Production and characterization of recombinant P1 adhesin essential for adhesion,gliding, and antigenic variation in the human pathogenic bacterium,Mycoplasma pneumoniae

Mycoplasma pneumoniae forms an attachment organelle at one cell pole, binds to the host cell surface, and glides via a unique mechanism. A 170-kDa protein, P1 adhesin, present on the organelle surface plays a critical role in the binding and gliding process. In this study, we obtained a recombinant P1 adhesin comprising 1476 amino acid residues, excluding the C-terminal domain of 109 amino acids that carried the transmembrane segment, that were fused to additional 17 amino acid residues carrying a hexa-histidine (6?×?His) tag using an Escherichia coli expression system. The recombinant protein showed solubility, and chirality in circular dichroism (CD). The results of analytical gel filtration, ultracentrifugation, negative-staining electron microscopy, and small-angle X-ray scattering (SAXS) showed that the recombinant protein exists in a monomeric form with a uniformly folded structure. SAXS analysis suggested the presence of a compact and ellipsoidal structure rather than random or molten globule-like conformation. Structure model based on SAXS results fitted well with the corresponding structure obtained with cryo-electron tomography from a closely related species, M. genitalium. This recombinant protein may be useful for structural and functional studies as well as for the preparation of antibodies for medical applications.     

Exercise ameliorates high-fat diet-induced impairment of differentiation of adipose-derived stem cells into neuron-like cells in rats

Adipose-derived stem cells (ADSCs) can differentiate into neurons under particular conditions. It remains largely unknown whether this differentiation potential is affected by physical conditions such as obesity, which modulates the functions of adipose tissue. In this study, we determined the impact of either a 9-week high-fat diet (60% fat; HFD) or 9-week exercise training on the differentiation potential of ADSCs into neuron-like cells in male Wistar rats. Rats were randomly assigned to a normal diet-fed (ND-SED) group, HFD-fed (HFD-SED) group, or exercise-trained HFD-fed group (HFD-EX). After a 9-week intervention, ADSCs from all groups differentiated into neuron-like cells. Expression of neuronal marker proteins (nestin, βIII-tubulin, and microtubule-associated protein 2 [MAP2]) and the average length of cell neurites were lower in cells from HFD-SED rats than in other groups. Instead, protein expression of COX IV and Cyt-c, the Bax/Bcl-2 and LC3-II/I ratio, and the malondialdehyde level in culture medium were higher in cells from HFD-SED rats. No significant difference between ND-SED and HFD-EX rats was observed, except for the average length of cell neurites in MAP2. Thus, HFD impaired the differentiation potential of ADSCs into neuron-like cells, which was accompanied by increases in apoptotic activity and oxidative stress. Importantly, exercise training ameliorated the HFD-induced impairment of neurogenesis in ADSCs. The adipose tissue microenvironment could influence the differentiation potential of ADSCs, a source of autologous stem cell therapy.     

Differences in biochemical properties of two 5′-nucleotidases from deep- and shallow-sea Shewanella species under various harsh conditions

Deep-sea Shewanella violacea 5′-nucleotidase (SVNTase) activity exhibited higher NaCl tolerance than that of a shallow-sea Shewanella amazonensis homologue (SANTase), the sequence identity between them being 70.4%. Here, SVNTase exhibited higher activity than SANTase with various inorganic salts, similar to the difference in their NaCl tolerance. In contrast, SVNTase activity decreased with various organic solvents, while SANTase activity was retained with the same concentrations of the solvents. Therefore, SVNTase is more robust than SANTase with inorganic salts, but more vulnerable with organic solvents. As to protein stability, SANTase was more stable against organic solvents and heat than SVNTase, which correlated with the differences in their enzymatic activities. We also found that SANTase retained higher activity for three weeks than SVNTase did in the presence of glycerol. These findings will facilitate further application of these enzymes as appropriate biological catalysts under various harsh conditions.

