Lactobacillus paracasei DSMZ16671 Reduces Mutans Streptococci: A Short-Term Pilot Study

Reducing the burden of pathogenic mutans streptococci is a goal of oral health. Lactobacillus paracasei DSMZ16671, even after heat-killing, specifically co-aggregates mutans streptococci in vitro and retains this activity in human saliva. In rats, it reduces mutans streptococcal colonization of teeth and caries scores. This pilot study sought to assess the potential of heat-killed L. paracasei DSMZ16671 (pro-t-action^®) to reduce levels of salivary mutans streptococci in humans, using sugar-free candies as a delivery vehicle. A randomized, placebo-controlled, double-blind in vivo study of three groups examined the short-term effect of sugar-free candies containing 0 (placebo), 1, or 2 mg/candy piece of heat-killed L. paracasei DSMZ16671 on the levels of salivary mutans streptococci determined before and after consumption of the candies. The candies were consumed 4 times during 1.5 consecutive days. Compared to the placebo group, the test groups’ saliva had significantly reduced mutans streptococci as an immediate effect. These results suggest the use of heat-killed L. paracasei DSMZ16671 in suckable candies as a method to reduce mutans streptococci in the mouth and, thereby, caries risk. We think this a new concept and strategy for caries prevention and management.     

Inactivation of the Lactobacillus leichmannii ribonucleoside triphosphate reductase by 2'-chloro-2'-deoxyuridine 5'-triphosphate: stoichiometry of inactivation, site of inactivation, and mechanism of the protein chromophore formation

The ribonucleoside triphosphate reductase (RTPR) of Lactobacillus leichmannii is inactivated by the substrate analogue 2'-chloro-2'-deoxyuridine 5'-triphosphate (ClUTP). Inactivation is due to alkylation by 2-methylene-3(2H)-furanone, a decomposition product of the enzymic product 3'-keto-2'-deoxyuridine triphosphate. The former has been unambiguously identified as 2-[(ethylthio)methyl]-3(2H)-furanone, an ethanethiol trapped adduct, which is identical by 1H NMR spectroscopy with material synthesized chemically. Subsequent to rapid inactivation, a slow process occurs that results in formation of a new protein-associated chromophore absorbing maximally near 320 nm. The terminal stages of the inactivation have now been investigated in detail. The alkylation and inactivation stoichiometries were studied as a function of the ratio of ClUTP to enzyme. At high enzyme concentrations (0.1 mM), 1 equiv of [5'-3H]ClUTP resulted in 0.9 equiv of 3H bound to protein and 83% inactivation. The amount of labeling of RTPR increased with increasing ClUTP concentration up to the maximum of approximately 4 labels/RTPR, yet the degree of inactivation did not increase proportionally. This suggests that (1) RTPR may be inactivated by alkylation of a single site and (2) decomposition of 3'-keto-dUTP is not necessarily enzyme catalyzed. The formation of the new protein chromophore was also monitored during inactivation and found to reach its full extent upon the first alkylation. Thus, out of four alkylation sites, only one appears capable of undergoing the subsequent reaction to form the new chromophore. While chromophore formation was prevented by NaBH4 treatment, the chromophore itself is resistant to reduction.(ABSTRACT TRUNCATED AT 250 WORDS)     

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A Tripartite SNARE-K+ Channel Complex Mediates in Channel-Dependent K+ Nutrition in Arabidopsis

A few membrane vesicle trafficking (SNARE) proteins in plants are associated with signaling and transmembrane ion transport, including control of plasma membrane ion channels. Vesicle traffic contributes to the population of ion channels at the plasma membrane. Nonetheless, it is unclear whether these SNAREs also interact directly to affect channel gating and, if so, what functional impact this might have on the plant. Here, we report that the Arabidopsis thaliana SNARE SYP121 binds to KC1, a regulatory K⁺ channel subunit that assembles with different inward-rectifying K⁺ channels to affect their activities. We demonstrate that SYP121 interacts preferentially with KC1 over other Kv-like K⁺ channel subunits and that KC1 interacts specifically with SYP121 but not with its closest structural and functional homolog SYP122 nor with another related SNARE SYP111. SYP121 promoted gating of the inward-rectifying K⁺ channel AKT1 but only when heterologously coexpressed with KC1. Mutation in any one of the three genes, SYP121, KC1, and AKT1, selectively suppressed the inward-rectifying K⁺ current in Arabidopsis root epidermal protoplasts as well as K⁺ acquisition and growth in seedlings when channel-mediated K⁺ uptake was limiting. That SYP121 should be important for gating of a K⁺ channel and its role in inorganic mineral nutrition demonstrates an unexpected role for SNARE–ion channel interactions, apparently divorced from signaling and vesicle traffic. Instead, it suggests a role in regulating K⁺ uptake coordinately with membrane expansion for cell growth.     

