Close association of centrosomes to the distal ends of the microbody during its growth, division and partitioning in the green alga Klebsormidium flaccidum

Summary. Division and partitioning of microbodies (peroxisomes) of the green alga Klebsormidium flaccidum, whose cells contain a single microbody, were investigated by electron microscopy. In interphase, the rod-shaped microbody is present between the nucleus and the single chloroplast, oriented perpendicular to the pole-to-pole direction of the future spindle. A centriole pair associates with one distal end of the microbody. In prophase, the microbody changes not only in shape, from a rodlike to a branched form, but also in orientation, from perpendicular to parallel to the future pole-to-pole direction. Duplicated centriole pairs are localized in close proximity to both distal ends of the microbody. In metaphase, the elongated microbody flanks the open spindle, with both distal ends close to the centriole pair at either spindle pole. The microbody further elongates in telophase and divides after septum formation (cytokinesis) has started. The association between the centrioles and both distal ends of the microbody is maintained throughout mitosis, resulting in the distal ends of the elongated microbody being fixed at the cellular poles. This configuration of the microbody may be favorable for faithful transmission of the organelle during cell division. After cytokinesis is completed, the microbody reverts to the perpendicular orientation by changing its shape. Microtubules radiating from the centrosomes flank the side of the microbody throughout mitosis. The close association of centrosomes and microtubules with the microbody is discussed in respect to the partitioning of the microbody in this alga. Correspondence: H. Hashimoto, Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Komaba, Meguro-ku, Tokyo 153-8902, Japan. Present address: M. Honda, Department of Computational Biology, Graduate School of Frontier Sciences, University of Tokyo, Kashiwa, Chiba, Japan.     

Design and synthesis of new potent dipeptidyl peptidase IV inhibitors with enhanced ex vivo duration

A series of 5beta-methylprolyl-2-cyanopyrrolidine analogs were synthesized and evaluated as DPP-IV inhibitors, and the duration of their ex vivo activity was assessed. Comparison of their potency and duration of action was done among three different species. The mode of binding was investigated, and the effect on the plasma glucose level was evaluated. Structure-activity relationships are also presented.     

L-lactic acid secreted from gastric mucosal cells enhances growth of Helicobacter pylori

BACKGROUND: Helicobacter pylori mainly inhabit the mucus layer in the gastric mucosa. However, mechanisms involving H. pylori colonization and proliferation in gastric mucosa are not well established. This study focuses on elucidating the role of gastric mucosal cells on growth of H. pylori. MATERIALS AND METHODS: H. pylori was co-cultured with the murine gastric surface mucosal cells (GSM06), and the growth of H. pylori on the cells was assessed by enumerating the colony-forming units (CFU). The H. pylori growth factor in the culture media conditioned by GSM06 cell was purified by HPLC, and the chemical structure of the growth factor was identified by analyses of (1)H- and (13)C-NMR spectra. RESULTS: A marked increase in the number of CFU of H. pylori was observed in the GSM06 cells. The enhanced H. pylori growth was also observed when indirectly incubated with GSM06 cells through semi-permeable membrane. In addition, culture media conditioned by GSM06 cell stimulated H. pylori growth approximately one thousand-fold. By bioassay-guided purification, the H. pylori growth factor was isolated from the conditioned medium of GSM06 cells and identified as L-lactic acid. The H. pylori growth-enhancing activity under microaerobic condition was well correlated with L-lactic acid concentrations in the conditioned media. CONCLUSIONS: This study demonstrates that L-lactic acid secreted by gastric mucosal cells enhances the growth of H. pylori, and this L-lactic acid-dependent growth of H. pylori may be important to the long-term colonization of H. pylori in the stomach.     

Identification and characterization of a dextranase gene of Streptococcus criceti

The dextranase gene, dex, was identified in Streptococcus criceti strain E49 by degenerate PCR and sequenced completely by the gene-walking method. A sequence of 3,960 nucleotides was determined. The dex gene encodes a 1,200-amino acid protein, which has a calculated molecular mass of 128,129.91 and pI of 4.15 and is predicted to be a cell-surface protein. The deduced amino acid sequence of dex showed homology to S. downei dextranase (63.9% identity). Phylogenetic analysis revealed the similarity of the deduced amino acid sequence of dextranases in S. criceti, S. sobrinus, and S. downei. A recombinant form of the protein with six histidine residues tagged in the C-terminus was partially purified and showed dextranase activity on blue-dextran sodium dodecyl sulfate-polyacrylamide gel electrophoresis (BD-SDSPAGE) followed by renaturation. We also detected dextranase activity in S. criceti cell extracts and culture supernatant by renatured BD-SDS-PAGE, whereas no dextranase activity of the cells was observed on blue-dextran brain heart infusion (BD-BHI) agar plates. Furthermore, PCR-based mutations of dextranase indicated that a deletion mutant of the C-terminal region could hydrolyze blue dextrans and that the D453E mutation, W793L mutation, and double mutations (W793L and deletion of the C-terminal region) resulted in a loss of dextranase activity. These findings suggest that Asp-453 and Trp-793 residues of S. criceti dextranase are critical to the enzyme's activity.     

