major conformational differences were detected between the different complexes . The 2,4-diaminopyrimidine-containing moieties in the three drugs bind essentially in the same binding pocket and the remaining parts of their molecules adapt their conformations such that they can make effective van der Waals interactions with essentially the same set of hydrophobic amino acids, the side-chain orientations and local conformations of which are not greatly changed in the different complexes (similar chi1 and chi2 values). Appl Microbiol Biotechnol, 1999 Feb, 51(2), 249 - 54 Influence of environmental factors on lipase production by Lactobacillus plantarum; Lopes Mde F et al.; A strain of Lactobacillus plantarum, DSMZ 12028 (Deutsch Sammlung von Mikroorganismen und Zellkulturen), isolated from a Portuguese dry fermented sausage, "chourico", was found to produce true lipase, producing free fatty acids from triolein (olive oil) . This enzymatic activity was found in whole cells, but was negligible in comparison to lipolytic activity in culture supernatant . Therefore, only extracellular activity was studied . The effect of pH, temperature and glucose concentration on extracellular lipase production was studied in continuously stirred tank reactors, the first time this technology has been used to study the production of this enzyme in lactobacilli . Maximum lipase production was achieved at a pH of 5.5 and 30 degrees C and was kept at a significant level over a wide range of dilution rates (0.05-0.4 h-1); the production of lipase was still significant for low pH values, temperature and glucose concentration, conditions that are close to the ones present during chourico ripening . The effect of glucose concentration was also studied in a batch system . The control of lipase production was found to be related both to glucose concentration in the medium and to the growth rate/dilution rate . Glucose concentration was found to be important for fast lipase production, although it did not influence the maximum lipase activity reached in a batch culture. J Food Prot, 1999 Mar, 62(3), 257 - 61 Application of reuterin produced by Lactobacillus reuteri 12002 for meat decontamination and preservation; El-Ziney MG et al.; Lactobacillus reuteri strain 12002 was used for reuterin production in the two-step fermentation process . A batch culture fermentation was used to produce a maximum biomass of L . reuteri . Then cells were harvested, resuspended in a glycerol-water solution, and anaerobically incubated to produce reuterin . The lyophilized supernatants (approximately 4000 activity units (AU) of reuterin per ml) were diluted in distilled water for decontamination and preservation trials . The MIC values of reuterin for Escherichia coli O157:H7 and Listeria monocytogenes were 4 and 8 AU/ml, respectively . In meat decontamination experiments, the surface of cooked pork was inoculated with either L . monocytogenes or E . coli O157:H7 at a level of approximately log10 5 CFU/cm2, incubated for 30 min at 7 degrees C, and decontaminated by exposure to reuterin (500 AU/ml) . The bactericidal effect of reuterin was analyzed 15 s and 24 h after exposure at 7 degrees C . After 15 s of exposure to reuterin, viable numbers decreased by 0.45 and 0.3 log10 CFU/cm2 for E . coli O157:H7 and L . monocytogenes, respectively . After 24 h the numbers decreased by 2.7 log10 CFU/cm2 for E . coli O157:H7 and by 0.63 log10 CFU/cm2 for L . monocytogenes . In the same experiment, the combined effect of reuterin and lactic acid was also investigated . Adding lactic acid (5%, vol/vol) to reuterin significantly enhanced (P < or = 0.05) the efficacy of reuterin . No additional effect (P < or = 0.05) was found when ethanol (40%) was added to the mixture of reuterin and lactic acid . To evaluate the preservative effect of reuterin during meat storage, reuterin was added to raw ground pork contaminated with E . coli O157:H7 or L . monocytogenes . Reuterin at a concentration of 100 AU/g resulted in a 5.0-log10 reduction of the viability of E . coli O157:H7 after 1 day of storage at 7 degrees C . Reuterin at a concentration of 250 AU/g reduced the number of the viable cells of L . monocytogenes by log10 3.0 cycles after 1 week of storage at 7 degrees C. Arch Biochem Biophys, 1999 Apr 1, 364(1), 115 - 21 Permeability and stability properties of membranes formed by lipids extracted from Lactobacillus acidophilus grown at different temperatures; Fernandez Murga ML et al.; Lactobacillus acidophilus CRL 640 grown at 25 and 37 degrees C showed a high content of cardiolipin, phosphatidylglycerol, and glycolipids . Cultures grown at 25 degrees C showed a twofold increase in glycolipids in relation to phospholipids, a twofold increase in the C16:0 and a fourfold increase in the C18:2 fatty acids . In contrast, the C19-cyc and the 10-hydroxy acid (C18:0-10 OH) species showed a noticeable decrease . Extracts of total lipids of bacteria grown at 25 and 37 degrees C dispersed in water yielded particles having a high negative surface potential as measured by electrophoretic mobility . Vesicles prepared by extrusion of these dispersions through polycarbonate membranes of 100-nm pore diameter showed high trapping of carboxyfluorescein (CF), which remained unchanged for at least 20 h . The fluorescence anisotropy measured with diphenylhexatriene (DPH) and the generalized polarization of Laurdan were significantly