Abbreviations: NTase: 5′-nucleotidase; SANTase: Shewanella amazonensis 5′-nucleotidase; SVNTase: Shewanella violacea 5′-nucleotidase; CD: circular dichroism     

Simultaneous determination of ascorbic acid and dehydroascorbic acid in fish tissues by high-performance liquid chromatography

An high-performance liquid chromatographic method with post-column derivatization has been developed for the simultaneous determination of ascorbic acid (AA) and dehydroascorbic acid (DHAA) in fish tissues. Extracted AA and DHAA were separated by a Shim-pack SCR-101H column within 20 min, reacted with sodium hydroxide containing sodium borohydride and monitored at 300 nm. The detection limits for both AA and DHAA were 0.1 μg/ml.     

Changes of protein profiles during pollen development in<Emphasis Type="Italic"> Lilium longiflorum</Emphasis>

Pollen proteins of Lilium longiflorum were examined at different developmental stages (young, mature and cultured) using two-dimensional differential gel electrophoresis. Quantitative changes of six proteins (MP1–MP6) during pollen development were observed in the acidic and low molecular weight region. After water absorption on the culture medium, the quantities of all six proteins were drastically changed. Mass spectrometric analysis revealed that MP2, MP3, MP4 and MP6 are late embryogenesis abundant (LEA) (D-7) protein, profilin 3, profilin 1 and enolase, respectively. The remaining two proteins (MP1 and MP5) could not be identified by mass spectrometric analysis. Immunogold electron microscopic examination showed the presence of these proteins in different regions: MP1 around lipid bodies, suggesting possible involvement in lipid metabolism, MP4 near actin in the cytoplasm, indicating the possibility of its interaction with actin in the regulatory pathways of pollen, and MP2 and MP6 in the cytoplasm.     

Six1 controls patterning of the mouse otic vesicle

Six1 is a member of the Six family homeobox genes, which function as components of the Pax-Six-Eya-Dach gene network to control organ development. Six1 is expressed in otic vesicles, nasal epithelia, branchial arches/pouches, nephrogenic cords, somites and a limited set of ganglia. In this study, we established Six1-deficient mice and found that development of the inner ear, nose, thymus, kidney and skeletal muscle was severely affected. Six1-deficient embryos were devoid of inner ear structures, including cochlea and vestibule, while their endolymphatic sac was enlarged. The inner ear anomaly began at around E10.5 and Six1 was expressed in the ventral region of the otic vesicle in the wild-type embryos at this stage. In the otic vesicle of Six1-deficient embryos, expressions of Otx1, Otx2, Lfng and Fgf3, which were expressed ventrally in the wild-type otic vesicles, were abolished, while the expression domains of Dlx5, Hmx3, Dach1 and Dach2, which were expressed dorsally in the wild-type otic vesicles, expanded ventrally. Our results indicate that Six1 functions as a key regulator of otic vesicle patterning at early embryogenesis and controls the expression domains of downstream otic genes responsible for respective inner ear structures. In addition, cell proliferation was reduced and apoptotic cell death was enhanced in the ventral region of the otic vesicle, suggesting the involvement of Six1 in cell proliferation and survival. In spite of the similarity of otic phenotypes of Six1- and Shh-deficient mice, expressions of Six1 and Shh were mutually independent.     

The Cerberus/Dan-family protein Charon is a negative regulator of Nodal signaling during left-right patterning in zebrafish

