In vitro evolution of a polyhydroxybutyrate synthase by intragenic suppression-type mutagenesis

In vitro evolution was applied to obtain highly active mutants of Ralstonia eutropha polyester synthase (PhbC(Re)), which is a key enzyme catalyzing the formation of polyhydroxybutyrate (PHB) from (R)-3-hydroxybutyryl-CoA (3HB-CoA). To search for beneficial mutations for activity improvement of this enzyme, we have conducted multi-step mutations, including activity loss and intragenic suppression-type activity reversion. Among 259 revertants, triple mutant E11S12 was obtained as the most active one via PCR-mediated secondary mutagenesis from mutant E11 with a single mutation (Ser to Pro at position 80), which exhibited reduced activity (as low as 27% of the wild-type level) but higher thermostability compared to the wild-type enzyme. Mutant E11S12 exhibited up to 79% of the wild-type enzyme activity. Mutation separation of E11S12 revealed that the replacement of Phe by Ser at position 420 (F420S), located in a highly conserved alpha/beta hydrolase fold region, of the E11S12 mutant contributes to the improvement of the enzyme activity. A purified sample of the genetically engineered mutant, termed E11S12-1, with the F420S mutation alone was found to exhibit a 2.4-fold increase in specific activity toward 3HB-CoA, compared to the wild-type.     

15-Deoxyspergualin inhibits Akt kinase activation and phosphatidylcholine synthesis

15-Deoxyspergualin (DSG) strongly inhibited growth of mouse EL-4 lymphoma cells in vitro and in vivo. It significantly prolonged the survival days of EL-4-transplanted mice. In vitro study revealed that its antiproliferative effect appeared only after 2 days of treatment. At that time, protein synthesis was significantly inhibited rather than DNA and RNA syntheses. Furthermore, DSG induced apoptosis without arresting the cell cycle. p70 S6 kinase (p70S6K), a key molecule in protein synthesis, was inhibited by 2 days of treatment of DSG. Akt, an upstream kinase of p70S6K, was also deactivated by 2 days of treatment of DSG. Hsp90 is reported to bind to and stabilize Akt kinase and also to bind to DSG. Yet DSG did not inhibit the binding of Hsp90 to Akt kinase. PI3-kinase, an activator of Akt, was not affected by DSG treatment. However, when we looked into phospholipid synthesis, we found that DSG inhibited phosphatidylcholine (PC) synthesis strongly rather than phosphatidylinositol even by 1 day of treatment. Moreover, DSG failed to inhibit Akt kinase activation and PC synthesis in DSG-less sensitive human K562 leukemia cells. These results demonstrate that DSG inhibits tumor cell growth through the inhibition of protein synthesis and induction of apoptosis, which is caused by the down-regulation of Akt kinase and p70S6K. It is also indicated that the down-regulation of Akt kinase by DSG should not depend on PI3-kinase and Hsp90. There might be possible involvement of PC in Akt kinase activity.     

The surface of lipid droplets is a phospholipid monolayer with a unique Fatty Acid composition

We found that caveolin-2 is targeted to the surface of lipid droplets (Fujimoto, T., Kogo, H., Ishiguro, K., Tauchi, K., and Nomura, R. (2001) J. Cell Biol. 152, 1079-1085) and hypothesized that the lipid droplet surface is a kind of membrane. To elucidate the characteristics of the lipid droplet surface, we isolated lipid droplets from HepG2 cells and analyzed them by cryoelectron microscopy and by mass spectrometry. By use of cryoelectron microscopy at the stage temperature of 4.2 K, the lipid droplet surface was observed as a single line without any fixation or staining, indicating the presence of a single layer of phospholipids. This result appeared consistent with the hypothesis that the lipid droplet surface is derived from the cytoplasmic leaflet of the endoplasmic reticulum membrane and may be continuous to it. However, mass spectrometry revealed that the fatty acid composition of phosphatidylcholine and lysophosphatidylcholine in lipid droplets is different from that of the rough endoplasmic reticulum. The ample presence of free cholesterol in lipid droplets also suggests that their surface is differentiated from the bulk endoplasmic reticulum membrane. On the other hand, although caveolin-2beta and adipose differentiation-related protein, both localizing in lipid droplets, were enriched in the low density floating fraction, the fatty acid composition of the fraction was distinct from lipid droplets. Collectively, the result indicates that the lipid droplet surface is a hemi-membrane or a phospholipid monolayer containing cholesterol but is compositionally different from the endoplasmic reticulum membrane or the sphingolipid/cholesterol-rich microdomain.     