lower in vesicles prepared with lipids containing the highest glycolipid ratio, in comparison to those of bacteria grown at 37 degrees C . No phase transition was detected between 5 and 50 degrees C as measured with both probes . In accordance with these results, no significant release of the trapped CF in this range of temperature was detected . Bile salts and NaCl promoted an increase in the fluorescence, which is interpreted as a change in the permeability properties of the membrane . This effect was lower with KCl, while CaCl2 did not cause any change . The greater permeability change was observed in vesicles with a low glycolipid/phospholipid ratio . NaCl did not affect the packing of the interface as measured with Laurdan, in contrast to CaCl2 . The action of Ca+2 may be ascribed to the binding to the negatively charged lipids, such as phosphatidyl glycerol and cardiolipin . It is concluded that the higher glycolipid/phospholipid ratio and the fatty acids C18:2 and C16:0 enhance the lipid membrane stability and decrease the organization in the interfacial and hydrocarbon zones . These results are congruent with the behavior of entire bacteria subject to osmotic and freeze/thaw stresses . Community Dent Oral Epidemiol, 1999 Feb, 27(1), 31 - 40 Prevention and reversal of dental caries: role of low level fluoride; Featherstone JD; Dental caries is a bacterially based disease that progresses when acid produced by bacterial action on dietary fermentable carbohydrates diffuses into the tooth and dissolves the mineral, that is, demineralization . Pathological factors including acidogenic bacteria (mutans streptococci and lactobacilli), salivary dysfunction, and dietary carbohydrates are related to caries progression . Protective factors which include salivary calcium, phosphate and proteins, salivary flow, and fluoride in saliva can balance, prevent or reverse dental caries . Fluoride works primarily via topical mechanisms which include (1) inhibition of demineralization at the crystal surfaces inside the tooth, (2) enhancement of remineralization at the crystal surfaces (the resulting remineralized layer is very resistant to acid attack), and (3) inhibition of bacterial enzymes . Fluoride in drinking water and in fluoride-containing products reduces tooth decay via these mechanisms . Low but slightly elevated levels of fluoride in saliva and plaque provided from these sources help prevent and reverse caries by inhibiting demineralization and enhancing remineralization . The level of fluoride incorporated into dental mineral by systemic ingestion is insufficient to play a significant role in caries prevention . The effect of systemically ingested fluoride on caries is minimal . Fluoride "supplements" can be best used as a topical delivery system by sucking or chewing tablets or lozenges prior to ingestion. FEMS Microbiol Lett, 1999 Feb 15, 171(2), 133 - 40 NADH reoxidation does not control glycolytic flux during exposure of respiring Saccharomyces cerevisiae cultures to glucose excess; Brambilla L et al.; Introduction of the Lactobacillus casei lactate dehydrogenase (LDH) gene into Saccharomyces cerevisiae under the control of the TPI1 promoter yielded high LDH levels in batch and chemostat cultures . LDH expression did not affect the dilution rate above which respiro-fermentative metabolism occurred (Dc) in aerobic, glucose-limited chemostats . Above Dc, the LDH-expressing strain produced both ethanol and lactate, but its overall fermentation rate was the same as in wild-type cultures . Exposure of respiring, LDH-expressing cultures to glucose excess triggered simultaneous ethanol and lactate production . However, the specific glucose consumption rate was not affected, indicating that NADH reoxidation does not control glycolytic flux under these conditions. Appl Microbiol Biotechnol, 1999 Jan, 51(1), 71 - 8 The expression signals of the Lactobacillus brevis slpA gene direct efficient heterologous protein production in lactic acid bacteria; Kahala M et al.; A cassette based on the expression signals of the Lactobacillus brevis surface (S)-layer protein gene (slpA) was constructed . The low-copy-number vector pKTH2095, derived from pGK12, was used as the cloning vector . The efficiency of slpA promoters in intracellular protein production was studied using three reporter genes, beta-glucuronidase (gusA), luciferase (luc) and aminopeptidase N (pepN) in three different lactic acid bacteria hosts: Lactococcus lactis, Lactobacillus plantarum and Lactobacillus gasseri . The S-layer promoters were recognized in each strain and especially L . lactis and Lb . plantarum exhibited high levels of transcripts . The production kinetics of reporter proteins was studied as a function of growth . The GusA, Luc and PepN activities varied considerably among the lactic acid bacterial strains studied . The highest levels of beta-glucuronidase and luciferase activity were obtained in L . lactis . The level of GusA obtained in L . lactis corresponded to over 15% of the total cellular proteins . The highest level of aminopeptidase N activity was achieved in Lb . plantarum where PepN corresponded up to 28% of the total cellular proteins at the late exponential phase of growth . This level of PepN activity is 30-fold higher than that in Lb . helveticus, which is the species from which the pepN gene originates. FEMS Immunol Med Microbiol, 1999 Feb, 23(2), 