We have isolated a novel gene, charon, that encodes a member of the Cerberus/Dan family of secreted factors. In zebrafish, Fugu and flounder, charon is expressed in regions embracing Kupffer's vesicle, which is considered to be the teleost fish equivalent to the region of the mouse definitive node that is required for left-right (L/R) patterning. Misexpression of Charon elicited phenotypes similar to those of mutant embryos defective in Nodal signaling or embryos overexpressing Antivin(Atv)/Lefty1, an inhibitor for Nodal and Activin. Charon also suppressed the dorsalizing activity of all three of the known zebrafish Nodal-related proteins (Cyclops, Squint and Southpaw), indicating that Charon can antagonize Nodal signaling. Because Southpaw functions in the L/R patterning of lateral plate mesoderm and the diencephalon, we asked whether Charon is involved in regulating L/R asymmetry. Inhibition of Charon's function by antisense morpholino oligonucleotides (MOs) led to a loss of L/R polarity, as evidenced by bilateral expression of the left side-specific genes in the lateral plate mesoderm (southpaw, cyclops, atv/lefty1, lefty2 and pitx2) and diencephalon (cyclops, atv/lefty1 and pitx2), and defects in early (heart jogging) and late (heart looping) asymmetric heart development, but did not disturb the notochord development or the atv/lefty1-mediated midline barrier function. MO-mediated inhibition of both Charon and Southpaw led to a reduction in or loss of the expression of the left side-specific genes, suggesting that Southpaw is epistatic to Charon in left-side formation. These data indicate that antagonistic interactions between Charon and Nodal (Southpaw), which take place in regions adjacent to Kupffer's vesicle, play an important role in L/R patterning in zebrafish.     

Determination of potato glycoalkaloids using high-pressure liquid chromatography-electrospray ionisation/mass spectrometry

A method for quantifying two toxic glycoalkaloids, alpha-solanine and alpha-chaconine, in potato (Solanum tuberosum) tuber tissue was developed using HPLC-electrospray ionisation (ESI)/MS. Potato samples were extracted with 5% aqueous acetic acid, and the extracts were subjected directly to HPLC-ESI/MS after filtration. By determining the intensities of the protonated molecules of alpha-solanine (m/z 868) and alpha-chaconine (m/z 852) using selected ion monitoring (positive ion mode), a sensitive assay was attained with detection limits of 38 and 14 ppb for the two glycoalkaloids, respectively. The high sensitivity and selectivity of MS detection effectively reduced the time of analysis thus enabling a high throughput assay of glycoalkaloids in potato tubers.     

Stereoselective synthesis of (22R)- and (22S)-castasterone/ponasterone A hybrid compounds and evaluation of their molting hormone activity

Two stereoisomers of a castasterone/ponasterone A hybrid compound, the (20R,22R) and (20R,22S)-isomers of 2alpha,3alpha,20,22-tetrahydroxy-5alpha-cholestan-6-one, were synthesized stereoselectively and their binding activity to the ecdysteroid receptor was determined. From the concentration-response curve for the inhibition of the incorporation of tritiated ponasterone A into ecdysteroid receptor containing insect cells, the concentration (IC50) required to inhibit 50% of the incorporation of radioactivity into cells was evaluated. The IC50 values of the (22R)- and (22S)-isomers were determined to be 0.30 and 38.9 microM against Kc cells, respectively, indicating that the (22R)-isomer is about 100 times more potent than the corresponding (22S)-isomer. IC50 values of these compounds against lepidopteran Sf-9 cells were determined to be 0.36 and 12.9 microM, respectively. The molting hormonal effect was examined in a Chilo suppressalis integument system and the 50% effective concentration for the stimulation of N-acetylglucosamine incorporation into the cultured integument was determined to be 2.7 microM for the (22R)-isomer, while the (22S)-isomer was inactive. On the other hand, both isomers did not show brassinolide-like activity in the rice lamina inclination assay.     

Recognition of virus infected cells by NK cells

NK cells show cytotoxicity against virus-infected cells and tumor cells and play an important role in host defense. Although mecheanism of target cell recognition by NK cells have been unclear for a long time, it has recently been elucidated that certain NK cell receptors specifically recognize virus products. Furthermore, expression pattern of NK cell receptors, which consist of activating and inhibitory receptors, determines susceptibility to virus-infection. Here, we review recent progress of mechanism of recognition of virus-infected by NK cells.