Mutual recognition between polymerized liposomes. IV. Polysaccharide-lectin system.

As a model of cell-cell recognition processes, the association processes of a polysaccharide (mannan)-carrying liposome with a lectin (Concanavalin A, Con A)-carrying polymerized liposome were followed by turbidimetry. The association process was strongly inhibited by the addition of a low molecular weight sugar, methyl-alpha-D-mannopyranoside, which shows that the association between the liposomes is due to the specific interaction between Con A and mannan. The association rate constant obtained was much smaller than the theoretical value for a diffusion-controlled binary association process. This implies that the association rate of liposomes is limited by the recognition between complementary ligands bound on the liposome surfaces. Another reason for the smaller association rate constant in the liposome-liposome system is the repulsive hydration effect. The effect of the surface density of the lectin immobilized on the liposome on the recognition was also examined.     

The extracellular matrix protein Del1 induces apoptosis via its epidermal growth factor motif

Mouse Del1 is an extracellular matrix protein mainly expressed in the developing embryo. Del1 has three EGF motifs and two discoidin domains. The second EGF motif reportedly contains an RGD sequence that binds to integrin receptors. Here, we provide evidence that Del1 protein induces cell death in vitro. Chromatin condensation and DNA laddering were observed, suggestive of apoptosis. The results of analysis using the TUNEL method and annexin V staining were also consistent with apoptosis. The apoptosis-inducing activity of Del1 could be mapped to the third EGF motif, which fitted the consensus sequence CX(D/N)XXXX(F/Y)XCXC, wherein the aspartic acid residue (D) could be β-hydroxylated. As little as twenty-five picomolar of recombinant E3 could induce apoptosis.     

Genetic mapping identifies a rice naringenin O-glucosyltransferase that influences insect resistance

Naringenin, the biochemical precursor for predominant flavonoids in grasses, provides protection against UV damage, pathogen infection and insect feeding. To identify previously unknown loci influencing naringenin accumulation in rice (Oryza sativa), recombinant inbred lines derived from the Nipponbare and IR64 cultivars were used to map a quantitative trait locus (QTL) for naringenin abundance to a region of 50 genes on rice chromosome 7. Examination of candidate genes in the QTL confidence interval identified four predicted uridine diphosphate-dependent glucosyltransferases (Os07g31960, Os07g32010, Os07g32020 and Os07g32060). In vitro assays demonstrated that one of these genes, Os07g32020 (UGT707A3), encodes a glucosyltransferase that converts naringenin and uridine diphosphate-glucose to naringenin-7-O-β-d -glucoside. The function of Os07g32020 was verified with CRISPR/Cas9 mutant lines, which accumulated more naringenin and less naringenin-7-O-β-d -glucoside and apigenin-7-O-β-d -glucoside than wild-type Nipponbare. Expression of Os12g13800, which encodes a naringenin 7-O-methyltransferase that produces sakuranetin, was elevated in the mutant lines after treatment with methyl jasmonate and insect pests, Spodoptera litura (cotton leafworm), Oxya hyla intricata (rice grasshopper) and Nilaparvata lugens (brown planthopper), leading to a higher accumulation of sakuranetin. Feeding damage from O. hyla intricata and N. lugens was reduced on the Os07g32020 mutant lines relative to Nipponbare. Modification of the Os07g32020 gene could be used to increase the production of naringenin and sakuranetin rice flavonoids in a more targeted manner. These findings may open up new opportunities for selective breeding of this important rice metabolic trait.     

Combining Machine Learning Systems and Multiple Docking Simulation Packages to Improve Docking Prediction Reliability for Network Pharmacology

Increased availability of bioinformatics resources is creating opportunities for the application of network pharmacology to predict drug effects and toxicity resulting from multi-target interactions. Here we present a high-precision computational prediction approach that combines two elaborately built machine learning systems and multiple molecular docking tools to assess binding potentials of a test compound against proteins involved in a complex molecular network. One of the two machine learning systems is a re-scoring function to evaluate binding modes generated by docking tools. The second is a binding mode selection function to identify the most predictive binding mode. Results from a series of benchmark validations and a case study show that this approach surpasses the prediction reliability of other techniques and that it also identifies either primary or off-targets of kinase inhibitors. Integrating this approach with molecular network maps makes it possible to address drug safety issues by comprehensively investigating network-dependent effects of a drug or drug candidate.     

Mating reaction inSaccharomyces cerevisiae

The effect of proteolytic enzymes on sexual agglutinability of haploid cells of the yeastSaccharomyces cerevisiae was examined. Sexual agglutinability of cells of botha and α types was lost on treatment with alkaline protease and two kinds of neutral proteases ofBacillus subtilis, pronase and α-chymotrypsin. Agglutinability of α type cells was lost after treatment with acid protease ofRhizopus chinensis and trypsin, but that ofa type cells was not. These results indicate that the sex-specific substance responsible for the sexual agglutination (agglutination factor) ina type cells differs from that in α type cells. Agglutination factors were solubilized from cell-wall fractions of both mating types by Glusulase treatment. These crude factors specifically inhibited the agglutinability of cells of the opposite mating type with little effect on the agglutinability of cells of the same mating type.     