87 - 92 Growth inhibition of Staphylococcus aureus by H2O2-producing Lactobacillus paracasei subsp . paracasei isolated from the human vagina; Ocana VS et al.; H2O2 production by certain Lactobacillus strains is one of the mechanisms that helps to regulate the vaginal ecosystem . This paper describes the kinetics of H2O2 production by two different strains of Lactobacillus paracasei subsp . paracasei under different culture conditions and the effect of this metabolite on the growth of Staphylococcus aureus . L . paracasei F2 produced 2.72 mmol 1-1 H2O2 while L . paracasei F28 produced 1.84 mmol l(-1), both in agitated cultures . Although L . paracasei F2 produced a higher H2O2 concentration than L . paracasei F28, H2O2 production per number of live bacterial cells was 10-fold higher for F28 . The latter also showed a faster decrease in viability during the stationary phase . There were no detectable levels of H2O2 in cultures without agitation . H2O2-producing lactobacilli inhibited growth of S . aureus in a plaque assay and in mixed cultures, depending on the initial inoculum of the pathogen. Microbiology, 1999 Feb, 145 ( Pt 2), 437 - 46 Characterization of a prolidase from Lactobacillus delbrueckii subsp . bulgaricus CNRZ 397 with an unusual regulation of biosynthesis; Morel F et al.; Lactobacillus delbrueckii subsp . bulgaricus CNRZ 397 (Lb . bulgaricus) is characterized by a high level of peptidase activities specific to proline-containing peptides . A prolidase (PepQ, EC 3.4.13.9) was purified to homogeneity and characterized as a strict dipeptidase active on X-Pro dipeptides, except Gly-Pro and Pro-Pro . The values for Km and Vmax were, respectively, 2.2 mM and 0.33 mmol min(-1) mg(-1), with Leu-Pro as the substrate . The enzyme exhibited optimal activity at 50 degrees C and pH 6.0, and required the presence of Zn2+ . Size exclusion chromatographies and SDS-PAGE analysis led to the conclusion that this prolidase was a homodimer . Antibodies raised against the purified protein allowed the detection of PepQ among several Lactobacillus species but not lactococci . The pepQ gene and the upstream region were isolated and sequenced . The deduced peptide sequence showed that PepQ belongs to the M24 family of metallopeptidases . The pepR1 gene is located immediately upstream of pepQ and its product is homologous to the transcription factor CcpA, which is involved in catabolite repression of catabolic operons from Gram-positive bacteria . The pepR1-pepQ intergenic region contains a consensus catabolite-responsive element (CRE) which could be a target for PepR1 protein . Moreover, in contrast to other proline-specific enzymes from Lb . bulgaricus, PepQ biosynthesis was shown to be dependent on the composition of the culture medium, but not on the peptide concentration . A possible regulation mechanism is discussed. J Dent, 1999 Feb, 27(2), 101 - 6 The effect of parabens in a mouthwash and incorporated into a sustained release varnish on salivary bacteria; Steinberg D et al.; OBJECTIVES: The purpose of this study was to evaluate the antimicrobial efficacy of parabens, in a mouthwash and in a slow release device, against cariogenic bacteria in the oral cavity . METHODS: Parabens were formulated in a mouthwash or in a topical slow release varnish preparation . In two separate studies, volunteers were asked to use the mouthwash or the slow release varnish applied in a personal night guard device . The levels of mutans streptococci (MS), Lactobacilli (LB) and total cultivated bacteria were measured before and after using the mouthwash or the slow release varnish . RESULTS: The mouthwash containing parabens had a minor and a short-term effect on MS and LB counts in human saliva . The use of slow release varnish containing parabens, applied in a night guard, had a prolonged antibacterial effect on both MS and LB counts . CONCLUSIONS: Parabens in a mouthwash had little effect on oral bacteria counts; however, paraben in the slow release device had a significant and an extended effect in reducing oral bacteria . Although substantial reductions in oral bacterial counts were recorded after the use of parabens in a slow release device, this effect could probably be enhanced by an improved pharmaceutical formulation. Mol Gen Genet, 1999 Feb, 261(1), 31 - 41 Isolation and characterization of a purC(orf)QLF operon from Lactococcus {correction of Lactobacillus} lactis MG1614; Peltonen T et al.; We have isolated genes encoding enzymes of the de novo purine nucleotide biosynthesis pathway from Lactococcus lactis MG1614 by colony hybridization using DIG-labeled DNA probes . The organization of the genes needed for the de novo biosynthesis of purine nucleotides in L . lactis differs from that found in other organisms . In L . lactis there is a gene cluster, which contains five out of the 11 genes needed for the de novo biosynthesis of IMP, namely purC, orf, purQ, purL and purF . These genes were shown to be transcribed as a single transcription unit by Northern hybridization analysis . The 5' end of the transcript of the purC(orf)QLF operon was determined by primer extension analysis using fluorescently end-labeled probes . The purC(orf)QLF operon of L . lactis is transcribed in Escherichia coli, and the gene product of the purF gene, glutamine phosphoribosylpyrophosphate amidotransferase (glutamine PRPP ATase, EC 2.4.2.14), can functionally complement the E . coli purF mutant strain TX158 . We also show that the promoter of the purC(orf)QLF operon is regulated in response to exogenously added purines. J Dairy Sci, 1999 Feb, 82(2), 243 - 8 Bile tolerance, taurocholate deconjugation, and binding of cholesterol by Lactobacillus gasseri strains; Usman et al.; Bile tolerance, deconjugation of sodium taurocholate, and the cholesterol-binding ability of 28 strains of Lactobacillus gasseri were examined . There was significant variation among strains in growth in media containing bile and also variation in the ability to bind cholesterol . Cultures grown for 12 h at 37 degrees C bound significantly more cholesterol than did cells from a 48-h incubation . Variation among strains in the ability to deconjugate sodium taurocholate was not significantly different . Maximal deconjugation of sodium taurocholate was achieved with the cells during the stationary phase of growth (12 h) . Statistical analysis showed no significant correlation between bile tolerance and sodium taurocholate deconjugation, bile tolerance and cholesterol-binding ability, or sodium taurocholate deconjugation and cholesterol-binding ability. J Am Coll Nutr, 1999 Feb, 18(1), 43 - 50 Effect of fermented milk (yogurt) containing Lactobacillus acidophilus L1 on serum cholesterol in hypercholesterolemic humans; Anderson JW et al.; OBJECTIVE: Two controlled clinical studies were performed to examine effects of consumption of one daily serving of fermented milk (FM) (yogurt) on serum lipids . METHODS: In the first study, subjects were randomly allocated to FM containing Lactobacillus acidophilus L1 of human origin or to FM containing L . acidophilus ATCC 43211 of swine origin . In this single-blind study, subjects consumed one 200 ml serving of FM daily for 3 weeks . The second study was a double-blind, placebo-controlled, cross-over study . Subjects completed a 4-week first treatment, had a 2-week washout, and completed a second 4-week treatment . In the second study subjects consumed FM containing L . acidophilus L1 or placebo FM over 4 weeks . RESULTS: In the first study, FM containing L . acidophilus L1 was accompanied by a 2.4% (p<0.05) reduction of serum cholesterol concentration . In the second study, strain L1 reduced serum cholesterol concentration by 3.2% (p<0.05) in the first treatment period . In the second treatment period there were no significant changes in serum cholesterol concentration . Combined analysis of the two L1 treatment studies demonstrated a 2.9% (p<0.01) reduction in serum cholesterol concentration . CONCLUSION: Since every 1% reduction in serum cholesterol concentration is associated with an estimated 2% to 3% reduction in risk for coronary heart disease, regular intake of FM containing an appropriate strain of L . acidophilus has the potential of reducing risk for coronary heart disease by 6 to 10%. J Biol Chem, 1999 Mar 12, 274(11), 7039 - 42 Binding of Cob(II)alamin to the adenosylcobalamin-dependent ribonucleotide reductase from Lactobacillus leichmannii . Identification of dimethylbenzimidazole as the axial ligand; Lawrence CC et al.; The ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii catalyzes the reduction of nucleoside 5'-triphosphates to 2'-deoxynucleoside 5'-triphosphates and uses coenzyme B12, adenosylcobalamin (AdoCbl), as a cofactor . Use of a mechanism-based inhibitor, 2'-deoxy-2'-methylenecytidine 5'-triphosphate, and isotopically labeled RTPR and AdoCbl in conjunction with EPR spectroscopy has allowed identification of the lower axial ligand of cob(II)alamin when bound to RTPR . In common with the AdoCbl-dependent enzymes catalyzing irreversible heteroatom migrations and in contrast to the enzymes catalyzing reversible carbon skeleton rearrangements, the dimethylbenzimidazole moiety of the cofactor is not displaced by a protein histidine upon binding to RTPR. Int J Food Microbiol, 1999 Jan 12, 46(1), 57 - 70 Shelf life of modified atmosphere packed cooked meat products: a predictive model; Devlieghere F et al.; The effect of temperature, concentration of dissolved CO2 and water activity on the growth of Lactobacillus sake was investigated by developing predictive models for the lag phase and the maximum specific growth rate of this specific spoilage organism for gas-packed cooked meat products . Two types of predictive model were compared: an extended Ratkowsky model and a response surface model . In general, response surface models showed a slightly better correlation, but the response surface model for the maximum specific growth rate showed illogical predictions at low water activities . The concentration of dissolved CO2 proved to be a significant independent variable for the maximum specific growth rate as well as for the lag phase of L . sake . Synergistic actions on the shelf life-extending effect were noticed between temperature and dissolved CO2, as well as between water activity and dissolved CO2 . The developed models were validated by comparison with the existing model of Kant-Muermans et al . (1997) and by means of experiments in gas-packed cooked meat products . Both developed models proved to be useful in the prediction of the microbial shelf life of gas-packed cooked meat products. Appl Environ Microbiol, 1999 Mar, 65(3), 1071 - 7 Cell surface-associated lipoteichoic acid acts as an adhesion factor for attachment of Lactobacillus johnsonii La1 to human enterocyte-like