Serine metabolism in Lactobacillus plantarum.

This study investigated the metabolism of (L-) serine by Lactobacillus plantarum B3089 isolated from cheese. Serine was deaminated by growing cells to ammonia with the corresponding formation of acetate and formate. Serine was also deaminated by non-growing cells to ammonia but with the formation of acetate only (no production of formate). Phosphoserine and threonine were not catabolised. It is proposed that serine was deaminated by serine dehydratase (deaminase) to ammonia and pyruvate. Pyruvate was further catabolised predominantly to acetate, carbon dioxide and formate in growing cells, catalysed by pyruvate-formate lyase and pyruvate oxidase; some of the pyruvate was converted to acetoin. In non-growing cells, however, pyruvate-formate lyase was inactive and pyruvate oxidase degraded the pyruvate to acetate and carbon dioxide. Serine dehydratase activity could not be detected in cell-free extracts, presumably because of enzyme instability. The growth of L. plantarum was neither enhanced nor stimulated by serine under the current conditions. Whereas there was little difference in serine utilisation between pH 7.0 and pH 5.8, serine utilisation was decreased by 30% at pH 5.0. NaCl of up to 4% (w/v) concentration had little effect on serine utilisation. Serine had no impact on lactose metabolism. Lactose was fermented mainly to lactate (73%) with the remainder converted to an unidentified polysaccharide (27%).

The potential of dairy lactic acid bacteria to metabolise amino acids via non-transaminating reactions and endogenous transamination.

The metabolism of amino acids by 22 starter and 49 non-starter lactic acid bacteria (LAB) was studied in a system consisting of amino acids and non-growing cells without added amino acceptors such as alpha-ketoglutarate. There were significant inter- and intra-species differences in the metabolism of amino acids. Some amino acids such as alanine, arginine, aspartate, serine and branched-chain amino acids (leucine, isoleucine and valine) were utilised, whereas other amino acids such as glycine, ornithine and citrulline were produced. Alanine and aspartate were utilised by some LAB and accumulated during the incubation of other LAB. Arginine was degraded not only by Lactococcus lactis subsp. lactis (the lactococcal subspecies known to catabolise arginine), but also by pediococci, heterofermentative lactobacilli (Lactobacillus brevis and Lb. fermentum) and some unidentified homofermentative lactobacilli. Serine was utilised predominantly by homofermentative Lb. paracasei subsp. paracasei, Lb. rhamnosus and Lb. plantarum. Of the LAB studied, Lb. brevis and Lb. fermentum were the most metabolically active, utilising alanine, arginine, aspartate, glutamate and branched-chain amino acids. Leuconostocs were the least metabolically active, showing little potential to metabolise amino acids. The formation of ammonia and acetate from amino acid metabolism varied both between species and between strains within species. These findings suggest that the potential of LAB for amino acid metabolism via non-transaminating reactions and endogenous transamination will impact both on the physiology of LAB and on cheese ripening, especially when transamination is rate-limiting in the absence of an exogenous amino acceptor such as alpha-ketoglutarate.

Ester synthesis in an aqueous environment by Streptococcus thermophilus and other dairy lactic acid bacteria.

The ability of Streptococcus thermophilus ST1 and 19 other dairy lactic acid bacteria (LAB) to synthesize esters was investigated in an aqueous environment. These LAB were able to synthesize esters from alcohols and glycerides via a transferase reaction (alcoholysis) in which fatty acyl groups from glycerides were transferred to alcohols. S. thermophilus ST1 was active on tributyrin and on di- or monoglycerides of up to C10 with ethanol as the acyl acceptor. This strain was also active on a diglyceride of C6 and monoglyceride of C8 with 2-phenyl ethanol as the acyl acceptor. Alcoholysis occurred preferentially over hydrolysis. S. thermophilus ST1 had an apparent K(m) value of 250 mM for ethanol and an apparent K(m) value of 1.3 mM for tributyrin, measured against whole cells. Around 80% of both the transferase activity and the esterase activity were detected in the cell-free extract (CFE) of strain ST1. Both activities in the CFEs of five LAB tested were, to a similar degree, enhanced slightly by growth in the presence of ethanol and tributyrin. Using tributyrin and ethanol as substrates, the transferase activities ranged over 0.006-1.37 units/mg cell dry weight among the LAB tested and were both species- and strain-dependent.