Clara cell secretory protein and phospholipase A2 activity modulate acute ventilator-induced lung injury in mice.

Lung vascular permeability is acutely increased by high-pressure and high-volume ventilation. To determine the roles of mechanically activated cytosolic PLA2 (cPLA2)and Clara cell secretory protein (CCSP), a modulator of cPLA2 activity, we compared lung injury with and without a PLA2 inhibitor in wild-type mice and CCSP-null mice (CCSP-/-) ventilated with high and low peak inflation pressures (PIP) for 2- or 4-h periods. After ventilation with high PIP, we observed significant increases in the bronchoalveolar lavage albumin concentrations, lung wet-to-dry weight ratios, and lung myeloperoxidase in both genotypes compared with unventilated controls and low-PIP ventilated mice. All injury variables except myeloperoxidase were significantly greater in the CCSP-/- mice relative to wild-type mice. Inhibition of cPLA2 in wild-type and CCSP-/- mice ventilated at high PIP for 4 h significantly reduced bronchoalveolar lavage albumin and total protein and lung wet-to-dry weight ratios compared with vehicle-treated mice of the same genotype. Membrane phospho-cPLA2 and cPLA2 activities were significantly elevated in lung homogenates of high-PIP ventilated mice of both genotypes but were significantly higher in the CCSP-/- mice relative to the wild-type mice. Inhibition of cPLA2 significantly attenuated both the phospho-cPLA2 increase and increased cPLA2 activity due to high-PIP ventilation. We propose that mechanical activation of the cPLA2 pathway contributes to acute high PIP-induced lung injury and that CCSP may reduce this injury through inhibition of the cPLA2 pathway and reduction of proinflammatory products produced by this pathway.     

Development of Biotin-Prototrophic and -Hyperauxotrophic Corynebacterium glutamicum Strains

To develop the infrastructure for biotin production through naturally biotin-auxotrophic Corynebacterium glutamicum, we attempted to engineer the organism into a biotin prototroph and a biotin hyperauxotroph. To confer biotin prototrophy on the organism, the cotranscribed bioBF genes of Escherichia coli were introduced into the C. glutamicum genome, which originally lacked the bioF gene. The resulting strain still required biotin for growth, but it could be replaced by exogenous pimelic acid, a source of the biotin precursor pimelate thioester linked to either coenzyme A (CoA) or acyl carrier protein (ACP). To bridge the gap between the pimelate thioester and its dedicated precursor acyl-CoA (or -ACP), the bioI gene of Bacillus subtilis, which encoded a P450 protein that cleaves a carbon-carbon bond of an acyl-ACP to generate pimeloyl-ACP, was further expressed in the engineered strain by using a plasmid system. This resulted in a biotin prototroph that is capable of the de novo synthesis of biotin. On the other hand, the bioY gene responsible for biotin uptake was disrupted in wild-type C. glutamicum. Whereas the wild-type strain required approximately 1 μg of biotin per liter for normal growth, the bioY disruptant (ΔbioY) required approximately 1 mg of biotin per liter, almost 3 orders of magnitude higher than the wild-type level. The ΔbioY strain showed a similar high requirement for the precursor dethiobiotin, a substrate for bioB-encoded biotin synthase. To eliminate the dependency on dethiobiotin, the bioB gene was further disrupted in both the wild-type strain and the ΔbioY strain. By selectively using the resulting two strains (ΔbioB and ΔbioBY) as indicator strains, we developed a practical biotin bioassay system that can quantify biotin in the seven-digit range, from approximately 0.1 μg to 1 g per liter. This bioassay proved that the engineered biotin prototroph of C. glutamicum produced biotin directly from glucose, albeit at a marginally detectable level (approximately 0.3 μg per liter).     

Comparative Study between Gas Chromatography and Ion-exchange Chromatography on Amino Acid Analysis of Foods

Lactobacillus brevis and Saccharomyces cerevisiae were completely sterilized by the supercritical (SC) CO2 micro-bubble method. Gaseous (G) and liquid (LQ) CO2 were used in a similar manner to compare the sterilizing effect. Among the three treatments, the microorganisms were only effectively sterilized by the SC CO2 treatment at 25 MPa and 35°C.     

Anti-barnacle activity of novel simple alkyl isocyanides derived from citronellol

Twenty novel simple alkyl isocyanides derived from citronellol were synthesized and evaluated for their antifouling activity and toxicity against cypris larvae of the barnacle, Balanus amphitrite. The anti-barnacle activity of the synthesized isocyanides was in the EC₅₀ range of 0.08–1.49 μg ml^?1. Simple isocyanides containing a benzoate and chloro group showed the most potent anti-barnacle activity. In addition, none of the synthesized compounds showed significant toxicity and LC₅₀ values were <10 μg ml^?1. The LC₅₀/EC₅₀ ratios of almost all of the synthesized compounds were >10². The results indicate that these simple isocyanides are promising low-toxicity antifouling agents.