Metabolome analysis of food-chain between plants and insects

Evolution has shown the co-dependency between host plants and predators (insects), especially inevitable dependency of predators on plant biomass for securing their energy sources. It was postulated that NAD⁺ source used for major energy producing pathway is the glycerol-3-phosphate shuttle in insects. Using high throughput metabolomics approach, we found that larva of leaf beetle (Gastrophysa atrocyanea), which feed oxalate-rich plants (Rumex obtusifolius), possessed a unique mechanism for accumulating unusually high amounts of lactate. Similarly, larva of butterfly (Papilio machaon) fed with fennel (Foeniculum vulgare) accumulated lactate. Same butterfly also showed the elevated level of glycerol-3-phosphate equivalent to lactate. These evidences provide new insights into the mechanism underlying metabolite alteration between host plants and insect herbivores.     

Low pressure condition of a lipid core in an eccentrically developed carotid atheromatous plaque: a static finite element analysis

Plaque ruptures in atherosclerotic carotid arteries cause cerebral strokes. Accumulation of lipoproteins in the deep intimal layer forms a lipid core (LC), whose progression may be enhanced by mechanical conditions on the arterial wall. In this study, we investigated the pressure conditions of a liquid LC through numerical simulations of a sliced segment finite element (FE) model and a three-dimensional (3D) symmetric FE model. A model of an LC filled with nearly incompressible fluid was compared with incompressible and soft neo-Hookean LC models in a static FE analysis. Material constants for a nonlinear hyperelastic model of the arterial wall were identified based on an inflation test using a tube specimen. The results from the FE analysis of a sliced segment model show an LC fluid pressure as low as 1.9 kPa at a blood pressure of 16 kPa. A neo-Hookean LC model with a Young’s modulus of 0.06 kPa produced an almost uniform pressure in the LC within an error of 1.3 %. The 3D model predicted a similar level of LC pressure. Such low fluid pressure in the LC region may enhance the infiltration of lipoproteins and other substances from the lumen and facilitate transport through microvessels from the adventitia to the LC.     

Direct Induction of Chondrogenic Cells from Human Dermal Fibroblast Culture by Defined Factors

The repair of large cartilage defects with hyaline cartilage continues to be a challenging clinical issue. We recently reported that the forced expression of two reprogramming factors (c-Myc and Klf4) and one chondrogenic factor (SOX9) can induce chondrogenic cells from mouse dermal fibroblast culture without going through a pluripotent state. We here generated induced chondrogenic (iChon) cells from human dermal fibroblast (HDF) culture with the same factors. We developed a chondrocyte-specific COL11A2 promoter/enhancer lentiviral reporter vector to select iChon cells. The human iChon cells expressed marker genes for chondrocytes but not fibroblasts, and were derived from non-chondrogenic COL11A2-negative cells. The human iChon cells formed cartilage but not tumors in nude mice. This approach could lead to the preparation of cartilage directly from skin in human, without going through pluripotent stem cells.     

Anisotropic intersubunit and inter-ring interactions revealed in the native bullet-shaped chaperonin complex from Thermus thermophilus

Background

The recent morphological studies on chaperonins have revealed that nearly equivalent amount of symmetric GroEL–(GroES)₂ (football-shaped) and asymmetric GroEL–GroES (bullet-shaped) complexes coexist during the chaperonin reaction cycle, which prompted us to reexamine the equatorial split observed for chaperonin from Thermus thermophilus by implementing semi-empirical molecular orbital (MO) calculations, since it is now believed that the symmetric formation is a precursor to the equatorial split.

Methods

Semi-empirical MO calculations were employed to investigate the intersubunit interactions within the bullet-shaped T. thermophilus chaperonin capturing the substrate of folding intermediates. Interaction energies between each cis-GroEL subunit and closely related remaining subunits in cis-GroEL ring, or in trans-GroEL ring across the equatorial plane, and further, interaction energies between each GroES subunit and adjacent subunits in the same GroES ring and in cis-GroEL ring were simulated.

Results

Anisotropic intensities and energy distribution of the subunits were revealed by the calculations, which are consistent with two conformations of the subunits forming cis-GroEL ring as revealed by X-ray crystal structure, and with an anisotropic critical binding site on cis-GroEL ring for chaperonin functioning.

Conclusions

This is the first application of semi-empirical MO calculations to the macromolecular complex of the native bullet-shaped chaperonin (GroEL–GroES–ADP homolog) from T. thermophilus.

General significance

The results also appear to support the occurrence of the equatorial split for T. thermophilus chaperonin observed via electron microscopy, but has not yet been fully observed for Escherichia coli GroEL–GroES system.     