Caco-2 cells; Granato D et al.; The influence of pH on the adhesion of two Lactobacillus strains to Caco-2 human intestinal cells was investigated . One strain, Lactobacillus johnsonii La1, was adherent at any pH between 4 and 7 . The other one, L . acidophilus La10, did not attach to this cell line under the same experimental conditions . On the basis of these results, we used the monoclonal antibody technique as a tool to determine differences on the surface of these bacteria and to identify a factor for adhesion . Mice were immunized with live La1, and the hybridomas produced by fusion of spleen cells with ONS1 cells were screened for the production of antibodies specific for L . johnsonii La1 . A set of these monoclonal antibodies was directed against a nonproteinaceous component of the L . johnsonii La1 surface . It was identified as lipoteichoic acid (LTA) . This molecule was isolated, chemically characterized, and tested in adhesion experiments in the same system . The adhesion of L . johnsonii La1 to Caco-2 cells was inhibited in a concentration-dependent way by purified LTA as well as by L . johnsonii La1 culture supernatant that contained LTA . These results showed that the mechanism of adhesion of L . johnsonii La1 to human Caco-2 cells involves LTA. Appl Environ Microbiol, 1999 Mar, 65(3), 974 - 81 Temperature and pH conditions that prevail during fermentation of sausages are optimal for production of the antilisterial bacteriocin sakacin K; Leroy F et al.; Sakacin K is an antilisterial bacteriocin produced by Lactobacillus sake CTC 494, a strain isolated from Spanish dry fermented sausages . The biokinetics of cell growth and bacteriocin production of L . sake CTC 494 in vitro during laboratory fermentations were investigated by making use of MRS broth . The data obtained from the fermentations was used to set up a predictive model to describe the influence of the physical factors temperature and pH on microbial behavior . The model was validated successfully for all components . However, the specific bacteriocin production rate seemed to have an upper limit . Both cell growth and bacteriocin activity were very much influenced by changes in temperature and pH . The production of biomass was closely related to bacteriocin activity, indicating primary metabolite kinetics, but was not the only factor of importance . Acidity dramatically influenced both the production and the inactivation of sakacin K; the optimal pH for cell growth did not correspond to the pH for maximal sakacin K activity . Furthermore, cells grew well at 35 degrees C but no bacteriocin production could be detected at this temperature . L . sake CTC 494 shows special promise for implementation as a novel bacteriocin-producing sausage starter culture with antilisterial properties, considering the fact that the temperature and acidity conditions that prevail during the fermentation process of dry fermented sausages are optimal for the production of sakacin K. J Immunol Methods, 1999 Feb 1, 223(1), 131 - 6 A rapid and safe plasmid isolation method for efficient engineering of recombinant lactobacilli expressing immunogenic or tolerogenic epitopes for oral administration; Maassen CB; Recombinant lactobacilli are being developed which can be used as expression and delivery vectors of heterologous antigens in oral vaccination and other therapeutic applications . Because most Lactobacillus strains do not accept ligation mixtures, sufficiently pure plasmid DNA needs to be isolated from Lactobacillus casei to transform other Lactobacillus strains . The isolation of plasmid DNA from Gram-positive lactobacilli is complicated by the resilience of the peptidoglycan layer . Here a rapid, safe and efficient method is described that combines enzymatic breakdown of the cell wall and purification of the plasmid by commercially available DNA-binding columns . For the lysis-resistant L . casei strain, this method yields high levels of pure plasmid DNA that can be used for common molecular techniques, such as digestion and transformation, with high efficiency. Sex Transm Dis, 1999 Feb, 26(2), 96 - 102 Race/ethnicity, vaginal flora patterns, and pH during pregnancy; Royce RA et al.; OBJECTIVES: To investigate the relationship between bacterial vaginosis during pregnancy and black race/ethnicity . STUDY DESIGN: Gram staining was used to evaluate vaginal flora in 842 women at 24 to 29 weeks' gestation . RESULTS: Overall, 22.3% of blacks and 8.5% of whites had bacterial vaginosis . Vaginal pH and flora differed significantly by race/ethnicity; blacks were more likely to have pH > or = 4.5, no lactobacilli, small gram-variable and -negative rods, and Mobiluncus compared with whites (odds ratios 1.6, 1.5, 1.4, and 10.6, respectively) . Quantity of morphotypes also differed, especially for Mobiluncus . Among women with Mobiluncus present (12.0% of blacks and 1.3% of whites), 73.3% of blacks compared with 40.0% of whites had the highest level . Adjustment for sociodemographics, sexual activity, sexually transmitted diseases, health behavior, and sexual hygiene did not explain these differences . CONCLUSION: We observed race/ethnicity differences in vaginal flora ecology . These differences may ultimately play a role in the larger proportion of preterm deliveries among black women compared with white women. Z Geburtshilfe Neonatol, 1998 Nov-Dec, 202(6), 247 - 50 {Initial results of