Micro-analysis of Amino Acids with Fluorescein-isothiocyanate

The separation and identification of fluorescein-thiocarbarnyl (FTC-) amino acid II were accomplished by one- and two-dimensional thin-layer chromatography. The authentic samples for identification of amino acids were synthesized with fluorescein-isothiocyanate II (FITC II) and 21 amino acids. These FTC-amino acids were studied spectrometrically.

For quantitative estimation of FTC-amino acids, the fluoroscopy was used. It was found that the fluorescence intensity was proportional to the concentration of FTC-amino acids in 2 pmole/ml to 20 nmole/ml range. Recovery of FTC-derivatives on silica gel plate was about 80%.     

Purification of the epidermal growth factor receptor by tyrosine-Sepharose affinity chromatography

The EGF receptor has been purified from human epidermoid carcinoma A431 cells by affinity chromatography on wheat germ agglutinin-agarose and tyrosine-Sepharose. The purified EGF receptor was shown to be homogeneous by SDS-polyacrylamide gel electrophoresis and possessed EGF-sensitive tyrosine kinase activity. Kinetic analysis of the autophosphorylation indicated that approximately 1.4 mol of phosphate was incorporated per mol of the EGF receptor. When a synthetic tyrosine-containing peptide was used as a phosphorylatable substrate, the specific activity of the EGF-stimulated kinase was 66 nmol/min/mg.     

Multi-scale computational modelling in biology and physiology

Recent advances in biotechnology and the availability of ever more powerful computers have led to the formulation of increasingly complex models at all levels of biology. One of the main aims of systems biology is to couple these together to produce integrated models across multiple spatial scales and physical processes. In this review, we formulate a definition of multi-scale in terms of levels of biological organisation and describe the types of model that are found at each level. Key issues that arise in trying to formulate and solve multi-scale and multi-physics models are considered and examples of how these issues have been addressed are given for two of the more mature fields in computational biology: the molecular dynamics of ion channels and cardiac modelling. As even more complex models are developed over the coming few years, it will be necessary to develop new methods to model them (in particular in coupling across the interface between stochastic and deterministic processes) and new techniques will be required to compute their solutions efficiently on massively parallel computers. We outline how we envisage these developments occurring.     

A CRISPR/Cas9-mediated gene knockout system in Aspergillus luchuensis mut. kawachii

ABSTRACT

We developed an approach to genome editing of the white koji fungus, Aspergillus luchuensis mut. kawachii using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system. Co-transformation of AMA1-based Cas9 and gRNA expression plasmids achieved efficient gene knockout in A. kawachii. The plasmids were easily lost when selective pressure was removed, allowing for successive rounds of genome editing.     

Lactobacillus helveticus SBT2171 Inhibits Lymphocyte Proliferation by Regulation of the JNK Signaling Pathway

Lactobacillus helveticus SBT2171 (LH2171) is a lactic acid bacterium with high protease activity and used in starter cultures in the manufacture of cheese. We recently reported that consumption of cheese manufactured using LH2171 alleviated symptoms of dextran sodium sulfate (DSS)-induced colitis in mice. In this study, we have examined whether LH2171 itself exerts an inhibitory effect on the excessive proliferation of lymphocytes. We found that LH2171 inhibited the proliferation of LPS-stimulated mouse T and B cells, and the human lymphoma cell lines, Jurkat and BJAB. Cell cycle analysis showed an accumulation of LH2171-treated BJAB cells in the G2/M phase. Further, phosphorylation of c-Jun N-terminal kinase (JNK) and c-Jun was reduced by LH2171 in BJAB cells. Subsequently, expression of cell division cycle 2 (CDC2), regulated by the JNK signaling pathway and essential for G2/M phase progression, was inhibited by LH2171. It was also demonstrated that intraperitoneal administration of LH2171 strongly alleviated symptoms of collagen-induced arthritis (CIA) in mice. These findings suggest that LH2171 inhibits the proliferation of lymphocytes through a suppression of the JNK signaling pathway and exerts an immunosuppressive effect in vivo.     