Characterization of Variant Creutzfeldt-Jakob Disease Prions in Prion Protein-humanized Mice Carrying Distinct Codon 129 Genotypes

To date, all clinical variant Creutzfeldt-Jakob disease (vCJD) patients are homozygous for methionine at polymorphic codon 129 (129M/M) of the prion protein (PrP) gene. However, the appearance of asymptomatic secondary vCJD infection in individuals with a PRNP codon 129 genotype other than M/M and transmission studies using animal models have raised the concern that all humans might be susceptible to vCJD prions, especially via secondary infection. To reevaluate this possibility and to analyze in detail the transmission properties of vCJD prions to transgenic animals carrying distinct codon 129 genotype, we performed intracerebral inoculation of vCJD prions to humanized knock-in mice carrying all possible codon 129 genotypes (129M/M, 129M/V, or 129V/V). All humanized knock-in mouse lines were susceptible to vCJD infection, although the attack rate gradually decreased from 129M/M to 129M/V and to 129V/V. The amount of PrP deposition including florid/amyloid plaques in the brain also gradually decreased from 129M/M to 129M/V and to 129V/V. The biochemical properties of protease-resistant abnormal PrP in the brain and transmissibility of these humanized mouse-passaged vCJD prions upon subpassage into knock-in mice expressing bovine PrP were not affected by the codon 129 genotype. These results indicate that individuals with the 129V/V genotype may be more susceptible to secondary vCJD infection than expected and may lack the neuropathological characteristics observed in vCJD patients with the 129M/M genotype. Besides the molecular typing of protease-resistant PrP in the brain, transmission studies using knock-in mice carrying bovine PrP may aid the differential diagnosis of secondary vCJD infection, especially in individuals with the 129V/V genotype.     

Chronic exposure to an extremely low‐frequency magnetic field induces depression‐like behavior and corticosterone secretion without enhancement of the hypothalamic–pituitary–adrenal axis in mice

An extremely low‐frequency magnetic field (ELF‐MF) is generated by power lines and household electrical devices. Many studies have suggested an association between chronic ELF‐MF exposure and anxiety and/or depression. The mechanism of these effects is assumed to be a stress response induced by ELF‐MF exposure. However, this mechanism remains controversial. In the present study, we investigated whether chronic ELF‐MF exposure (intensity, 3 mT; total exposure, 200 h) affected emotional behavior and corticosterone synthesis in mice. ELF‐MF‐treated mice showed a significant increase in total immobility time in a forced swim test and showed latency to enter the light box in a light–dark transition test, compared with sham‐treated (control) mice. Corticosterone secretion was significantly high in the ELF‐MF‐exposed mice; however, no changes were observed in the amount of the adrenocorticotropic hormone and the expression of genes related to stress response. Quantification of the mRNA levels of adrenal corticosteroid synthesis enzymes revealed a significant reduction in Cyp17a1 mRNA in the ELF‐MF‐exposed mice. Our findings suggest the possibility that high intensity and chronic exposure to ELF‐MF induces an increase in corticosterone secretion, along with depression‐ and/or anxiety‐like behavior, without enhancement of the hypothalamic–pituitary–adrenal axis. Bioelectromagnetics 34:43–51, 2013. © 2012 Wiley Periodicals, Inc.     

The sulfamide moiety affords higher inhibitory activity and oral bioavailability to a series of coumarin dual selective RAF/MEK inhibitors

Introducing a sulfamide moiety to our coumarin derivatives afforded enhanced Raf/MEK inhibitory activity concomitantly with an acceptable PK profile. Novel sulfamide 17 showed potent HCT116 cell growth inhibition (IC₅₀ = 8 nM) and good PK profile (bioavailability of 51% in mouse), resulting in high in vivo antitumor efficacy in the HCT116 xenograft (ED₅₀ = 4.8 mg/kg). We confirmed the sulfamide moiety showed no negative impact on tests run on the compound to evaluate DMPK (PK profiles in three animal species, CYP inhibition and CYP induction) and the safety profile (hERG and AMES tests). Sulfamide 17 had favorable properties that warranted further preclinical assessment     

An Improved Assay Method for Antifouling Substances Using the Blue Mussel,Mytilus edulis

The restriction endonuclease AatII was purified from cell-free extracts of Acetobacter aceti IFO 3281 by streptomycin treatment, ammonium sulfate fractionation, combined column chromatographies on DEAE-Toyopearl 650S, heparin-Sepharose CL-6B and DEAE-Sepharose CL-6B and FPLC on Mono Q and on Superose 12 (gel filtration). The purified enzyme was homogeneous on SDS-polyacrylamide gel disk electrophoresis. The relative molecular mass of the purified enzyme was 190,000 daltons by gel filtration. The SDS-polyacrylamide gel disk electrophoresis gave the relative molecular mass of 47,500 daltons. These data indicated that the purified, native enzyme is a tetramer (190,000 daltons) composed of four 47,500-dalton subunits. The isoelectric point of the enzyme was 6.0. The purified enzyme was intensely activated by manganese ion (50-fold increase or more when compared with magnesium ion). The enzyme worked best at 37°C and pH 8.5 in a reaction mixture (50 μl) containing 1.0 μg λDNA, 10 mm Tris-HCl, 7 mm 2-mercaptoethanol, 7 mm MnCl₂ and 50 mm NaCl. The enzyme recognizes the same palindromic hexanucleotide sequence 5′-GACGTC-3′, cuts between T and C and produces a 3′-tetranucleotide extension in the presence of MnCl₂, as it does in the presence of MgCl₂.