Evaluation of three individual glucosyltransferases produced by Streptococcus mutans using monoclonal antibodies

Abstract We previously established murine hybridomas producing a monoclonal antibody monospecific against three glucosyl-transferases (I, SI and S) of Streptococcus mutans which contribute to dental caries formation. Here, we developed a new immunochemical technique (cross-dot system) with which individual levels of glucosyltransferases expressed by S. mutans can be evaluated. We also examined glucosyltransferase production and in vitro artificial plaque formation by a reference strain and several clinical isolates of S. mutans . The findings indicate that the levels of glucosyltransferases produced greatly vary with the cells and the culture medium, and that the cells producing high levels of both glucosyltransferase-SI and glucosyltransferase-I enzymes may possess high in vitro artificial plaque forming ability. We suggest that the cross-dot system will be useful for estimating the cariogenic potential of S. mutans isolates.     

Ultrasonic studies of lipid bilayer. Phase transition in synthetic phosphatidylcholine liposomes

The ultrasonic velocity at 3 MHz and the density in the nonsonicated and sonicated liposomes of dipalmitoylphosphatidylcholine have been measured in the temperature range from 0 degrees C to 55 degrees C. The results indicate that nonsonicated multilamellar vesicles undergo a weak first order transition which is analogous to the nematic-isotropic transition of liquid crystals. A sharp change in the ultrasonic velocity associated with the first order transition disappears when the multilamellar vesicles are sonicated. The bulk modulus of the lipid bilayer calculated from the ultrasonic velocity and the density of sonicated liposomes has a value of 3.0 X 10(10) dyne/cm2 at 20 degrees C, reaches a minimum value of 2.1 X 10(10) dyne/cm2 at its transition temperature and increases slightly to 2.2 X 10(10) dyne/cm2 at 50 degrees C.     

A C57BL/KsJ mouse model of Niemann-Pick disease (spm) belongs to the same complementation group as the major childhood type of Niemann-Pick disease type C

A cell line (SPM-3T3) derived from a C57BL/ KsJ mouse model of Niemann-Pick disease type C (NP-C) shows biochemical abnormalities similar to those in fibroblasts derived from NP-C. Somatic cell hybridization analysis of the SPM-3T3 cells and five fibroblast strains derived from NP-C patients (four childhood cases and one adult case) was carried out. The criterion for complementation was the restoration of a normal intracellular fluorescent pattern in multinucleated cells stained with filipin to demonstrate cholesterol accumulation. These cells can be assigned to two complementation groups. The SPM-3T3 cells did not complement cell strains derived from childhood-type NP-C, while they complemented a cell strain derived from an adult patient. Our results suggest that SPM-3T3 represents a genetically authentic model of a major complementation group of NP-C, and that NP-C consists of genetically heterogeneous groups. Received: 5 March 1996     

Shear viscoelasticity of suspensions of biological cells with viscoelastic membrane II

To discuss the relaxation phenomena of biological cell suspensions, we calculate the complex intrinsic viscosity of dispersions of spherical cells with viscoelastic membrane as a function of the frequency taking account of interfacial tension at both the interfaces of the membrane. The Maxwell model and two kinds of the three-parameter models are used to describe the viscoelasticity of the cell membrane. The results are computed mainly for the Maxwell model similarly in case of the Voigt Model (Abe, K., Takano, Y. and Sakanishi, A. Biorheology 21 405-414, 1984). The computed results of the four models, the Voigt, the Maxwell and the two kinds of the three-parameter viscoelastic models, are compared with the experimental data.     

Ultrasound: mechanical gene transfer into plant cells by sonoporation

Development of nonviral gene transfer methods would be a valuable alternative of gene therapy or transformation. Ultrasound can produce a variety of nonthermal bioeffects via acoustic cavitation. Cavitation bubbles can induce cell death or transient membrane permeabilization (sonoporation) on cells. Application of sonoporation for gene transfer into cells or tissues develops quickly in recent years. Many studies have been performed in vitro exposure systems to a variety of cell lines transfected successfully. In vivo, cavitation initiation and control are more difficult, but can be enhanced by ultrasound contrast agents (microbubbles). The use of ultrasound for nonviral gene delivery has been applied for mammalian systems, which provides a fundamental basis and strong promise for development of new gene therapy methods for clinical medicine. In this paper, ultrasound applied to plant cell transformation or gene transfer is reviewed. Recently, most researches are focused on sonication-assisted Agrobacterium-mediated transformation (SAAT) in plant cells or tissues. Microbubbles are also proposed to apply to gene transfer in plant cells and tissues.     

N-Benzylideneaniline and N-Benzylaniline are Potent Inhibitors of Lignostilbene-α,β-dioxygenase,a Key Enzyme in Oxidative Cleavage of the Central Double Bond of Lignostilbene

Lignostilbene-α,β-dioxygenase (LSD, EC 1.13.11.43) is involved in oxidative cleavage of the central double bond of lignostilbene to form the corresponding aldehydes by a mechanism similar to those of 9-cis-epoxycarotenoid dioxygenase and β-carotene 15,15′-dioxygenase, key enzymes in abscisic acid biosynthesis and vitamin A biosynthesis, respectively. In this study, several N-benzylideneanilines and amine were synthesized and examined for their efficacy as inhibitors of LSD. N-(4-Hydroxybenzylidene)-3-methoxyaniline was found to be a potent inhibitor with IC₅₀ = 0.3 µM and N-(4-hydroxybenzyl)-3-methoxyaniline was also active with IC₅₀ = 10 µM. The information obtained from the structure-activity relationships study here can aid in discovering inhibitors of both abscisic acid and vitamin A biosynthesis.     