Extrem disharmonische Genom-Plastom-Kombinationen und v?terliche Plastidenvererbung beiOenothera

Zusammenfassung Ähnlich wie bei den Bastarden zwischen Euoenotheren und Raimannien gibt es auch innerhalb der UntergattungEuoenothera extrem disharmonische Genom-Plastom-Kombinationen, bei denen die Entwicklung der Embryonen unterbunden wird. Ihre besondere Bedeutung bekommen diese Kombinationen in Kreuzungen, bei denen die inkompatible Plastidensorte durch die Mutter und eine normal verträgliche durch den Vater übertragen werden. Aus derartig gemischten Zygoten entwickeln sich nicht nur früh absterbende Embryonen (durch taube Samen repräsentiert), sondern in nicht geringer Zahl auch Keimlinge, die im Extremfall ausschließlich die Plastidensorte des Vaters besitzen. In weniger extremen Fällen trägt noch ein geringer Anteil mütterlicher Plastiden zu einer Weißscheckung der Nachkommenschaft bei. Derartige Chimären entwickeln außerdem in bestimmten Sektoren grüne Blätter von irregulater Gestalt. Die irregulaten Sproßabschnitte sind von einer nicht normal entwickelten Epidermis bedeckt. WährendSchwemmle undSimon (1956) vermuteten, daß die irregulaten Sektoren aus dauermodifiziertem grünem Gewebe bestehen, konnten wir in einzelnen Fällen nachweisen, daß eine chimärische Struktur vorliegt: Eine gehemmte Epidermis, welche die disharmonische Genom-Plastom-Kombination mit den Plastiden der Mutter besitzt, umschließt das genetisch unveränderte Gewebe der harmonischen Genom-Plastom-Kombination mit den Plastiden des Vaters.Die rein väterliche Plastidenvererbung in bestimmtenOenothera-Kreuzungen kann verschiedenen Problemstellungen nutzbar gemacht werden; wir denken an Mutationsversuche mit Pollenplastiden, Untersuchungen über Korrelationen zwischen Plastiden- und Zellvermehrung und das Studium der Entmischungsvorgänge bei der Embryoentwicklung.Mit 1 Textabbildung     

Induction of synchronous differentiation (encystment) in Physarum polycephalum myxamoebae

Encystment of Physarum polycephalum myxamoebae, grown under nearly identical physiological conditions as plasmodia is induced by transfer to a salts medium containing 0.5 M mannitol or mannose. After 24 h induction approximately 50% of amoebae had differentiated to cells which were identified to be young cysts by light and electron microscopy. Several other polyols, sugars, biogenic amines, and a starvation period from 24 h to one week caused no reproducible cyst formation. In contrast to the formation of dormant forms in the plasmodial stage of the life cycle, the induction of cysts and their germination to amoebae are not inhibited neither by actinomycin C nor by cycloheximide. In addition, the isoenzyme spectra of aminopeptidases and acid proteases remain nearly identical in growing and differentiating amoebae.Abbreviations SD semi-defined BSS basal salts solution The investigation is a part of the Ph. D. thesis of A. Haars, Göttingen, 1976     

Importance of the cyanobacterial Gun4 protein for chlorophyll metabolism and assembly of photosynthetic complexes

