Improved polyhydroxybutyrate (PHB) production in transgenic tobacco by enhancing translation efficiency of bacterial PHB biosynthetic genes

Polyhydroxybutyrate [P(3HB)] was produced in the transgenic tobacco harboring the genes encoding acetoacetyl-CoA reductase (PhaB) and polyhydroxyalkanoate synthase (PhaC) from Ralstonia eutropha (Cupriavidus necator) with optimized codon usage for expression in tobacco. P(3HB) contents in the transformants (0.2mg/g dry cell weight in average) harboring the codon-optimized phaB gene was twofold higher than the control transformants harboring the wild-type phaB gene. The immunodetection revealed an increased production of PhaB in leaves, indicating that the enhanced expression of PhaB was effective to increase P(3HB) production in tobacco. In contrast, codon-optimization of the phaC gene exhibited no apparent effect on P(3HB) production. This result suggests that the efficiency of PhaB-catalyzed reaction contributed to the flux toward P(3HB) biosynthesis in tobacco leaves.     

Evaluation of MPN method combined with PCR procedure for detection and enumeration of Vibrio parahaemolyticus in seafood

Vibrio parahaemolyticus densities in spiked and naturally contaminated seafood samples were enumerated by the MPN method combined with a PCR procedure (MPN-PCR method) targeting the species-specific thermolabile hemolysin gene (tlh), and by the MPN method using subcultivation of alkaline-peptone-water (APW) enrichment culture on thiosulfate-citrate-bile-sucrose (TCBS) agar (MPN-TCBS method). In the samples spiked with both V. parahaemolyticus and V. alginolyticus, the numbers of V. parahaemolyticus enumerated by the MPN-PCR method were similar to, or higher than the numbers of spiked cells, whereas those enumerated by the MPN-TCBS method were below the numbers of spiked cells. In naturally contaminated seafood samples, the numbers of V. parahaemolyticus enumerated by the MPN-PCR method were higher than those by the MPN-TCBS method. In the case of the MPN-TCBS method, isolation of V. parahaemolyticus from some APW cultures was difficult because of the overgrowth of many colonies other than V. parahaemolyticus (e.g., V. alginolyticus) on TCBS agar. In contrast, the PCR technique could detect tlh from APW culture without isolation of V. parahaemolyticus, so the possibility of failing to obtain a positive result in APW culture by the MPN-PCR method was considered to be lower than that by the MPN-TCBS method. Furthermore, utilization of the PCR technique reduces the time and labor required for the biochemical identification tests used in the MPN-TCBS method. For the detection and enumeration of V. parahaemolyticus in seafood, especially for samples that show many colonies other than V. parahaemolyticus on TCBS agar, the MPN-PCR method may be more convenient and reliable than the MPN-TCBS method.     

Mumefural and related HMF derivatives from Japanese apricot fruit juice concentrate show multiple inhibitory effects on pandemic influenza A (H1N1) virus

Fruit-juice concentrate of Japanese apricot (Prunus mume Sieb. et Zucc.) has been shown to be effective against influenza A infection in MDCK cells. In this study, we isolated five components from the fruit-juice concentrate of Japanese apricot, 5-(hydroxymethyl)-2-formylfuran (HMF), 1-[5-(2-formylfuryl)methyl]dihydrogen 2-hydroxypropane-1,2,3-tricarboxylate (mumefural, MF), 2-[5-(2-formylfuryl)methyl]dihydrogen 2-hydroxypropane-1,2,3-tricarboxylate (MF‘), 1-[5-(2-formylfuryl)methyl]hydrogen 1-hydroxyethane-1,2-dicarboxylate (MA1) and 2-[5-(2-formylfuryl)methyl]hydrogen 1-hydroxyethane-1,2-dicarboxylate (MA2), and investigated their inhibitory activities against the novel influenza A/Narita/1/2009 (H1N1) pandemic virus hemagglutinin and neuraminidase functions, which are essential for viral attachment and budding, respectively. An hemagglutination inhibition assay indicated that MF and MF‘ were effective at minimum hemagglutination concentrations of 3.1 and 6.3 mM, respectively. An inhibition study for sialidase activity of the neuraminidase spike showed that MF was the most active anti-sialidase compound with an IC₅₀ value of 0.21 ± 0.01 mM, followed by MA2 (IC₅₀, 0.71 ± 0.09 mM), MA1 (IC₅₀, 1.64 ± 0.31 mM) and MF‘(IC₅₀, 1.62 ± 0.22 mM). Furthermore, MF was shown to inhibit the growth of the pandemic virus in a dose-dependent manner (62 ± 3% inhibition at 5 mM). The results suggest that MF, a citric acid ester linked to HMF at the 1-position of the propane backbone, might be a lead compound for the development of anti-influenza A inhibitors.     

