High-throughput fluorescence labelling of full-length cDNA products based on a reconstituted translation system

Although recent advances in fluorescence-based technologies, such as protein microarrays, have made it possible to analyse more than 10,000 proteins at once, there is a bottleneck in the step of preparation of large numbers of fluorescently labelled proteins for the comprehensive analysis of protein-protein interactions. Here we describe two independent methods for high-throughput fluorescence-labelling of full-length cDNA products at their C-termini using a reconstituted translation system containing fluorescent puromycin. For the first method, release factor-free systems were used. For the second method, stop codons were excluded from cDNAs by using a common mismatch primer in mutagenic PCR. These methods yielded large numbers of labelled proteins from cDNA sets of various organisms, such as mouse, yeast and Escherichia coli.     

Elastic behavior of RecA-DNA helical filaments

Escherichia coli RecA protein forms a right-handed helical filament with DNA molecules and has an ATP-dependent activity that exchanges homologous strands between single-stranded DNA (ssDNA) and duplex DNA. We show that the RecA-ssDNA filamentous complex is an elastic helical molecule whose length is controlled by the binding and release of nucleotide cofactors. RecA-ssDNA filaments were fluorescently labelled and attached to a glass surface inside a flow chamber. When the chamber solution was replaced by a buffer solution without nucleotide cofactors, the RecA-ssDNA filament rapidly contracted approximately 0.68-fold with partial filament dissociation. The contracted filament elongated up to 1.25-fold when a buffer solution containing ATPgammaS was injected, and elongated up to 1.17-fold when a buffer solution containing ATP or dATP was injected. This contraction-elongation behavior was able to be repeated by the successive injection of dATP and non-nucleotide buffers. We propose that this elastic motion couples to the elastic motion and/or the twisting rotation of DNA strands within the filament by adjusting their helical phases.     

Discovery of a novel azetidine scaffold for colony stimulating factor-1 receptor (CSF-1R) Type II inhibitors by the use of docking models

We report the discovery of a novel azetidine scaffold for colony stimulating factor-1 receptor (CSF-1R) Type II inhibitors by using a structure-based drug design (SBDD) based on a docking model. The work leads to the representative compound 4a with high CSF-1R inhibitory activity (IC₅₀?=?9.1?nM). The obtained crystal structure of an azetidine compound with CSF-1R, which matched our predicted docking model, demonstrates that the azetidine compounds bind to the DFG-out conformation of the protein as a Type II inhibitor.     

l-Lactate Production from Biodiesel-Derived Crude Glycerol by Metabolically Engineered Enterococcus faecalis: Cytotoxic Evaluation of Biodiesel Waste and Development of a Glycerol-Inducible Gene Expression System

Biodiesel waste is a by-product of the biodiesel production process that contains a large amount of crude glycerol. To reuse the crude glycerol, a novel bioconversion process using Enterococcus faecalis was developed through physiological studies. The E. faecalis strain W11 could use biodiesel waste as a carbon source, although cell growth was significantly inhibited by the oil component in the biodiesel waste, which decreased the cellular NADH/NAD⁺ ratio and then induced oxidative stress to cells. When W11 was cultured with glycerol, the maximum culture density (optical density at 600 nm [OD₆₀₀]) under anaerobic conditions was decreased 8-fold by the oil component compared with that under aerobic conditions. Furthermore, W11 cultured with dihydroxyacetone (DHA) could show slight or no growth in the presence of the oil component with or without oxygen. These results indicated that the DHA kinase reaction in the glycerol metabolic pathway was sensitive to the oil component as an oxidant. The lactate dehydrogenase (Ldh) activity of W11 during anaerobic glycerol metabolism was 4.1-fold lower than that during aerobic glycerol metabolism, which was one of the causes of low l-lactate productivity. The E. faecalis pflB gene disruptant (Δpfl mutant) expressing the ldhL1_LP gene produced 300 mM l-lactate from glycerol/crude glycerol with a yield of >99% within 48 h and reached a maximum productivity of 18 mM h⁻¹ (1.6 g liter⁻¹ h⁻¹). Thus, our study demonstrates that metabolically engineered E. faecalis can convert crude glycerol to l-lactate at high conversion efficiency and provides critical information on the recycling process for biodiesel waste.     

Postural Abnormality as a Risk Marker for Leg Deep Venous Thrombosis in Parkinson’s Disease

Background

Pulmonary thromboembolism is a common cause of death in patients with autopsy-confirmed Parkinsonism. This study investigated the incidence of leg deep vein thrombosis in Parkinson’s disease and relationships between deep vein thrombosis and clinical/laboratory findings, including postural abnormalities as assessed by photographic measurements.

