Effects of Lectins on initial attachment of cariogenic <Emphasis Type="Italic">Streptococcus mutans</Emphasis>

Oral bacteria initiate biofilm formation by attaching to tooth surfaces via an interaction of a lectin-like bacterial protein with carbohydrate chains on the pellicle. This study aimed to find naturally derived lectins that inhibit the initial attachment of a cariogenic bacterial species, Streptococcus mutans (S. mutans), to carbohydrate chains in saliva in vitro. Seventy kinds of lectins were screened for candidate motifs that inhibit the attachment of S. mutans ATCC 25175 to a saliva-coated culture plate. The inhibitory effect of the lectins on attachment of the S. mutans to the plates was quantified by crystal violet staining, and the biofilm was observed under a scanning electron microscope (SEM). Surface plasmon resonance (SPR) analysis was performed to examine the binding of S. mutans to carbohydrate chains and the binding of candidate lectins to carbohydrate chains, respectively. Moreover, binding assay between the biotinylated-lectins and the saliva components was conducted to measure the lectin binding. Lectins recognizing a salivary carbohydrate chain, Galβ1-3GalNAc, inhibited the binding of S. mutans to the plate. In particular, Agaricus bisporus agglutinin (ABA) markedly inhibited the binding. This inhibition was confirmed by SEM observation. SPR analysis indicated that S. mutans strongly binds to Galβ1-3GalNAc, and ABA binds to Galβ1-3GalNAc. Finally, the biotinylated Galβ1-3GalNAc-binding lectins including ABA demonstrated marked binding to the saliva components. These results suggest that ABA lectin inhibited the attachment of S. mutans to Galβ1-3GalNAc in saliva and ABA can be useful as a potent inhibitor for initial attachment of oral bacteria and biofilm formation.     

A Pichia pastoris single-cell biosensor for detection of enzymatically produced methanol

Applied Microbiology and Biotechnology - We conducted single-cell analyses of the methylotrophic yeast Pichia pastoris to develop a biosensor for the detection of methanol produced by heterologous...     

Involvement of Acidic Polysaccharide Ph-PS-2 and Protein in Initiation of Coccolith Mineralization,as Demonstrated by In Vitro Calcification on the Base Plate

Coccolithophorids, unicellular marine microalgae, have calcified scales with elaborate structures, called coccoliths, on the cell surface. Coccoliths generally comprise a base plate, CaCO₃, and a crystal coat consisting of acidic polysaccharides. In this study, the in vitro calcification conditions on the base plate of Pleurochrysis haptonemofera were examined to determine the functions of the base plate and acidic polysaccharides (Ph-PS-1, -2, and -3). When EDTA-treated coccoliths (acidic polysaccharide-free base plates) or low pH-treated coccoliths (whole acidic polysaccharide-containing base plates) were used, mineralization was not detected on the base plate. In contrast, in the case of coccoliths which were decalcified by lowering of the pH and then treated with urea (Ph-PS-2-containing base plates), distinct aggregates, probably containing CaCO₃, were observed only on the rim of the base plates. Energy dispersive X-ray spectroscopy (EDS) confirmed that the aggregates contained Ca and O, although X-ray diffraction analysis did not reveal any evidence of crystalline materials. Also, in vitro mineralization experiments performed on EDTA-treated coccoliths using isolated acidic polysaccharides demonstrated that the Ca-containing aggregates were markedly formed only in the presence of Ph-PS-2. Furthermore, in vitro mineralization experiments conducted on protein-extracted base plates suggested that the coccolith-associated protein(s) are involved in the Ca deposition. These findings suggest that Ph-PS-2 associated with the protein(s) on the base plate rim initiates Ca²⁺ binding at the beginning of coccolith formation, and some other factors are required for subsequent calcite formation.     

Dielectric relaxation of collagen in aqueous solutions

The complex dielectric constant of collagen in aqueous solutions (polymer concentration, C_p = 0.02–0.2%) was measured at 10°C in the frequency range from 3 Hz to 30 kHz. The loss peak for C_p = 0.02% is located at 90 Hz and the dielectric relaxation time τ_D is estimated to be 1.8 ± 0.3 msec. The τ_D agrees well with the rotational relaxation time estimated from the reduced viscosity, and the relaxation is ascribed to the end-over-end rotation of the molecule. The C_p dependence of τ_D and the dielectric increment Δε are interpreted in terms of the aggregation of molecules. The dipole moment of a molecule, obtained from Δε at C_p = 0.02% and pH 6.5, is (5.2 ± 0.2) × 10⁴D, which is explained by the asymmetrical distribution of the ionized side chains of the molecule.     

Acute effects of thixotropy conditioning of inspiratory muscles on end-expiratory chest wall and lung volumes in normal humans.