Lincomycin at Subinhibitory Concentrations Potentiates Secondary Metabolite Production by Streptomyces spp.

Antibiotics have either bactericidal or bacteriostatic activity. However, they also induce considerable gene expression in bacteria when used at subinhibitory concentrations (below the MIC). We found that lincomycin, which inhibits protein synthesis by binding to the ribosomes of Gram-positive bacteria, was effective for inducing the expression of genes involved in secondary metabolism in Streptomyces strains when added to medium at subinhibitory concentrations. In Streptomyces coelicolor A3(2), lincomycin at 1/10 of its MIC markedly increased the expression of the pathway-specific regulatory gene actII-ORF4 in the blue-pigmented antibiotic actinorhodin (ACT) biosynthetic gene cluster, which resulted in ACT overproduction. Intriguingly, S. lividans 1326 grown in the presence of lincomycin at a subinhibitory concentration (1/12 or 1/3 of its MIC) produced abundant antibacterial compounds that were not detected in cells grown in lincomycin-free medium. Bioassay and mass spectrometry analysis revealed that some antibacterial compounds were novel congeners of calcium-dependent antibiotics. Our results indicate that lincomycin at subinhibitory concentrations potentiates the production of secondary metabolites in Streptomyces strains and suggest that activating these strains by utilizing the dose-response effects of lincomycin could be used to effectively induce the production of cryptic secondary metabolites. In addition to these findings, we also report that lincomycin used at concentrations for markedly increased ACT production resulted in alteration of the cytoplasmic protein (F_oF₁ ATP synthase α and β subunits, etc.) profile and increased intracellular ATP levels. A fundamental mechanism for these unique phenomena is also discussed.     

A Novel Antibody Engineering Strategy for Making Monovalent Bispecific Heterodimeric IgG Antibodies by Electrostatic Steering Mechanism

Producing pure and well behaved bispecific antibodies (bsAbs) on a large scale for preclinical and clinical testing is a challenging task. Here, we describe a new strategy for making monovalent bispecific heterodimeric IgG antibodies in mammalian cells. We applied an electrostatic steering mechanism to engineer antibody light chain-heavy chain (LC-HC) interface residues in such a way that each LC strongly favors its cognate HC when two different HCs and two different LCs are co-expressed in the same cell to assemble a functional bispecific antibody. We produced heterodimeric IgGs from transiently and stably transfected mammalian cells. The engineered heterodimeric IgG molecules maintain the overall IgG structure with correct LC-HC pairings, bind to two different antigens with comparable affinity when compared with their parental antibodies, and retain the functionality of parental antibodies in biological assays. In addition, the bispecific heterodimeric IgG derived from anti-HER2 and anti-EGF receptor (EGFR) antibody was shown to induce a higher level of receptor internalization than the combination of two parental antibodies. Mouse xenograft BxPC-3, Panc-1, and Calu-3 human tumor models showed that the heterodimeric IgGs strongly inhibited tumor growth. The described approach can be used to generate tools from two pre-existent antibodies and explore the potential of bispecific antibodies. The asymmetrically engineered Fc variants for antibody-dependent cellular cytotoxicity enhancement could be embedded in monovalent bispecific heterodimeric IgG to make best-in-class therapeutic antibodies.     