Eruption of third permanent molars after the extraction of second permanent molars. Part 1: Assessment of third molar position and size.

The eruptive path of third molars after extraction of second molars was examined in 63 patients. Panoramic radiographs from the start and the end of active treatment and 3 or more years after treatment were assessed. Study models were used to compare the size of the second and third molars and to assess the final position of the third molars. All third molars erupted; none became impacted. During eruption, maxillary third molar crowns uprighted and maintained their angulation as they came into occlusion. Mandibular third molar crowns continued to upright significantly mesiodistally after active treatment, with space closure the result of horizontal translation rather than mesial tipping. Further uprighting occurred once occlusion was established, although few became as upright as the second molars they replaced. However, mandibular third molar roots were frequently curved distally, thus the third molar crown position was invariably better than the overall tooth angulation would suggest, by 16.5 degrees on average. Model analysis (Richardsons' scoring system) showed 96% of mandibular and 99% of maxillary third molars erupted into a good or acceptable position. Limitations of this scoring system are discussed. The mesiodistal size of third molars was suitable to replace second molars; on average, mandibular third molars were 0.55 mm larger and maxillary third molars were 0.7 mm smaller than second molars.

Assessment of clinical case presentations for the Membership in Orthodontics, Royal College of Surgeons of England 1995, 1996.

The cases presented and treated at successive examinations by the candidates for the Membership Examination in Orthodontics in 1995 and 1996 at The Royal College of Surgeons of England, were of a very high standard and demonstrated a wide range of treatment modalities. All cases had fixed appliances, predominantly with pre-adjusted Edgewise appliances. IOTN confirmed that most cases were in great need of treatment, with PAR scores showing them to be treated to a high standard.

Bonded retainers 'hands free'.

A simple technique is described to position and maintain a wire retained during direct bonding using a custom made acrylic carrier.

The assessment of crowding without the need to record arch perimeter. Part II: Crowded and irregular arches.

A mathematically determined technique which seemed to be valid for the calculation of crowding and spacing in arches with acceptable alignment, was tested on a series of arches with varying degrees of crowding and irregularity. Upper and lower study casts of 30 individuals with sufficient malalignment and crowding to warrant orthodontic treatment with fixed appliances were examined. Models were measured by three operators, using a reflex microscope interfaced to an IBM compatible PC and a customized calculation program. The degree of crowding was compared with a "clinical' assessment of each arch, in which the irregularity was measured directly using a steel ruler. Both the computer calculated and clinical values varied between observers, but the agreement between observers was better using the reflex microscope. Correlation coefficients ranged from 0.92 to 0.96 for the reflex microscope, and between 0.86 and 0.91 for the clinical assessment. Inter-observer agreement for the three operators was high: 0.93 for the reflex microscope and 0.87 where a ruler was used. This suggests that the computer program used to calculate the crowding was valid for the crowded arches under consideration.

Ex vivo shear bond strength of fibreglass reinforced aesthetic brackets.

Fibreglass reinforced brackets (FGBs) and ceramic brackets were bonded to the enamel of extracted premolars to evaluate their shear bond strength and fracture mode. One-hundred extracted premolars were divided into five groups, and combinations of two-paste and no-mix composite, and plastic bracket conditioner were used in the test groups. Bond strength testing was carried out 24 hours after direct bonding. The shear strength of ceramic brackets with a two-paste composite was significantly higher than FGBs bonded with either two-mix or no-mix composite ( P < 0.05). Ceramic brackets bonded with two-mix and no-mix composite demonstrated a 30 and a 10 percent enamel fracture rate, respectively. There were no enamel or bracket fractures in any of the FGB groups.

Characterization of Loosely Associated Material from the Cell Surface of Lactococcus lactis subsp. cremoris E8 and Its Phage-Resistant Variant Strain 398.