Thixotropy conditioning of inspiratory muscles consisting of maximal inspiratory effort performed at an inflated lung volume is followed by an increase in end-expiratory position of the rib cage in normal human subjects. When performed at a deflated lung volume, conditioning is followed by a reduction in end-expiratory position. The present study was performed to determine whether changes in end-expiratory chest wall and lung volumes occur after thixotropy conditioning. We first examined the acute effects of conditioning on chest wall volume during subsequent five-breath cycles using respiratory inductive plethysmography (n = 8). End-expiratory chest wall volume increased after conditioning at an inflated lung volume (P < 0.05), which was attained mainly by rib cage movements. Conditioning at a deflated lung volume was followed by reductions in end-expiratory chest wall volume, which was explained by rib cage and abdominal volume changes (P < 0.05). End-expiratory esophageal pressure decreased and increased after conditioning at inflated and deflated lung volumes, respectively (n = 3). These changes in end-expiratory volumes and esophageal pressure were greatest for the first breath after conditioning. We also found that an increase in spirometrically determined inspiratory capacity (n = 13) was maintained for 3 min after conditioning at a deflated lung volume, and a decrease for 1 min after conditioning at an inflated lung volume. Helium-dilution end-expiratory lung volume increased and decreased after conditioning at inflated and deflated lung volumes, respectively (both P < 0.05; n = 11). These results suggest that thixotropy conditioning changes end-expiratory volume of the chest wall and lung in normal human subjects.     

Decrease of hepatic delta-aminolevulinate dehydratase activity in an animal model of fatigue

Fatigue can be defined physiologically as inability to maintain the expected power output. At present, no standard of fatigue are yet available. In order to find biomarkers of fatigue, we investigated the level of delta-aminolevulinic acid (ALA), the first intermediate metabolite in the heme biosynthetic pathway, in the plasma and urine of an animal model of fatigue. To prepare fatigued animals, we kept rats for 5 days in a cage filled with water to a height of 1.5 cm. As a result, the plasma and urinary ALA levels were increased in the fatigued animals as compared with those in the control animals. One day after the rats had been returned to their normal cages, these increased levels were restored to the control ones. We also examined the activity of the enzyme ALA dehydratase (ALAD), which is the second enzyme in the heme biosynthetic pathway, and ALAD gene expression during the fatigue and its recovery sessions. The ALAD activity, as well as its gene expression, in the liver of the fatigued animals was decreased as compared with those of the control animals. Both activity and gene expression of ALAD were recovered to their respective control levels after the rats had been allowed to rest in their normal cages for 1 day. Furthermore, the activity of ALA synthase (ALAS), the rate-limiting enzyme in the heme biosynthesis, in the liver was increased after the fatigue session for 5 days. Although this level of increase in the plasma concentration of ALA may not induce fatigue, increase in plasma and urinary ALA levels can be biomarkers of fatigue.     

A method for the synthesis of an oseltamivir PET tracer

A protocol applicable for the synthesis of an oseltamivir positron emission tomography (PET) tracer was developed. Acetylation of amine 3 with CH(3)COCl, followed by deprotection and aqueous workup, produced oseltamivir 4 from 3 within 10 min. The obtained 4 was sufficiently pure for PET studies. This method can be extended to PET tracer synthesis using CH(3)(11)COCl.     

Cloning and genetic analyses of the bacteriocin 41 determinant encoded on the Enterococcus faecalis pheromone-responsive conjugative plasmid pYI14: a novel bacteriocin complemented by two extracellular components (lysin and activator)

The conjugative plasmid pYI14 (61 kbp) was isolated from Enterococcus faecalis YI714, a clinical isolate. pYI14 conferred a pheromone response on its host and encoded bacteriocin 41 (bac41). Bacteriocin 41 (Bac41) only showed activity against E. faecalis. Physical mapping of pYI14 showed that it consisted of EcoRI fragments A to P. The clone pHT1100, containing EcoRI fragments A (12.6 kbp) and H (3.5 kbp), conferred the bacteriocin activity on E. faecalis strains. Genetic analysis showed that the determinant was located in a 6.6-kbp region within the EcoRI AH fragments. Six open reading frames (ORFs) were identified in this region and designated ORF7 (bacL₁) ORF8 (bacL₂), ORF9, ORF10, ORF11 (bacA), and ORF12 (bacI). They were aligned in this order and oriented in the same direction. ORFs bacL₁, bacL₂, bacA, and bacI were essential for expression of the bacteriocin in E. faecalis. Extracellular complementation of bacteriocin expression was possible for bacL₁ and -L₂ and bacA mutants. bacL₁ and -L₂ and bacA encoded bacteriocin component L and activator component A, respectively. The products of these genes are secreted into the culture medium and extracellularly complement bacteriocin expression. bacI encoded immunity, providing the host with resistance to its own bacteriocin activity. The bacL₁-encoded protein had significant homology with lytic enzymes that attack the gram-positive bacterial cell wall. Sequence data for the deduced bacL₁-encoded protein suggested that it has a domain structure consisting of an N-terminal signal peptide, a second domain with the enzymatic activity, and a third domain with a three-repeat structure directing the proenzyme to its cell surface receptor.