Effects of different temperatures, velocities and loads on the gliding resistance of flexor digitorum profundus tendons in a human cadaver model

The purpose of this study was to investigate the effects of temperature, velocity and load on the gliding resistance (GR) of flexor digitorum profundus (FDP) tendons in a human cadaver model. A total of 40 FDP tendons from the index through small digits of ten human cadavers were tested to assess the effect of temperature (4, 23 or 36 °C), velocity (2, 4, 6, 8, 10 or 12 mm/s) and load (250, 500, 750, 1000, 1250 and 1500 g) on GR. The mean GR at 4 °C was significantly higher than the mean GR at 36 °C (p<0.0066). There was no significant difference in the mean GR of the tested velocities. The mean GR was proportional to load, with each successive load having significantly higher GR than the loads before it (all p<0.001). There was no significant difference in the mean GR by digit. In this in vitro model, we have demonstrated that tendon gliding resistance is proportional to load, independent of velocity and somewhat affected by temperature. We conclude that it is important to specify these conditions when reporting gliding resistance, especially load and temperature.     

Kawasaki Disease-Specific Molecules in the Sera Are Linked to Microbe-Associated Molecular Patterns in the Biofilms

Background

Kawasaki disease (KD) is a systemic vasculitis of unknown etiology. The innate immune system is involved in its pathophysiology at the acute phase. We have recently established a novel murine model of KD coronary arteritis by oral administration of a synthetic microbe-associated molecular pattern (MAMP). On the hypothesis that specific MAMPs exist in KD sera, we have searched them to identify KD-specific molecules and to assess the pathogenesis.

Methods

We performed liquid chromatography-mass spectrometry (LC-MS) analysis of fractionated serum samples from 117 patients with KD and 106 controls. Microbiological and LC-MS evaluation of biofilm samples were also performed.

Results

KD samples elicited proinflammatory cytokine responses from human coronary artery endothelial cells (HCAECs). By LC-MS analysis of KD serum samples collected at 3 different periods, we detected a variety of KD-specific molecules in the lipophilic fractions that showed distinct m/z and MS/MS fragmentation patterns in each cluster. Serum KD-specific molecules showed m/z and MS/MS fragmentation patterns almost identical to those of MAMPs obtained from the biofilms formed in vitro (common MAMPs from Bacillus cereus, Yersinia pseudotuberculosis and Staphylococcus aureus) at the 1^st study period, and from the biofilms formed in vivo (common MAMPs from Bacillus cereus, Bacillus subtilis/Bacillus cereus/Yersinia pseudotuberculosis and Staphylococcus aureus) at the 2^nd and 3^rd periods. The biofilm extracts from Bacillus cereus, Bacillus subtilis, Yersinia pseudotuberculosis and Staphylococcus aureus also induced proinflammatory cytokines by HCAECs. By the experiments with IgG affinity chromatography, some of these serum KD-specific molecules bound to IgG.

Conclusions

We herein conclude that serum KD-specific molecules were mostly derived from biofilms and possessed molecular structures common to MAMPs from Bacillus cereus, Bacillus subtilis, Yersinia pseudotuberculosis and Staphylococcus aureus. Discovery of these KD-specific molecules might offer novel insight into the diagnosis and management of KD as well as its pathogenesis.     

Production of ethanol from potato pulp: Investigation of the role of the enzyme from Acremonium cellulolyticus in conversion of potato pulp into ethanol

In this study, the saccharification and fermentation of the by-product of starch manufacture, potato pulp, were investigated. Analytic results of the components show that the potato pulp contains large amounts of starch, cellulose, and pectin. A commercial enzyme from Acremonium cellulolyticus was found to be highly efficient in the saccharification of potato pulp, since it exhibited high pectinase, α-amylase, and cellulase activities. Hydrothermal treatment of the potato pulp increased the saccharification rate, with a corresponding glucose concentration of 114 g/L and yield of 68% compared to the glucose concentration of 47 g/L and yield of 28% in the untreated case. The hydrolyzate could be used as both nitrogen and carbon sources for ethanol fermentation, showing that bioconversion of potato pulp to ethanol is feasible.     

Interferon-gamma-induced apoptosis and activation of THP-1 macrophages

Apoptotic macrophages are frequently observed in human atherosclerotic lesions, and are considered to be involved in plaque instability in atherosclerosis. However, the molecular mechanism that promotes programmed cell death of macrophages in atherosclerosis remains to be elucidated. In this study, we investigated the effects of interferon-gamma (IFN-gamma), a cytokine secreted by activated T helper 1 (Th1) lymphocytes, on apoptotic cell death of THP-1 macrophages. Further we studied whether these apoptotic macrophages could be simultaneously activated in vitro and subsequently overgenerate monocyte chemoattractant protein-1 (MCP-1). When THP-1 macrophages were cultured with various concentrations of IFN-gamma, DNA synthesis was significantly decreased. IFN-gamma was found significantly to induce apoptotic cell death in THP-1 macrophages. RNase protection assay revealed that IFN-gamma up-regulated the mRNA levels of two pro-apoptotic molecules, tumor necrosis factor-alpha receptor 1 (TNFR1) and caspase-8, in THP-1 cells. Furthermore, TNF-alpha antibodies were found completely to neutralize the IFN-gamma-induced inhibition in DNA synthesis as well as apoptotic cell death in macrophages. IFN-gamma was found to activate these macrophages to stimulate MCP-1 production. The results suggest that IFN-gamma not only exerted apoptotic effects on macrophages, but also activated them and subsequently overgenerated MCP-1, and was thus involved in the development and progression of atherosclerosis.