Elevation of antioxidant potency in mice brain by low-dose X-ray irradiation and its effect on Fe-NTA-induced brain damage

The increase in lipid peroxide levels in mice brain following Fe3+ administration was about 50% of that when 1-methyl-4-phenyl 1,2,3,6-tetrahydropyridine (MPTP) was administered. This may be due to excessive oxidation by Fe3+, and was supported by the decrease in activities of antioxidant enzymes, such as superoxide dismutase (SOD), catalase and glutathione peroxidase (GPX), Na+,K(+)-ATPase activity and membrane fluidity after Fe3+ administration. Relatively low-dose X-ray irradiation (0.5 Gy) inhibited lipid peroxidation associated with Fe3+ administration and restored the decreased activities of the above antioxidant enzymes and Na+,K(+)-ATPase, and membrane fluidity to the levels in the non-Fe(3+)-administered group. In the purine metabolism system, uric acid decreased after Fe3+ administration, which may be due to transient impairment of the system for production of uric acid from xanthine by excessive oxidation by Fe3+. However, 0.5 Gy irradiation inhibited this decrease in uric acid, increasing its level to that in the non Fe(3+)-administrated group. This may be due to factors such as rapid recovery of the activities of the above antioxidant enzymes and Na+,K(+)-ATPase, and membrane fluidity after 0.5 Gy irradiation. In addition, since no changes were observed in xanthine and uric acid, increased inosine and hypoxanthine may have advanced to a salvage pathway leading to not xanthine but inosine 5'-monophosphate (IMP).     

A simple exploratory algorithm for the accurate and fast detection of spontaneous synaptic events

We have developed a program for the fast and accurate detection of spontaneous synaptic events. The algorithm identifies each event of which the slope and amplitude which meet criteria. The significant feature of this algorithm is its stepwise and exploratory search for the onset and the peak points. During the first step, the program employing the algorithm makes a rough estimate of the candidate for a synaptic event, and determines a 'temporary' onset data point. The next step is the detection of the true onset data point and 'temporary' peak data point, which probably exist several points after the temporary onset data point. The third step is a backward search to detect the true peak data point. The final step is to check whether the amplitude of the detected event exceeds the threshold. This stepwise and shuttlewise search allows for the accurate detection of the peak points. Using this program, we succeeded in detecting an increased frequency and amplitude of spontaneous excitatory postsynaptic currents in chick cerebral neurons following the application of 12-O-tetradecanoyl-phorbol-13-acetate (TPA). In addition, we demonstrated that the program employing the algorithm was able to be used for the detection of extracellular action potentials.     

Expression of biologically active recombinant equine interferon-gamma by two different baculovirus gene expression systems using insect cells and silkworm larvae

The full-length equine interferon-gamma (eIFN-gamma) cDNA, including the secretion signal peptide coding region, was recloned into baculovirus transfer vector pAcYM1. This vector was co-transfected with Autographa californica nuclear polyhedrosis virus DNA or hybrid nuclear polyhedrosis virus DNA into Spodoptera frugiperda cells. The recombinant viruses, named AcEIFN-gamma and HyEIFN-gamma, were then recovered. Recombinant eIFN-gamma (reIFN-gamma) was accumulated in the culture fluid of the AcEIFN-gamma or HyEIFN-gamma infected Tricoplusia ni -derived cell line, BTI TN 5B1-4, and hemolymph of HyEIFN-gamma infected silkworm larvae. These reIFN-gamma forms were shown to be 14, 16, 18 and 20kDa proteins, and glycosylated as confirmed by SDS-PAGE and tunicamycin treatment. Both reIFN-gamma proteins, showed high-level biological activities to vesicular stomatitis virus by cytopathic effect reduction assay, and MHC class II antigen induction on the equine fetal kidney-78 cell line.     

Protective effect of colored rice over white rice on Fenton reaction-based renal lipid peroxidation in rats

Rice has been one of the most important grains. While polished white rice is favored, colored strains of rice, red, or black, have been maintained for religious purposes in Japan. We studied whether feeding of unpolished colored rice instead of white rice ameliorates oxidative renal tubular damage in rats induced by ferric nitrilotriacetate. Whereas renal lipid peroxidation was exacerbated in white rice-fed group in comparison with standard chow group, this exacerbation was not observed in red or black rice-fed groups. These changes were dependent on the proportion of colored rice to standard chow in the diet. Cyanidin 3- O - β- d -glucoside was detectable neither in the serum nor kidney after one week of colored rice diet, but serum protocatechuic acid was significantly increased after black rice diet. There was a generalized decrease in the renal glutathione peroxidase activity in rice diet groups. Renal enzymatic activities of superoxide dismutase, glutathione S -transferase and NAD(P)H quinone reductase were not associated with the levels of lipid peroxidation. However, renal catalase activity was significantly increased in black rice-fed groups. These may partly explain the antioxidative effect. Furthermore, colored strains of rice are rich in proteins. Thus, our data warrants further investigation of the antioxidative effect of colored rice.     