Ab initio simulation of a 57-residue protein in explicit solvent reproduces the native conformation in the lowest free-energy cluster

An enhanced conformational sampling method, multicanonical molecular dynamics (McMD), was applied to the ab intio folding of the 57-residue first repeat of human glutamyl- prolyl-tRNA synthetase (EPRS-R1) in explicit solvent. The simulation started from a fully extended structure of EPRS-R1 and did not utilize prior structural knowledge. A canonical ensemble, which is a conformational ensemble thermodynamically probable at an arbitrary temperature, was constructed by reweighting the sampled structures. Conformational clusters were obtained from the canonical ensemble at 300 K, and the largest cluster (i.e., the lowest free-energy cluster), which contained 34% of the structures in the ensemble, was characterized by the highest similarity to the NMR structure relative to all alternative clusters. This lowest free-energy cluster included native-like structures composed of two anti-parallel α-helices. The canonical ensemble at 300 K also showed that a short Gly-containing segment, which adopts an α-helix in the native structure, has a tendency to be structurally disordered. Atomic-level analyses demonstrated clearly that inter-residue hydrophobic interactions drive the helix formation of the Gly-containing segment, and that increasing the hydrophobic contacts accompanies exclusion of water molecules from the vicinity of this segment. This study has shown, for the first time, that the free-energy landscape of a structurally well-ordered protein of about 60 residues is obtainable with an all atom model in explicit water without prior structural knowledge.     

Soluble interleukin-18 receptor complex is a novel biomarker in rheumatoid arthritis

Introduction

There has been no report in the literature of a soluble form of interleukin (IL)-18 receptor α (IL-18Rα). In this study, we evaluated the levels and characteristics of soluble IL-18Rα (sIL-18Rα) in the sera of patients with rheumatoid arthritis (RA) and compared these results to control populations.

Methods

The sIL-18Rα complex was isolated from pooled human blood serum using an anti-IL-18Rα monoclonal antibody affinity column. The purified sIL-18Rα was then examined using Western blot analysis and used in experiments to evaluate the effects on an IL-18-responsive natural killer (NK) human cell line, NK0. An enzyme-linked immunosorbent assay was developed, and sera from 145 patients with RA, 6 patients with adult-onset Still's disease, 31 patients with osteoarthritis (OA), 39 patients with systemic lupus erythematosus (SLE) and 67 controls were tested, along with levels of immunoglobulin M, rheumatoid factor, anticyclic citrullinated peptide antibody, IL-18, IL-13 and interferon (IFN)-γ. Area under the receiver operating characteristic curve (ROC-AUC) analysis was used to evaluate the diagnostic utility of the sIL-18Rα complex.

Results

The isolated sIL-18Rα complex can be associated with IL-18 and the soluble form of the IL-18Rβ chain. The sIL-18Rα complex bound to the surface to the NK0 cell line, antagonized the stimulatory effects of IL-18 and IL-2 on the NK0 cell line and inhibited IFN-γ production by the cells. The serum levels of sIL-18Rα complex in RA (186.0 ± 33.5 ng/mL, n = 145) and adult-onset Still's disease (98.2 ± 8.9 ng/mL, n = 6) were significantly (P < 0.001) higher than those in the healthy controls (52.3 ± 8.5 ng/mL, n = 67), OA (38.6 ± 5.4 ng/mL, n = 31), SLE (44.6 ± 3.2 ng/mL, n = 39). The serum level of sIL-18Rα complex was not significantly different between RA and adult-onset Still's disease patients. The serum levels of IL-18, IL-13 and IFN-γ in the RA patients were significantly (P < 0.01) higher than in OA and SLE patients as well as healthy controls. ROC-AUC analysis of the serum concentration of sIL-18Rα indicated that it was significantly diagnostic of RA. Moreover, a tumor necrosis factor inhibitor, etanercept, significantly (P < 0.0001) decreased levels of sIL-18Rα in the sera of 29 RA patients 6 months after treatment.

Conclusions

The sIL-18Rα complex could be a potentially useful biomarker for the diagnosis of RA.     

Bioluminescence characteristics of the fruiting body of Mycena chlorophos

Bioluminescent fungi are widely distributed on land and most belong to the class Basidomycetes. Light of about 530 nm wavelength maximum is emitted continuously. The molecular basis for the light‐emitting process remains unclear. We investigated the characteristics of the bioluminescence using cultivated fruiting bodies of M. chlorophos. Only fresh fruiting bodies exhibited long‐lasting light emission; rapid decay of light emission was observed with frozen and freeze‐dried samples. Freeze‐dried samples can be stored at room temperature under dry conditions and may be useful for the isolation of luciferin. The light emission of the fresh fruiting bodies was maintained in various buffers at varying pH; it could be stopped with pH 4 acetate buffer and could be recovered at pH 6. The isolation of luciferin from the fresh fruiting bodies might be possible by the control of buffer pH. The effect of temperature on the light emission of fruiting bodies indicated that bioluminescence in M. chlorophos may involve enzymatic reaction(s). The solubilization of bioluminescent components from the fruiting bodies could not be achieved with various surfactants. Copyright © 2011 John Wiley & Sons, Ltd.