Asymmetry of the GroEL-GroES complex under physiological conditions as revealed by small-angle x-ray scattering

Despite the well-known functional importance of GroEL-GroES complex formation during the chaperonin cycle, the stoichiometry of the complex has not been clarified. The complex can occur either as an asymmetric 1:1 GroEL-GroES complex or as a symmetric 1:2 GroEL-GroES complex, although it remains uncertain which type is predominant under physiological conditions. To resolve this question, we studied the structure of the GroEL-GroES complex under physiological conditions by small-angle x-ray scattering, which is a powerful technique to directly observe the structure of the protein complex in solution. We evaluated molecular structural parameters, the radius of gyration and the maximum dimension of the complex, from the x-ray scattering patterns under various nucleotide conditions (3 mM ADP, 3 mM ATPγS, and 3 mM ATP in 10 mM MgCl₂ and 100 mM KCl) at three different temperatures (10°C, 25°C, and 37°C). We then compared the experimentally observed scattering patterns with those calculated from the known x-ray crystallographic structures of the GroEL-GroES complex. The results clearly demonstrated that the asymmetric complex must be the major species stably present in solution under physiological conditions. On the other hand, in the presence of ATP (3 mM) and beryllium fluoride (10 mM NaF and 300 μM BeCl₂), we observed the formation of a stable symmetric complex, suggesting the existence of a transiently formed symmetric complex during the chaperonin cycle.     

Preliminary study of combination therapy with interferon-α and zinc in chronic hepatitis C patients with genotype 1b

We have evaluated the efficacy of interferon-α (IFN-α) plus zinc therapy in hepatitis C patients with genotype 1b, poor responders for IFN alone. Ten patients were injected with 10 MU of IFN-α every day for 4 wk, followed by three times a week for 20 wk (control group). Nine patients took 300 mg of zinc sulfate a day orally during IFN-α therapy (zinc sulfate group), and 15 patients took IFN-α and 150 mg of polaprezinc (polaprezinc group). On the d 8 of IFN therapy, circadian zinc levels in serum elevated significantly in the polaprezinc group compared to the zinc sulfate group or control group. Serum ALT levels normalized in 73.3% of the polaprezinc group, 55.6% of the zinc sulfate group, and 40.0% of the control group at 6 mo after the end of IFN therapy. Sustained eradication for the hepatitis C virus RNA judged at the end of the 6-mo follow-up period was higher in the polaprezinc group than in the zinc sulfate group (53.3% vs 11.1%, p<0.05) or the control group (20.0%). No clinical side effects of zinc were observed at the dose used. The data suggest that polaprezinc is expected to increase the therapeutic response of IFN-α for chronic hepatitis C with genotype 1b.     

Lactobacillus gasseri SBT2055 Induces TGF-β Expression in Dendritic Cells and Activates TLR2 Signal to Produce IgA in the Small Intestine

Probiotic bacteria provide benefits in enhancing host immune responses and protecting against infection. Induction of IgA production by oral administration of probiotic bacteria in the intestine has been considered to be one reason for this beneficial effect, but the mechanisms of the effect are poorly understood. Lactobacillus gasseri SBT2055 (LG2055) is a probiotic bacterium with properties such as bile tolerance, ability to improve the intestinal environment, and it has preventive effects related to abdominal adiposity. In this study, we have found that oral administration of LG2055 induced IgA production and increased the rate of IgA⁺ cell population in Peyer''s patch and in the lamina propria of the mouse small intestine. The LG2055 markedly increased the amount of IgA in a co-culture of B cells and bone marrow derived dendritic cells (BMDC), and TLR2 signal is critical for it. In addition, it is demonstrated that LG2055 stimulates BMDC to promote the production of TGF-β, BAFF, IL-6, and IL-10, all critical for IgA production from B cells. Combined stimulation of B cells with BAFF and LG2055 enhanced the induction of IgA production. Further, TGF-β signal was shown to be critical for LG2055-induced IgA production in the B cell and BMDC co-culture system, but TGF-β did not induce IgA production in a culture of only B cells stimulated with LG2055. Furthermore, TGF-β was critical for the production of BAFF, IL-6, IL-10, and TGF-β itself from LG2055-stimulated BMDC. These results demonstrate that TGF-β was produced by BMDC stimulated with LG2055 and it has an autocrine/paracrine function essential for BMDC to induce the production of BAFF, IL-6, and IL-10.