the Erfurt Prevention of Prematurity Campaign}; Hoyme UB et al.; OBJECTIVES: Genital infection particularly bacterial vaginosis (BV) increases the relative risk of prematurity . Detection of disturbances of vaginal milieu at an early stage and the use of suitable countermeasures such as intervention with antimicrobial substances, e.g . clindamycin, can reduce the preterm birth rate, provided the diagnosis is made early enough . STUDY DESIGN: Since October 1996 pregnant women being given prenatal care in 16 of the 29 outpatient offices in Erfurt, have been informed about the Prematurity Prevention Programme and have been offered to take part and to perform self-measurements of their vaginal pH twice a week in order to screen for any disturbances in the vaginal milieu . Special CarePlan-VpH gloves (Selfcare, Oberhaching) were used to identify patients a risk (pH > 4.7) . The pregnant women taking part in the programme were instructed to see their physician immediately, if abnormal values were present, in order to get them confirmed and to start lactobacillus acidophilus therapy (Gynoflor, Nourypharma, Oberschleissheim) or, in case of BV, to treat with clindamycin cream (Sobelin, Upjohn, Erlangen) i.vag . Patients being given prenatal care in the 13 outpatient offices not participating and other pregnant women in Erfurt who were not interested in the programme served as control group . RESULTS: Up to now 59 out of 314 women in the intervention group have been identified as risk cases (p > or = 4.7) . 52 of them were treated with a lactobacillus preparation, and 19 additionally with clindamycin cream, 3 patients refused to have any therapy . In this ongoing study the prematurity rate was 8.3% in the self-measurement/intervention group vs . 13.0% in the control group (n = 1,842); 0.3% vs . 3.3% of the neonates belonged to the group of very early prematures with a gestational age of < 32 + 0 weeks (p < 0.01) . PROM was registered in 22.3% vs . 32.1% (p < 0.001) respectively . CONCLUSION: Self-measurement of vaginal pH at close intervals, as recommended by Saling, leads to the early identification of women at risk for prematurity . Earliest possible intervention by the obstetrician appears to result in reducing the rate of prematures and in particular of very early prematures (< 32 + 0 weeks). Int J Syst Bacteriol, 1999 Jan, 49 Pt 1, 217 - 21 Phenotypic and phylogenetic characterization of a novel Lactobacillus species from human sources: description of Lactobacillus iners sp . nov; Falsen E et al.; Eleven strains of a hitherto undescribed Gram-positive, catalase-negative, facultatively anaerobic rod-shaped bacterium from human sources and medical care products were characterized by phenotypic and molecular taxonomic methods . The phenotypic properties of the bacterium were consistent with its assignment to the genus Lactobacillus but it was readily distinguished from all currently described species of this genus by its biochemical characteristics and by SDS-PAGE analysis of its cellular proteins . Comparative 16S rRNA gene sequence analysis demonstrated that the unknown bacterium was a member of rRNA group I Lactobacillus which includes Lactobacillus delbrueckii, the type species of the genus, and close relatives . Lactobacillus gasseri and Lactobacillus johnsonii were the nearest phylogenetic relatives of the unknown bacterium, but 16S rRNA sequence divergence values of > 4% clearly showed that it represents a distinct species . Based on both phylogenetic and phenotypic evidence, it is proposed that the unknown bacterium should be classified in the genus Lactobacillus, as Lactobacillus iners sp . nov . The type strain of Lactobacillus iners is CCUG 28746T. Biochemistry, 1999 Feb 16, 38(7), 2127 - 34 1H/15N HSQC NMR studies of ligand carboxylate group interactions with arginine residues in complexes of brodimoprim analogues and Lactobacillus casei dihydrofolate reductase; Morgan WD et al.; 1H and 15N NMR studies have been undertaken on complexes of Lactobacillus casei dihydrofolate reductase (DHFR) formed with analogues of the antibacterial drug brodimoprim (2,4-diamino-5-(3', 5'-dimethoxy-4'-bromobenzyl)pyrimidine) in order to monitor interactions between carboxylate groups on the ligands and basic residues in the protein . These analogues had been designed by computer modeling with carboxylated alkyl chains introduced at the 3'-O position in order to improve their binding properties by making additional interactions with basic groups in the protein . Specific interactions between ligand carboxylate groups and the conserved Arg57 residue have been detected in studies of 1H/15N HSQC spectra of complexes of DHFR with both the 4-carboxylate and the 4, 6-dicarboxylate brodimoprim analogues . The spectra from both complexes showed four resolved signals for the four NHeta protons of the guanidino group of Arg57, and this is consistent with hindered rotation in the guanidino group resulting from interactions with the 4-carboxylate group in each analogue . In the spectra of each complex, one of the protons from each of the two NH2 groups and both nitrogens are considerably deshielded compared to the shielding values normally observed for such nuclei . This pattern of deshielding is that expected for a symmetrical end-on interaction of the carboxylate oxygens with the NHeta12 and NHeta22 guanidino protons . The differences in the degree of deshielding between the complexes of the two structurally similar brodimoprim analogues and the methotrexate indicates that the shielding is very sensitive to geometry, most probably to hydrogen bond lengths . The 1H/15N HSQC spectrum of the DHFR complex with the brodimoprim-6-carboxylate analogue does not feature any deshielded Arg NHeta protons and this argues against a similar interaction with the Arg57 in this case . It has not proved possible to determine whether the 6-carboxylate in this analogue is interacting directly with any residue in the protein . 