Loosely associated material (LAM) was isolated by gentle extraction procedures from the cell surface of Lactococcus lactis subsp. cremoris E8 and its phage-resistant variant strain 398. LAM from both strains was chemically characterized, and its role in the adsorption of three small isometric bacteriophages, phi 618, phi 833, and phi 852, to the cell surface of the two strains was investigated. The phage-resistant strain (strain 398) produced LAM which differed significantly from the material produced by the parent strain. The total yield of LAM from strain 398 was two- to threefold higher than that from strain E8, and the material contained fivefold more rhamnose and twofold more galactose. Polyacrylamide gel electrophoretic analysis showed that LAM from strain 398 lacked a 21-kDa protein which was present in LAM from the parent strain. Inhibition studies of phage binding by using isolated LAM from two strains showed that although LAM from strain E8 reduced the titer of phi 618 and phi 852 by 53 and 82% respectively, LAM from strain 398 had no effect on the plaque-forming ability of any of the three phages tested. Treatment of LAM from strain E8 with sodium metaperiodate destroyed its ability to bind with phi 618 and phi 852. Phenotypically, strain 398 differed from its parent strain E8 in that it was more prone to cell lysis and required an osmotically adjusted buffer system for the extraction of LAM.

Streptococcus mutans serotype c tagatose 6-phosphate pathway gene cluster.

DNA cloned into Escherichia coli K-12 from a serotype c strain of Streptococcus mutans encodes three enzyme activities for galactose utilization via the tagatose 6-phosphate pathway: galactose 6-phosphate isomerase, tagatose 6-phosphate kinase, and tagatose-1,6-bisphosphate aldolase. The genes coding for the tagatose 6-phosphate pathway were located on a 3.28-kb HindIII DNA fragment. Analysis of the tagatose proteins expressed by recombinant plasmids in minicells was used to determine the sizes of the various gene products. Mutagenesis of these plasmids with transposon Tn5 was used to determine the order of the tagatose genes. Tagatose 6-phosphate isomerase appears to be composed of 14- and 19-kDa subunits. The sizes of the kinase and aldolase were found to be 34 and 36 kDa, respectively. These values correspond to those reported previously for the tagatose pathway enzymes in Staphylococcus aureus and Lactococcus lactis.

Properties of 2,3-Butanediol Dehydrogenases from Lactococcus lactis subsp. lactis in Relation to Citrate Fermentation.

Two 2,3-butanediol dehydrogenases (enzymes 1 and 2; molecular weight of each, 170,000) have been partially purified from Lactococcus lactis subsp. lactis (Streptococcus diacetylactis) D10 and shown to have reductase activity with either diacetyl or acetoin as the substrate. However, the reductase activity with 10 mM diacetyl was far greater for both enzymes (7.0- and 4.7-fold for enzymes 1 and 2, respectively) than with 10 mM acetoin as the substrate. In contrast, when acetoin and diacetyl were present together, acetoin was the preferred substrate for both enzymes, with enzyme 1 showing the more marked preference for acetoin. meso-2,3-Butanediol was the only isomeric product, with enzyme 1 independent of the substrate combinations. For enzyme 2, both the meso and optical isomers of 2,3-butanediol were formed with acetoin as the substrate, but only the optical isomers were produced with diacetyl as the substrate. With batch cultures of strain D10 at or near the point of citrate exhaustion, the main isomers of 2,3-butanediol present were the optical forms. If the pH was sufficiently high (>pH 5), acetoin reduction occurred over time and was followed by diacetyl reduction, and meso-2,3-butanediol became the predominant isomer. Interconversion of the optical isomers into the meso isomer did occur. The properties of 2,3-butanediol dehydrogenases are consistent with diacetyl and acetoin removal and the appearance of the isomers of 2,3-butanediol.

Magnets and orthodontics.

The first part of this paper is a literature review of magnets and their uses in orthodontics. The biological safety of magnets is considered and a report is given of experiments carried out on rat osteosarcoma cell line UMR-106. The second part of the paper describes a case where neodynium-iron-boron magnets were used to assist eruption of an unerupted, vertically impacted upper right canine. Previously, space was available for this tooth, but it failed to show signs of eruption. Following surgical attachment of a magnet, and the use of a second magnet attached to an upper removable appliance, rapid eruption occurred producing a favourable position for bonding.

Polysaccharide Production by Propionibacteria during Lactose Fermentation.

The fermentation products from 10 strains of propionibacteria accounted for only 72% (average value) of the lactose carbon utilized. The balance of the carbon was accounted for by the production of a polysaccharide containing methylpentose (the major component), glucose, and galactose. The presence of methylpentose explained the low ratios of propionate to acetate (<2:1).

Citrate Cycle Intermediates in the Metabolism of Aspartate and Lactate by Propionibacterium freudenreichii subsp. shermanii.