In vivo visual reporter system for detection of estrogen-like substances by transgenic medaka

Detection of endocrine disrupting chemicals, in particular, environmental estrogens with living organisms, has many advantages if compared to chemical analysis. The screening of novel pollutants with meaningful endpoints, the integration of uptake, bioconcentration, and excretion as well as the evaluation of endocrine disrupting effects with respect to toxicity require in vivo biotests for estrogen-like substances (ELSs). Critical disadvantages of whole organism biotests are their low sensitivity and the need for laborious and time-consuming work. To overcome these problems, we have developed a transgenic medaka strain harboring the green fluorescence protein (GFP) gene driven by choriogenin H gene regulatory elements. Choriogenin H is an egg envelope protein induced by estrogens in the liver. With yolk sac larvae of this strain, GFP induction in liver was observed 24 h after onset of aqueous exposure to 0.63 nM 17beta-estradiol (E2), 0.34 nM ethynylestradiol, or 14.8 nM estrone. Furthermore, concentrated sewage treatment effluent induced GFP expression. Comparison of E2 equivalents estimated by GFP-induction in transgenic medaka, a YES assay, and GC/MS showed detection limits in the same order of magnitude. These results indicated that the sensitivity of the transgenic medaka strain was sufficient for application as an alternative model in monitoring environmental water samples for ELSs.     

Intramammary challenge of lipopolysaccharide stimulates secretion of lingual antimicrobial peptide into milk of dairy cows

Lingual antimicrobial peptide (LAP) belongs to the β-defensin family in cattle and is found in bovine milk. However, it is unclear whether LAP is involved in the early immune response to mammary infection. The aim of the study was to investigate the changes of LAP concentration in milk after intramammary challenge with lipopolysaccharide (LPS), the gram-negative bacteria cell membrane component, in dairy cows. Milk was collected before and after LPS or phosphate-buffered saline (control) challenge every hour for 12 h on d 0 and twice daily from d 1 to 7. Somatic cell count (SCC), LAP concentration, and lactoperoxidase (LPO) activity in the milk were measured. Somatic cell count started to increase at 2 h postchallenge and remained high until d 5 (694 ± 187 × 10³ to >1,000 ± 0 × 10³ cells/mL at d 0; >1,000 ± 0 × 10³ cells/mL at d 1 to 3; 684 ± 194 × 10³ to 829 ± 108 × 10³ cells/mL at d 4; 527 ± 197 × 10³ to 656 ± 145 × 10³ cells/mL at d 5). Somatic cell count increased in the control cows, although the levels were lower compared with those in the LPS challenge group. The LAP concentration in milk increased significantly at 2 h post-LPS-challenge and was maintained at high levels until d 2 (8.6 ± 0.6 to 17.5 ± 2.3 nM). In the control cow infused with phosphate-buffered saline, there was no increase of LAP concentration in milk (5.1 ± 0.6 to 7.2 ± 0.8 nM). Increase of LPO activity in the milk was observed at 6 h after LPS challenge and continued until d 3 (4.7 ± 0.3 to 9.4 ± 1.1 U). No increase of LPO activity was observed in the milk of control cows. The increase and subsequent decrease in LAP concentration after LPS challenge occurred earlier than those of LPO activity. In multiparous cows with LPS infusion, there was a significantly negative relationship between the days leading to the basal levels in LAP concentration and LPO activity (r = −0.75). These results suggest that LPS induces secretion of LAP into milk within hours and that LPO may have a synergistic antimicrobial function with LAP in mammary glands of dairy cows.     

Low‐Energy Electron Beam Induced Charging and Secondary Electron Emission Properties of FEP Film Used on Satellite Surfaces

Many kinds of insulating materials are used outside a spacecraft. They include FEP films, polyimide films, and so on, and are used as thermal control materials. These materials are exposed to a charged‐particle environment around the spacecraft. Thus then become charged due to charged particles, especially electrons. It has been pointed out that charging of these materials is likely to cause discharges on the surfaces. From this viewpoint, we investigated the charging potential characteristics of 127‐μm‐thick FEP film, a typical thermal control material, by exposing it to electron irradiation at various energies below 20 keV. In the dependence of the charging potential on the electron energy, we found that the electron energy at which no charge‐up occurs is about 2.7 keV. This appears to be the energy at the which secondary electron emission yield becomes unity. This indicates that electron irradiation of FEP film with energies lower than 2.7 keV induces positive charging. From the charge decay characteristics after electron irradiation, the volume resistivity of the film was also obtained as a function of the electric fields in the bulk of the FEP film.     