1H/15N HSQC spectra have been fully assigned for the complexes with the three brodimoprim analogues and chemical shift mapping used to explore interactions in the binding site . The 1H signals of the bound ligands for all three brodimoprim analogues have been assigned . Their 1H chemical shifts were found to be fairly similar in the different complexes indicating that the 2, 4-diaminopyrimidine and the benzyl ring are binding in essentially the same binding sites and with the same overall conformation in the different complexes . The rotation rate about the NepsilonCzeta bond in the brodimoprim-4,6-dicarboxylate complex with DHFR has been determined from a zz-HSQC exchange experiment, and its value is quite similar to that observed in the DHFR.methotrexate complex (24 +/- 10 s-1 at 8 degrees C and 50 +/- 10 s-1 at 15 degrees C, respectively) . The 1H and 15N chemical shift differences of selected amide and guanidino NH groups, measured between the DHFR complexes, provided further evidence about the interactions involving Arg57 with the 4-carboxylate and 4,6-dicarboxylate brodimoprim analogues. Proteins, 1999 Feb 15, 34(3), 356 - 68 Effect of amino acid substitutions at the subunit interface on the stability and aggregation properties of a dimeric protein: role of Arg 178 and Arg 218 at the Dimer interface of thymidylate synthase; Prasanna V et al.; The significance of two interface arginine residues on the structural integrity of an obligatory dimeric enzyme thymidylate synthase (TS) from Lactobacillus casei was investigated by thermal and chemical denaturation . While the R178F mutant showed apparent stability to thermal denaturation by its decreased tendency to aggregate, the Tm of the R218K mutant was lowered by 5 degrees C . Equilibrium denaturation studies in guanidinium chloride (GdmCl) and urea indicate that in both the mutants, replacement of Arg residues results in more labile quaternary and tertiary interactions . Circular dichroism studies in aqueous buffer suggest that the protein interior in R218K may be less well-packed as compared to the wild type protein . The results emphasize that quaternary interactions may influence the stability of the tertiary fold of TS . The amino acid replacements also lead to notable alteration in the ability of the unfolding intermediate of TS to aggregate . The aggregated state of partially unfolded intermediate in the R178F mutant is stable over a narrower range of denaturant concentrations . In contrast, there is an exaggerated tendency on the part of R218K to aggregate in intermediate concentrations of the denaturant . The 3 A crystal structure of the R178F mutant reveals no major structural change as a consequence of amino acid substitution . The results may be rationalized in terms of mutational effects on both the folded and unfolded state of the protein . Site specific amino acid substitutions are useful in identifying specific regions of TS involved in association of non-native protein structures. Clin Exp Allergy, 1998 Dec, 28(12), 1474 - 9 Probiotic bacteria down-regulate the milk-induced inflammatory response in milk-hypersensitive subjects but have an immunostimulatory effect in healthy subjects; Pelto L et al.; BACKGROUND: Probiotic bacteria can influence immune responses both specifically by stimulating antibody production and nonspecifically by enhancing phagocytosis of pathogens and modifying cytokine production . OBJECTIVE: The authors hypothesized that probiotic bacteria can alleviate hypersensitivity by influencing phagocytes . The modulation of phagocytes may be different in healthy subjects compared with hypersensitive subjects . SUBJECTS AND METHODS: In a double-blind, cross-over study, challenges with milk in milk-hypersensitive and healthy adults with or without an intestinal bacterial strain, Lactobacillus GG (ATCC 53103) were performed . The challenge-induced immunoinflammatory response was recorded by measuring the expression of phagocytosis receptors prior to and after the challenge using flow cytometry . RESULTS: In milk-hypersensitive subjects, milk challenge increased significantly the expression of CR1, FcgammaRI and FcalphaR in neutrophils and CR1, CR3 and FcalphaR in monocytes . Milk with Lactobacillus GG prevented the increase of the receptor expression . In healthy subjects, milk challenge did not influence receptor expression while milk with Lactobacillus GG increased significantly the expression of CR1, CR3, FcgammaRIII and FcalphaR in neutrophils . CONCLUSION: Probiotic bacteria appear to modulate the nonspecific immune response differently in healthy and hypersensitive subjects . This is seen as an immunostimulatory effect in healthy subjects, and as a down-regulation of immunoinflammatory response in milk-hypersensitive subjects. J Dairy Sci, 1999 Jan, 82(1), 23 - 31 Isolation and characterization of acid- and