Electron-beam sterilization of laboratory animal diets--sterilizing effect of 10-MeV electrons from a linear accelerator.

Electron beam sterilization for laboratory animal diets was examined as an alternative to 60Co gamma rays. Solid, powder diets for "mice and rats" and solid diets for "rabbits and guinea pigs" which are the main products sterilized by 60Co gamma rays were irradiated with 10-MeV electrons from a linear accelerator at the Research Institute for Advanced Science and Technology, Osaka Prefecture University. At least 20 kGy was required to sterilize the samples irrespective of solid or powder diets, which was in good accordance with the results for 60Co gamma rays. Using a set dose of 30 kGy, a thickness of 45 mm for solid diets and 30 mm for powder diets could be sterilized by "one-sided" irradiation. "Dual-sided" irradiation could sterilize all the solid diets and the powder diets contained in the thicknesses of 90 mm and 75 mm, respectively. Irradiation effects of 10-MeV electrons on the nutrient quality of each diet were almost equivalent to those of 60Co gamma rays. These results suggest that commercially adopted sterilization doses for 60Co gamma rays are applicable to electron sterilization without modification if the depth-dose profile and the minimum dose of irradiated samples are precisely assessed.     

Impaired neurite development associated with mitochondrial dysfunction in dopaminergic neurons differentiated from exfoliated deciduous tooth-derived pulp stem cells of children with autism spectrum disorder

Autism spectrum disorder (ASD) is a highly heterogeneous neurodevelopmental disorder characterized by impaired social interactions, restrictive interests, and repetitive stereotypic behaviors. Among the various mechanisms underlying the pathogenesis of ASD, dysfunctions of dopaminergic signaling and mitochondria have been hypothesized to explain the core symptoms of children with ASD. However, only a few studies focusing on the pathological association between dopaminergic neurons (DN) and mitochondria in ASD have been performed using patient-derived stem cells and in vitro differentiated neurons. Stem cells from human exfoliated deciduous teeth (SHED) are neural crest-derived mesenchymal stem cells present in the dental pulp of exfoliated deciduous teeth; these cells can differentiate into dopaminergic neurons (DN) in vitro. This study aimed to investigate the pathological association between development of DN and mitochondria in ASD by using SHED as a disease- or patient-specific cellular model. The SHED obtained from three children with ASD and three typically developing children were differentiated into DN, and the neurobiology of these cells was examined. The DN derived from children with ASD showed impaired neurite outgrowth and branching, associated with decreased mitochondrial membrane potential, ATP production, number of mitochondria within the neurites, amount of mitochondria per cell area and intracellular calcium level. In addition, impaired neurite outgrowth and branching of ASD-derived DN were not improved by brain-derived neurotrophic factor (BDNF), suggesting impairment of the BDNF signaling pathway in ASD. These results imply that intracerebral dopamine production may have decreased in these children. The earliest age at which deciduous teeth spontaneously exfoliate in humans, and SHED can be noninvasively collected, is approximately 6 years. Our results suggest that in vitro analysis of SHED-derived DN obtained from children with ASD provides neurobiological information that may be useful in determining treatment strategies in the early stages of ASD.     

Transformation of lithocholic acid to a new bile acid, 3α, 15β-dihydroxy-5β-cholanic acid by Cunninghamella blakesleeana ST-22

Summary A fungus identified as Cunninghamella blakesleeana (Lendner) can carry out 15-hydroxylation of lithocholic acid to a new bile acid (3,15-dihydroxy-5-cholanic acid). By optimizing the fermentation conditions, the amount of the product increased from 0.17 g/l to 1.2 g/l. Hydrophilicity measurements and in vitro cholesterol solubilization tests showed that 3, 15-dihydroxy-5-cholanic acid was as effective as ursodeoxycholic acid in cholesterol solubilization.Abbreviations LCA lithocholic acid (3-hydroxy-5-cholanic acid) - 3, 15-DHC (3, 15-dihydroxy-5-cholanic acid) - DMSO dimethyl sulfoxide - CHES 2-[N-cyclohexylamino]ethanesulfonic acid