Propionibacterium freudenreichii subsp. shermanii metabolized 7 mol of aspartate to 6 mol of succinate, 4 mol of CO(2), and 7 mol of ammonia. When lactate, sparged with 100% CO(2), was fermented at pH 5.5, unexpectedly high ratios of propionate to acetate were obtained (i.e., 3.2 to 3.8:1). Citrate cycle intermediates may be involved in these fermentations.

Properties of Alanine Dehydrogenase and Aspartase from Propionibacterium freudenreichii subsp. shermanii.

During lactate fermentation by Propionibacterium freudenreichii subsp. shermanii ATCC 9614, the only amino acid metabolized was aspartate. After lactate exhaustion, alanine was one of the two amino acids to be metabolized. For every 3 mol of alanine metabolized, 2 mol of propionate, 1 mol each of acetate and CO(2), and 3 mol of ammonia were formed. The specific activity of alanine dehydrogenase was 0.08 U/mg of protein during lactate fermentation, and it increased to 0.9 U/mg of protein after lactate exhaustion. Alanine dehydrogenase and aspartase, key enzymes in the metabolism of alanine and aspartate, respectively, were partially purified, and some of their properties were studied. Alanine dehydrogenase had a pH optimum of 9.2 to 9.6 and high K(m) values for both NAD (1 to 4 mM) and alanine (7 to 20 mM). Activity was inhibited by low concentrations of pyruvate and NADH. The pH optimum of aspartase decreased from approximately 7.5 to approximately 6.4 when the MgCl(2) and aspartate concentrations were decreased. Plots of aspartate concentration versus activity showed either hyperbolic or sigmoidal kinetics (interaction coefficient, up to a value of 3.1), depending on pH and MgCl(2) concentration. MgCl(2) was either an activator or an inhibitor, depending on pH and its concentration. Aspartase activity was inhibited by low concentrations of fumarate. The properties of alanine dehydrogenase and aspartase are consistent with the finding that aspartate is metabolized during lactate fermentation, while alanine is only fermented after lactate exhaustion and then at a slow rate.

Utilization of Lactate Isomers by Propionibacterium freudenreichii subsp. shermanii: Regulatory Role for Intracellular Pyruvate.

Five strains of Propionibacterium freudenreichii subsp. shermanii utilized the l-(+) isomer of lactate at a faster rate than they did the d-(-) isomer when grown with a mixture of lactate isomers under a variety of conditions. ATCC 9614, grown anaerobically in defined medium containing 160 mM dl-lactate, utilized only 4 and 15% of the d-(-)-lactate by the time 50 and 90%, respectively, of the l-(+)-lactate was used. The intracellular pyruvate concentration was high (>100 mM) in the initial stages of lactate utilization, when either dl-lactate or the l-(+) isomer was the starting substrate. The concentration of this intermediate dropped during dl-lactate fermentation such that when only d-(-)-lactate remained, the concentration was <20 mM. When only the d-(-) isomer was initially present, a similar relatively low concentration of intracellular pyruvate was present, even at the start of lactate utilization. The NAD-independent lactate dehydrogenase activities in extracts showed different kinetic properties with regard to pyruvate inhibition, depending upon the lactate isomer present. Pyruvate gave a competitive inhibitor pattern with l-(+)-lactate and a mixed-type inhibitor pattern with d-(-)-lactate. It is suggested that these properties of the lactate dehydrogenases and the intracellular pyruvate concentrations explain the preferential use of the l-(+) isomer.

Metabolism of Aspartate by Propionibacterium freudenreichii subsp. shermanii: Effect on Lactate Fermentation.

More than 90% of the aspartate in a defined medium was metabolized after lactate exhaustion such that 3 mol of aspartate and 1 mol of propionate were converted to 3 mol of succinate, 3 mol of ammonia, 1 mol of acetate, and 1 mol of CO(2). This pathway was also evident when propionate and aspartate were the substrates in complex medium in the absence of lactate. In complex medium with lactate present, about 70% of the aspartate was metabolized to succinate and ammonia during lactate fermentation, and as a consequence of aspartate metabolism, more lactate was fermented to acetate and CO(2) than was fermented to propionate. The conversion of aspartate to fumarate and ammonia by the enzyme aspartase and subsequent reduction of fumarate to succinate occurred in the five strains of Propionibacterium freudenreichii subsp. shermanii studied. The ability to metabolize aspartate in the presence of lactate appeared to be related to aspartase activity. The specific activity of aspartase increased during and after lactate utilization, and the levels of this enzyme were lower in cells grown in defined medium than levels in those cells grown in complex medium. Under the conditions used, no other amino acids were readily metabolized in the presence of lactate. The possibility that aspartate metabolism by propionibacteria in Swiss cheese has an influence on CO(2) production is discussed.