Methods

This cross-sectional study assessed the presence of deep vein thrombosis using bilateral leg Doppler ultrasonography in 114 asymptomatic outpatients with Parkinson’s disease.

Results

Deep vein thrombosis was detected in 23 patients (20%) with Parkinson’s disease. Deep vein thrombosis was located in the distal portion in 18 patients and in the proximal portion in 5 patients. No significant differences in age, sex, body mass index, disease duration, Hoehn-Yahr stage, anti-Parkinson’s drugs, or daily levodopa-equivalent dose were seen between deep vein thrombosis-positive and -negative groups. Univariate analysis for developing deep vein thrombosis in patients with Parkinson’s disease identified the following markers: long-term wheelchair use, bent knee, bent spine, and D-dimer elevation. Bending angles were significantly greater in the deep vein thrombosis-positive group at the knee and spine than in the deep vein thrombosis-negative group. Half of Parkinson’s disease patients with camptocormia had deep vein thrombosis. Among diabetes mellitus cases, long-term wheelchair use, bent knee over 15°, camptocormia, D-dimer elevation, the more risk markers were associated with a higher incidence of DVT. The presence of risk markers contributed to the development of deep vein thrombosis. On multivariate logistic regression analysis, a bent knee posture was strongly associated with an increased risk of deep vein thrombosis.

Conclusion

Presence of leg deep vein thrombosis correlated with postural abnormalities in Parkinson’s disease. We recommend non-invasive ultrasonographic screening for leg deep vein thrombosis in these high-risk patients with Parkinson’s disease.     

Development of stapled short helical peptides capable of inhibiting vitamin D receptor (VDR)–coactivator interactions

We synthesized stapled helical leucine-based peptides (DPI-01-07) containing 2-aminoisobutyric acid and a covalent cross-linked unit as inhibitors of vitamin D receptor (VDR)–coactivator interactions. The effects of these peptides on the human VDR were examined in an inhibition assay based on the receptor cofactor assay system, and one of them, DPI-07, exhibited potent inhibitory activity (IC₅₀: 3.2 μM).     

Specific and sensitive detection of H. pylori in biological specimens by real-time RT-PCR and in situ hybridization

PCR detection of H. pylori in biological specimens is rendered difficult by the extensive polymorphism of H. pylori genes and the suppressed expression of some genes in many strains. The goal of the present study was to (1) define a domain of the 16S rRNA sequence that is both highly conserved among H. pylori strains and also specific to the species, and (2) to develop and validate specific and sensitive molecular methods for the detection of H. pylori. We used a combination of in silico and molecular approaches to achieve sensitive and specific detection of H. pylori in biologic media. We sequenced two isolates from patients living in different continents and demonstrated that a 546-bp domain of the H. pylori 16S rRNA sequence was conserved in those strains and in published sequences. Within this conserved sequence, we defined a 229-bp domain that is 100% homologous in most H. pylori strains available in GenBank and also is specific for H. pylori. This sub-domain was then used to design (1) a set of high quality RT-PCR primers and probe that encompassed a 76-bp sequence and included at least two mismatches with other Helicobacter sp. 16S rRNA; and (2) in situ hybridization antisense probes. The sensitivity and specificity of the approaches were then demonstrated by using gastric biopsy specimens from patients and rhesus monkeys. This H. pylori-specific region of the 16S rRNA sequence is highly conserved among most H. pylori strains and allows specific detection, identification, and quantification of this bacterium in biological specimens.     

Histochemical study on the bile duct system of normal Mongolian gerbils

The bile duct system of normal Mongolian gerbils was examined histochemically. The luminal surface membrane and apical cytoplasm of the biliary and gallbladder epithelial cells were stained with periodic acid-Schiff (PAS), alcian blue, pH 2.5 (AB) and high iron diamine (HID)-AB, and many epithelial cells of the common bile duct and gallbladder had weakly PAS-positive granular material in their supranuclear cytoplasm. Lectin-histochemically, these cells had binding sites to Concanavalia ensiformis (ConA), Dolichos biflorus (DBA), Glycine max (SBA), Ulex europeas-I (UEA-I), and Triticum vulgaris (WGA). On the other hand, the periductal glandular epithelial cells were not stained by any histochemical stainings. In addition to these light microscopic findings, the electron microscopic findings based on the periodic acid-silver methenamine method and avidin-biotin colloidal gold method for DBA and WGA suggested that the biliary and gallbladder epithelial cells of Mongolian gerbils secreted mucin with terminal sialic and sulfonic acid residues and that the lectin binding activity of mucin secreted from these cells was similar to that of mucin secreted from the periductal glandular epithelial cells of mice and rats.