bile-tolerant isolates from strains of Lactobacillus acidophilus; Chou LS et al.; Lactic acid bacteria have been reported to be useful as a health adjunct and are commonly added to food as the delivery mechanism . The literature contains many conflicting observations for their proposed benefits, and the mechanism of action is undefined . One source of variation is the large number of strains used without proper controls supplemented . Additionally, many of the organisms are not characterized for their acid shock response or the acid-tolerance response, which are known to vary among bacterial species . Our objective was to isolate acid-resistant and bile-resistant variants of Lactobacillus acidophilus and to determine the phenotypic changes . The acid- and bile-tolerant isolates were obtained using natural selection techniques after sequential exposure to hydrochloric acid (pH 3.5 to 7.0) and mixed bile salts . The acid- and bile-tolerant isolates retained their ability to grow at pH 3.5 with 0.3% bile after the selective pressure was removed and reapplied . Isolates varied from their parents for stability in freezing, lactose utilization, protease activity, aminopeptidase activity, plasmid profile, and cell-wall fatty acid profile . These data suggest that the isolated acid- and bile-tolerant isolates possess growth advantages over that of the parents under stress conditions and may be considered as candidates for probiotic strains after further characterization with animal models. Biol Pharm Bull, 1999 Jan, 22(1), 11 - 5 Lectin-like protein fractions in lactic acid bacteria isolated from chickens; Gusils C et al.; Agglutination of fifty bacterial strains isolated from gastric-intestinal tract of chickens with yeasts treated with glutaraldehyde and glycine was assayed to study possible adhesion mechanisms to epithelia . It was found that Lactobacillus fermentum, Lactobacillus fermentum subsp . cellobiosus and Lactobacillus animalis agglutinated yeasts massively and this agglutination was inhibited in each strain by different representative sugars: agglutination was inhibited in three selected strains with 0.2 M mannose . The three strains agglutinated chicken red blood cells, but did not agglutinate human red blood cells, whereas the agglutinating capacity for other animal erythrocytes was variable . The lectins were partially purified on a Sephadex G-75 column and affinity chromatography . The molecular weight of each of the protein subunits was estimated as about 50-67 kDa using electrophoretic techniques . After treatment with lipase and metaperiodate modifications bacterial superficies and/or liberation of some structures produced an unspecific interaction with increase in the yeast agglutination and hemagglutination . The optimal conditions of work were: pH 6-7, Ca2+ 1 mM, Mg2+ 2 mM and 37-42 degrees C. Biochim Biophys Acta, 1999 Jan 11, 1429(2), 516 - 20 Cloning of a novel prolidase gene from Aureobacterium esteraromaticum; Kabashima T et al.; The prolidase gene from Aureobacterium esteraromaticum was cloned and expressed in Escherichia coli . The cloned enzyme had the same enzymatic properties as the wild-type enzyme . Kinetic analysis of the enzyme indicated that the best substrate was Pro-Hyp, which was not hydrolyzed by other prolidases . Interestingly, there was no homology between the deduced amino acid sequence of A . esteraromaticum prolidase and those of the other sources such as human E . coli and Lactobacillus . However, homology was seen with the yeast hypothetical protein YJL213w, the function of which is unknown . These findings indicate that the A . esteraromaticum prolidase is a novel enzyme different from other prolidases reported to date. Biochim Biophys Acta, 1999 Jan 11, 1429(2), 501 - 5 The prolyl aminopeptidase from Lactobacillus delbrueckii subsp . bulgaricus belongs to the alpha/beta hydrolase fold family; Morel F et al.; Prolyl aminopeptidase (PepIP) of Lactobacillus delbrueckii subsp . bulgaricus displays the Gly-x-Ser-x-Gly-Gly consensus motif surrounding the catalytic serine of the prolyl oligopeptidases family . Sequence comparison revealed that this motif and two other domains appear well conserved among bacterial PepIPs and members of the alpha/beta hydrolase fold family . Secondary structural predictions of PepIP were performed from amino acid sequence and corroborated by circular dichroism analysis . These predictions well matched the core structure of alpha/beta hydrolases organised in eight beta-sheets connected by alpha-helices . We obtained 26 mutants of PepIP by chemical or site-directed mutagenesis . Most substitutions associated with stable and inactive mutant proteins were mainly located in the three conserved boxes (including the catalytic serine motif) . Taken together, our results strongly suggest that PepIP belongs to the alpha/beta hydrolase fold family and that Ser107, Asp246 and His273 constitute the catalytic triad of the enzyme. J Clin Microbiol, 1999 Mar, 37(3), 729 - 33 Lactobacillus species identification, H2O2 production, and antibiotic resistance and correlation with human clinical status; Felten A et al.; Lactobacilli recovered from the blood, cerebrospinal fluid, respiratory tract, and gut of 20 hospitalized immunocompromised septic patients were analyzed . Biochemical carbohydrate fermentation and total soluble cell protein profiles were used to identify the species .