Enterococcus faecium WB2000 Inhibits Biofilm Formation by Oral Cariogenic Streptococci

This study investigated the inhibitory effect of probiotic Enterococcus faecium WB2000 on biofilm formation by cariogenic streptococci. The ability of E. faecium WB2000 and JCM5804 and Enterococcus faecalis JCM5803 to inhibit biofilm formation by seven laboratory oral streptococcal strains and 13 clinical mutans streptococcal strains was assayed. The Enterococcal strains inhibited biofilm formation in dual cultures with the mutans streptococcal strains Streptococcus mutans Xc and Streptococcus sobrinus JCM5176 (P < 0.05), but not with the noncariogenic streptococcal strains. Enterococcus faecium WB2000 inhibited biofilm formation by 90.0% (9/10) of the clinical S. mutans strains and 100% (3/3) of the clinical S. sobrinus strains. After culturing, the pH did not differ between single and dual cultures. The viable counts of floating mutans streptococci were lower in dual cultures with E. faecium WB2000 than in single cultures. Enterococcus faecium WB2000 acted as a probiotic bacterial inhibitor of cariogenic streptococcal biofilm formation

Equatorial assembly of the cell-division actomyosin ring in the absence of cytokinetic spatial cues

The position of the division site dictates the size and fate of daughter cells in many organisms. In animal cells, division-site placement involves overlapping mechanisms, including signaling from the central spindle microtubules, astral microtubules, and spindle poles and through polar contractions [1, 2, 3]. In fission yeast, division-site positioning requires overlapping mechanisms involving the anillin-related protein Mid1 and the tip complex (comprising the Kelch-repeat protein Tea1, the Dyrk-kinase Pom1, and the SH3-domain protein Tea4) [4, 5, 6, 7, 8, 9, 10, 11]. In addition to these factors, cell shape has also been shown to participate in the maintenance of the position of the actomyosin ring [12, 13, 14]. The first principles guiding actomyosin ring placement, however, have not been elucidated in any organism. Because actomyosin ring positioning, ring assembly, and cell morphogenesis are genetically separable in fission yeast, we have used it to derive actomyosin ring placement mechanisms from first principles. We report that, during ring assembly in the absence of cytokinetic cues (anillin-related Mid1 and tip-complex proteins), actin bundles follow the path of least curvature and assemble actomyosin rings in an equatorial position in spherical protoplasts and along the long axis in cylindrical cells and compressed protoplasts. The equatorial position of rings is abolished upon treatment of protoplasts with an actin-severing compound or by slowing down actin polymerization. We propose that the physical properties of actin filaments/bundles play key roles in actomyosin ring assembly and positioning, and that key cytokinetic molecules may modulate the length of actin filaments to promote ring assembly along the short axis

Class I/II hybrid inhibitory oligodeoxynucleotide exerts Th1 and Th2 double immunosuppression

We designed class I/II hybrid inhibitory oligodeoxynucleotides (iODNs), called iSG, and found that the sequence 5′-TTAGGG-3′, which has a six-base loop head structure, and a 3′-oligo (dG)3–5 tail sequence are important for potent immunosuppressive activity. Interestingly, splenocytes isolated from ovalbumin (OVA)-immunized mice and treated with iSG3 showed suppression of not only interleukin (IL)-6, IL-12p35, IL-12p40, and interferon (IFN) γ mRNA expression, but also IL-4 and IL-13 mRNA expression. Thus, both Th2 and Th1 immune responses can be strongly suppressed by iODNs in splenocytes from allergen-immunized mice, suggesting usefulness in the treatment of diseases induced by over-active immune activation.ArticleFEBS Open Bio. 3:41-45 (2013)journal articl

A Case Report of Tooth Wear Associated with a Patient's Inappropriate Efforts to Reduce Oral Malodor Caused by Endodontic Lesion

Here, we report a case of severe tooth wear associated with a patient's inappropriate efforts to reduce oral malodor. A 72-year-old male patient visited our breath clinic complaining of strong breath odor. Former dentists had performed periodontal treatments including scaling and root planing, but his oral malodor did not decrease. His own subsequent breath odor-reducing efforts included daily use of lemons and vinegar to reduce or mask the odor, eating and chewing hard foods to clean his teeth, and extensive tooth brushing with a hard-bristled toothbrush. Oral malodor was detected in our breath clinic by several tests, including an organoleptic test, portable sulphide monitor, and gas chromatography. Although patient's oral hygiene and periodontal condition were not poor on presentation, his teeth showed heavy wear and hypersensitiving with an unfitted restoration on tooth 16. Radiographic examination of the tooth did not reveal endodontic lesion, but when the metal crown was removed, severe pus discharge and strong malodor were observed. When this was treated, his breath odor was improved. After dental treatment and oral hygiene instruction, no further tooth wear was observed; he was not concerned about breath odor thereafter

Development and Characterization of Novel Molecular Probes for Ca2+/Calmodulin-Dependent Protein Kinase Kinase, Derived from STO-609

Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) activates particular multifunctional kinases, including CaMKI, CaMKIV, and 5′AMP-activated protein kinase (AMPK), resulting in the regulation of various Ca2+-dependent cellular processes, including neuronal, metabolic, and pathophysiological pathways. We developed and characterized a novel pan-CaMKK inhibitor, TIM-063 (2-hydroxy-3-nitro-7H-benzo[de]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one) derived from STO-609 (7H-benzimidazo[2,1-a]benz[de]isoquinoline-7-one-3-carboxylic acid), and an inactive analogue (TIM-062) as molecular probes for the analysis of CaMKK-mediated cellular responses. Unlike STO-609, TIM-063 had an inhibitory activity against CaMKK isoforms (CaMKKα and CaMKKβ) with a similar potency (Ki = 0.35 μM for CaMKKα, and Ki = 0.2 μM for CaMKKβ) in vitro. Two TIM-063 analogues lacking a nitro group (TIM-062) or a hydroxy group (TIM-064) completely impaired CaMKK inhibitory activities, indicating that both substituents are necessary for the CaMKK inhibitory activity of TIM-063. Enzymatic analysis revealed that TIM-063 is an ATP-competitive inhibitor that directly targets the catalytic domain of CaMKK, similar to STO-609. TIM-063 suppressed the ionomycin-induced phosphorylation of exogenously expressed CaMKI, CaMKIV, and endogenous AMPKα in HeLa cells with an IC50 of ∼0.3 μM, and it suppressed CaMKK isoform-mediated CaMKIV phosphorylation in transfected COS-7 cells. Thus, TIM-063, but not the inactive analogue (TIM-062), displayed cell permeability and the ability to inhibit CaMKK activity in cells. Taken together, these results indicate that TIM-063 could be a useful tool for the precise analysis of CaMKK-mediated signaling pathways and may be a promising lead compound for the development of therapeutic agents for the treatment of CaMKK-related diseases

N-terminal acetylation and arginylation of actin determines the architecture and assembly rate of linear and branched actin networks

The great diversity in actin network architectures and dynamics is exploited by cells to drive fundamental biological processes, including cell migration, endocytosis and cell division. While it is known that this versatility is the result of the many actin-remodeling activities of actin-binding proteins, such as Arp2/3 and Cofilin, recent work also implicates post-translational acetylation or arginylation of the actin N-terminus itself as an equally important regulatory mechanism. However, the molecular mechanisms by which acetylation and arginylation alter the properties of actin are not well understood. Here, we directly compare how processing and modification of the N-terminus of actin affects its intrinsic polymerization dynamics and its remodeling by actin-binding proteins that are essential for cell migration. We find that in comparison to acetylated actin, arginylated actin reduces intrinsic as well as formin-mediated elongation and Arp2/3-mediated nucleation. By contrast, there are no significant differences in Cofilin-mediated severing. Taken together, these results suggest that cells can employ these differently modified actins to regulate actin dynamics. In addition, unprocessed actin with an N-terminal methionine residue shows very different effects on formin-mediated-elongation, Arp2/3-mediated nucleation, and severing by Cofilin. Altogether, this study shows that the nature of the N-terminus of actin can promote distinct actin network dynamics, which can be differentially used by cells to locally finetune actin dynamics at distinct cellular locations, such as at the leading edge

Rapid production of pure recombinant actin isoforms in Pichia pastoris

Actins are major eukaryotic cytoskeletal proteins, which perform many important cell functions, including cell division, cell polarity, wound healing, and muscle contraction. Despite obvious drawbacks, muscle actin, which is easily purified, is used extensively presently for biochemical studies of actin cytoskeleton from other organisms / cell types. Here we report a rapid and cost-effective method to purify heterologous actins expressed in the yeast Pichia pastoris. Actin is expressed as a fusion with the actin-binding protein thymosin β4 and purified using an affinity tag introduced in the fusion. Following cleavage of thymosin β4 and the affinity tag, highly purified functional full-length actin is liberated. We purify actins from S. cerevisiae, S. pombe, and the β- and γ- isoforms of human actin. We also report a modification of the method that facilitates expression and purification of arginylated actin, a form of actin thought to regulate actin dendritic networks in mammalian cells. The methods we describe can be performed in all laboratories equipped for molecular biology, and should greatly facilitate biochemical and cell biological studies of the actin cytoskeleton

Actin turnover maintains actin filament homeostasis during cytokinetic ring contraction

Cytokinesis in many eukaryotes involves a tension-generating actomyosin-based contractile ring. Many components of actomyosin rings turn over during contraction, although the significance of this turnover has remained enigmatic. Here, using Schizosaccharomyces japonicus, we investigate the role of turnover of actin and myosin II in its contraction. Actomyosin ring components self-organize into ∼1-µm-spaced clusters instead of undergoing full-ring contraction in the absence of continuous actin polymerization. This effect is reversed when actin filaments are stabilized. We tested the idea that the function of turnover is to ensure actin filament homeostasis in a synthetic system, in which we abolished turnover by fixing rings in cell ghosts with formaldehyde. We found that these rings contracted fully upon exogenous addition of a vertebrate myosin. We conclude that actin turnover is required to maintain actin filament homeostasis during ring contraction and that the requirement for turnover can be bypassed if homeostasis is achieved artificially