Gun4 is a porphyrin-binding protein that activates magnesium chelatase, a multimeric enzyme catalyzing the first committed step in chlorophyll biosynthesis. In plants, GUN4 has been implicated in plastid-to-nucleus retrograde signaling processes that coordinate both photosystem II and photosystem I nuclear gene expression with chloroplast function. In this work we present the functional analysis of Gun4 from the cyanobacterium Synechocystis sp. PCC 6803. Affinity co-purification of the FLAG-tagged Gun4 with the ChlH subunit of the magnesium chelatase confirmed the association of Gun4 with the enzyme in cyanobacteria. Inactivation of the gun4 gene abolished photoautotrophic growth of the resulting gun4 mutant strain that exhibited a decreased activity of magnesium chelatase. Consequently, the cellular content of chlorophyll-binding proteins was highly inadequate, especially that of proteins of photosystem II. Immunoblot analyses, blue native polyacrylamide gel electrophoresis, and radiolabeling of the membrane protein complexes suggested that the availability of the photosystem II antenna protein CP47 is a limiting factor for the photosystem II assembly in the gun4 mutant.     

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The Ralstonia eutropha PhaR Protein Couples Synthesis of the PhaP Phasin to the Presence of Polyhydroxybutyrate in Cells and Promotes Polyhydroxybutyrate Production

Polyhydroxyalkanoates (PHAs) are polyoxoesters that are produced by many bacteria and that accumulate as intracellular granules. Phasins (PhaP) are proteins that accumulate during PHA synthesis, bind PHA granules, and promote further PHA synthesis. Interestingly, PhaP accumulation seems to be strictly dependent on PHA synthesis, which is catalyzed by the PhaC PHA synthase. Here we have tested the effect of the Ralstonia eutropha PhaR protein on the regulation of PhaP accumulation. R. eutropha strains with phaR, phaC, and/or phaP deletions were constructed, and PhaP accumulation was measured by immunoblotting. The wild-type strain accumulated PhaP in a manner dependent on PHA production, and the phaC deletion strain accumulated no PhaP, as expected. In contrast, both the phaR and the phaR phaC deletion strains accumulated PhaP to higher levels than did the wild type. This result implies that PhaR is a negative regulator of PhaP accumulation and that PhaR specifically prevents PhaP from accumulating in cells that are not producing PHA. Transfer of the R. eutropha phaR, phaP, and PHA biosynthesis (phaCAB) genes into a heterologous system, Escherichia coli, was sufficient to reconstitute the PhaR/PhaP regulatory system, implying that PhaR both regulates PhaP accumulation and responds to PHA directly. Deletion of phaR caused a decrease in PHA yields, and a phaR phaP deletion strain exhibited a more severe PHA defect than a phaP deletion strain, implying that PhaR promotes PHA production and does this at least partially through a PhaP-independent pathway. Models for regulatory roles of PhaR in regulating PhaP and promoting PHA production are presented.     

Location of the redox-active thiols of ribonucleotide reductase: sequence similarity between the Escherichia coli and Lactobacillus leichmannii enzymes

The redox-active thiols of Escherichia coli ribonucleoside diphosphate reductase and of Lactobacillus leichmannii ribonucleoside triphosphate reductase have been located by a procedure involving (1) prereduction of enzyme with dithiothreitol, (2) specific oxidation of the redox-active thiols by treatment with substrate in the absence of exogenous reductant, (3) alkylation of other thiols with iodoacetamide, and (4) reduction of the disulfides with dithiothreitol and alkylation with [1-14C]iodoacetamide. The dithiothreitol-reduced E. coli B1 subunit is able to convert 3 equiv of CDP to dCDP and is labeled with 5.4 equiv of 14C. Sequencing of tryptic peptides shows that 2.8 equiv of 14C is on cysteines-752 and -757 at the C-terminus of B1, while 1.0-1.5 equiv of 14C is on cysteines-222 and -227. It thus appears that two sets of redox-active dithiols are involved in substrate reduction. The L. leichmannii reductase is able to convert 1.1 equiv of CTP to dCTP and is labeled with 2.1 equiv of 14C. Sequencing of tryptic peptides shows that 1.4 equiv of 14C is located on the two cysteines of C-E-G-G-A-C-P-I-K. This peptide shows remarkable and unexpected similarity to the thiol-containing region of the C-terminal peptide of E. coli B1, C-E-S-G-A-C-K-I.