Kinetics of activation of L-lactate dehydrogenase from Streptococcus lactis by fructose 1,6-bisphosphate.

A lag is observed before the steady state during pyruvate reduction catalysed by lactate dehydrogenase from Streptococcus lactis. The lag is abolished by preincubation of enzyme with the activator fructose 1,6-bisphosphate before mixing with the substrates. The rate constants for the lag phase showed a linear dependence on fructose-1,6-bisphosphate concentration, with a second-order rate constant of 2.0 X 10(4) M-1 s-1, but were independent of enzyme concentration. Binding of fructose 1,6-bisphosphate produces a decrease in the protein fluorescence of the enzyme. The second-order rate constant for the fluorescence change is twice that for the lag in pyruvate reduction. The results suggest that binding of fructose 1,6-bisphosphate induces a conformational change in the enzyme, producing a form with reduced protein fluorescence and increased activity towards pyruvate reduction.

Regulation of product formation during glucose or lactose limitation in nongrowing cells of Streptococcus lactis.

Nongrowing cells of Streptococcus lactis in a pH-stat were dosed with sugar to allow fermentation at the maximum rate or were fed a continuous supply of sugar at rates less than the maximum. Under anaerobic conditions, rapid fermentation of either glucose or lactose was essentially homolactic. However, with strain ML3, limiting the fermentation rate diverted approximately half of the pyruvate to formate, acetate, and ethanol. At limiting glucose fermentation rates, cells contained lower concentrations of lactate dehydrogenase activator (fructose 1,6-diphosphate) and pyruvate formate-lyase inhibitors (triose phosphates). As a result, pyruvate formate-lyase and pyruvate dehydrogenase play a greater role in pyruvate metabolism. In contrast to strain ML3, strain ML8 did not give the same diversion of products under anaerobic conditions, and cells retained higher concentrations of the above effector compounds. Lactose metabolism under aerobic conditions resulted in pyruvate excretion by both S. lactis ML3 and ML8. At 7% of the maximum utilization rate, pyruvate accounted for 69 and 35% of the lactose metabolized by ML3 and ML8, respectively. Acetate was also a major product, especially with ML8. The data suggest that NADH oxidase is involved in coenzyme recycling in the presence of oxygen and that pyruvate formate-lyase is inactivated, but the pyruvate dehydrogenase complex still functions.

Selection of Galactose-Fermenting Streptococcus thermophilus in Lactose-Limited Chemostat Cultures.

Stock cultures of Streptococcus thermophilus are essentially galactose negative (Gal). Although both galactose 1-phosphate uridyl transferase and uridine-5-diphospho-glucose 4-epimerase are present, suggesting that the genes for the Leloir pathway exist, cells cannot induce high levels of galactokinase. Therefore, galactose is largely excreted when cultures are grown on lactose, and most strains cannot be readily adapted to grow on free galactose. Gal cultures were grown in a chemostat under lactose limitation in which high concentrations of residual galactose were present. Under this selection pressure, Gal organisms eventually took over the culture with all four strains examined. Gal cells had induced galactokinase, and three of the four strains grew on free galactose with doubling times of 40 to 50 min. When Gal organisms were grown on lactose in batch culture, the galactose moiety was only partially utilized while lactose was still present. As lactose was exhausted, and catabolite repression was lifted, the Leloir pathway enzymes (especially galactokinase) were induced and the residual galactose fermented. Neither phospho-beta-galactosidase activity nor the enzymes of the d-tagatose 6-phosphate pathway were detected in S. thermophilus. In contrast to Streptococcus cremoris and Streptococcus lactis, fermentation was homolactic with galactose in batch cultures and with lactose limitation in the chemostat. When mixed Gal-Gal cultures were repeatedly transferred in milk, the Gal cells became the dominant cell type. The Gal phenotype of stock cultures probably reflects their prolonged maintenance in milk.