Photoacoustic Spectroscopy of Some Energetic Materials

To find an effective laser source to ignite energetic materials, the absorption spectra of some energetic materials are obtained by means of a photoacoustic spectroscopy (PAS). In this experiment, PAS covers the wavelength region of 400 nm-1600 nm in which no other conventional method can take absorption spectra for powdered energetic materials. Photoacoustic spectra of 18 energetic materials are reported. In general, energetic materials tested showed peaks in 600 nm–800 nm and 1400 nm–1600 nm ranges. It is found that the energy required to initiate explosives in the case of ruby laser initiation were correlated with their photoacoustic signal intensities.     

Characteristics of a-Si solar cells prepared by the super chamber at a high substrate temperature

A new approach to high-performance a-Si solar cells was studied. a-Si films prepared at a high substrate temperature (> 250°C) have a higher absorption coefficient and a low Si H₂ bond density. the effect of deposition temperature on the open-circuit voltage (V_oc) has been investigated systematically for glass/SnO2 Ipin/metal and glass/metal/nip/indium tin oxide (ITO) structure a-Si solar cells. The V_oc is found to depend strongly on the thermal history of the p/i interface. A short-circuit current of 19.5 mA/cm⁻⁻² was achieved for an a-Si solar cell using an a-Si i-layer with a thickness of 4000 Å, which was prepared at a substrate temperature of 270°C.     

Decomposition characteristics of biodegradable plastics made from sago starch‐extraction residue

Biodegradable plastics were synthesized for the effective use of sago starch‐extraction residue, which has been discarded as a waste. Two types of esterified sago starch‐extraction residue, P‐SP and L‐SP, were obtained. It had black color for P‐SP₁₆₀ (esterified by palm oil) to light yellow color for L‐SP₈₀ (esterified by lauric acid) and showed high carbon content, ranging from 399.3 to 537.1 g kg⁻¹. Biodegradable plastics from the residue, which had high esterification degree showed thermoplasticity and slower decomposition in Andisols in Japan and Inceptisols in Philippines. The esterification degrees of P‐SP₁₆₀ and L‐SP were 3.23 and 2.95 to 5.18 mmol g⁻¹, respectively. In addition, L‐SP₈₀ exhibited the most appropriate thermal softening behavior by heating. The cumulative decomposition of P‐SP₁₆₀ in Andisols and Inceptisols showed 16.7 and 32.8% of total carbon during 31 day of the incubation. On the other hand, the decomposition rates of L‐SP₈₀ in Andisols and Inceptisols were less than 10% of total carbon during 31 day of the incubation. The addition of triacetin as plasticizer to P‐SP₁₆₀ and L‐SP₈₀ remarkably influenced the decomposition rate of both molded P‐SP₁₆₀ and L‐SP₈₀. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

The Association of Cholesterol Uptake and Synthesis with Histology and Genotype in Cortisol-Producing Adenoma (CPA)

Cortisol-producing adenoma (CPA) is composed of clear and compact cells. Clear cells are lipid abundant, and compact ones lipid poor but associated with higher production of steroid hormones. PRKACA mutation (PRKACA mt) in CPA patients was reported to be associated with more pronounced clinical manifestation of Cushing’s syndrome. In this study, we examined the association of histological features and genotypes with cholesterol uptake receptors and synthetic enzymes in 40 CPA cases, and with the quantitative results obtained by gas chromatography-mass spectrometry (GC-MS) analysis in 33 cases to explore their biological and clinical significance. Both cholesterol uptake receptors and synthetic enzymes were more abundant in compact cells. GC-MS analysis demonstrated that the percentage of compact cells was inversely correlated with the concentrations of cholesterol and cholesterol esters, and positively with the activity of cholesterol biosynthesis from cholesterol esters. In addition, hormone-sensitive lipase (HSL), which catalyzes cholesterol biosynthesis from cholesterol esters, tended to be more abundant in compact cells of PRKACA mt CPAs. These results demonstrated that both cholesterol uptake and biosynthesis were more pronounced in compact cells in CPA. In addition, more pronounced HSL expression in compact cells of PRKACA mt CPA could contribute to their more pronounced clinical manifestation.