Yeast whole-cell biocatalyst constructed by intracellular overproduction of Rhizopus oryzae lipase is applicable to biodiesel fuel production

Yeast whole-cell biocatalysts for lipase-catalyzed reactions were constructed by intracellularly overproducing Rhizopus oryzae lipase (ROL) in Saccharomvces cerevisiae MT8-1. The gene encoding lipase from R. orvzae IFO4697 was cloned, and intracellular overproduction systems of a recombinant ROL with a pro-sequence (rProROL) were constructed. When rProROL from R. oryzae IFO4697 was produced under the control of the 5'-upstream region of the isocitrate lyase gene of Candida tropicalis (UPR-ICL) at 30 degrees C for 98 h by two-stage cultivation using SDC medium (SD medium with 2% casamino acids) containing 2.0% and 0.5% glucose, intracellular lipase activity reached levels up to 474.5 IU/l. These whole-cell biocatalysts were permeabilized by air-drying and used for the synthesis of methyl esters (MEs), a potential biodiesel fuel, from plant oil and methanol in a solvent-free and water-containing system. The ME content in the reaction mixture was 71 wt% after a 165-h reaction at 37 degrres C with stepwise addition of methanol. These results indicate that an efficient whole-cell biocatalyst can be prepared by intracellular overproduction of lipase in yeast cells and their permeabilization.     

Development of a quantification method for digoxin, a typical P-glycoprotein probe in clinical and non-clinical studies, using high performance liquid chromatography-tandem mass spectrometry: the usefulness of negative ionization mode to avoid competitive adduct-ion formation

Highly sensitive and accurate liquid chromatography-tandem mass spectrometry (LC/MS/MS) methods have been developed and validated for measuring digoxin (DGX), a typical P-glycoprotein probe, in human plasma, rat plasma, and rat brain. We extracted DGX and deuterium-labeled DGX (as internal standard) from sample fluids under basic conditions using acetonitrile and sodium chloride-saturated 0.1 mol/L sodium hydroxide. The upper organic layer was diluted with distilled water, and the resulting solution was injected into an LC/MS/MS system in negative ionization mode. Chromatographic separation was achieved on a C(18)-ODS column in the gradient mobile phase, which comprised 0.05% (w/v) ammonium carbonate (pH 9.0) and methanol at a flow rate of 0.7 mL/min. Regardless of the type of biological matrix, intra-day and inter-day validation tests demonstrated good linearity of calibration curves within ranges of 0.1-10 ng/mL for plasma and 0.5-50 ng/g for rat brain and gave excellent accuracy and precision of quality control samples at 4 concentration levels. Unlike existing methods, our approach uses negative ionization to avoid competitive adduct formation of DGX. Our method showed higher sensitivity and wider applicability to various types of biological matrices than existing methods. Our method will support clinical and preclinical investigation of in vivo P-glycoprotein functionality using DGX.     

Regulatory processes and population cyclicity in laboratory populations ofAnagasta kühniella (Zeller) (Lepidoptera: Phycitidae) IV. The sequential steps in the behavioral process of host finding and parasitization by the entomophagous parasite,Venturia canescens (Gravenhorst) (Hymenoptera: Ichneumonidae)

Summary The sequential steps in the behavioral process of a California stock of the entomophagous parasite,Venturia canescens (Grav.), parasitizingAnagasta kühniella (Zeller) was studied. In host-habitat finding, food media infested with hosts were very attractive to the parasites. Host finding was not covered in detail in this paper as it is presented in subsequent papers. Briefly, in all experiments host density was the most influential factor affecting the efficiency of the parasite. When three age stages of the host were exposed to a parasite, all tests showed that the large, last instar larvae was the preferred age stage but it was not the most suitable (when parasitized) for successful parasite development. Small larvae were less preferred but more suitable for parasite development when accepted. These studies were conducted as a partial fulfillment in the Ph. D. program of one of us (B. M. Matsumoto) and form a part of a broad investigation into the processes operating in the dynamics of arthropod populations under grants toC. B. Huffaker from the U. S. Public Health Service, National Institutes of Health and the U. S. Department of Agriculture.     

The effect of deoxyadenosine plus deoxycoformycin on replicative and repair synthesis of DNA in human lymphoblasts and isolated nuclei

Deoxyadenosine plus deoxycoformycin (dCf) causes increased DNA breaks in lymphoid cells. This study explored the possible inhibition of repair synthesis of DNA by dAdo plus dCf as a cause of DNA breakage. It was shown that DNA breaks accumulated in a human T-lymphoblast cell line, CCRF-CEM, following incubation with dAdo plus dCf and were not fully repaired 20 h after their removal. Analysis of the density distribution of radiolabeled DNA on alkaline CsCl gradient showed that incubation of CCRF-CEM cells with dAdo plus dCf caused inhibition of semiconservative, but not repair synthesis of DNA. Semiconservative synthesis of DNA was also inhibited in CCRF-CEM nuclei isolated from cells pretreated with dAdo and dCf, suggesting damage to DNA replicative machinery. However, no such inhibition was observed in the nuclei of a similarly treated CCRF-CEM mutant that was deficient in adenosine kinase and deoxycytidine kinase. This suggests that dAdo must be phosphorylated in intact cells to exert its effect. Using [3H]dTTP incorporation in isolated CCRF-CEM nuclei to measure DNA synthesis, it was found that a high concentration (greater than 100 microM) of dATP inhibits semiconservative but not repair synthesis of DNA. The present studies thus indicate that accumulation of DNA strand breaks induced by dAdo plus dCf is not the consequence of inhibition of repair DNA synthesis. This implies the mechanism may involve perturbation of DNA ligation or activation of a certain process which causes DNA strand breaks. In addition, dATP may interfere with some steps of semiconservative DNA synthesis, but not the repair synthesis of DNA.     

Expression of normal cellular prion protein (PrP(c)) on T lymphocytes and the effect of copper ion: Analysis by wild-type and prion protein gene-deficient mice

The purpose of this report was to determine the effect of prion protein (PrP) gene disruption on T lymphocyte function. Previous studies have suggested that normal cellular prion protein (PrP(c)) binds to copper and Cu(2+) is essential for interleukin-2 (IL-2) mRNA synthesis. In this study, IL-2 mRNA levels in a copper-deficient condition were investigated using T lymphocytes from prion protein gene-deficient (PrP(0/0)) and wild-type mice. Results showed that Cu(2+) deficiency had no effect on PrP(c) expression in Con A-activated splenocytes. However, a delay in IL-2 gene expression was observed in PrP(0/0) mouse T lymphocyte cultures using Con A and Cu(2+)-chelator. These results suggest that PrP(c) expression may play an important role in rapid Cu(2+) transfer in T lymphocytes. The rapid transfer of Cu(2+) in murine T lymphocytes could be one of the normal functions of PrP(c).     

The functions and possible significance of Kremen as the gatekeeper of Wnt signalling in development and pathology

Kremen (Krm) was originally discovered as a novel transmembrane protein containing the kringle domain. Both Krm1 (the first identified Krm) and its relative Krm2 were later identified to be the high-affinity receptors for Dickkopf (Dkk), the inhibitor of Wnt/beta-catenin signalling. The formation of a ternary complex composed of Krm, Dkk, and Lrp5/6 (the coreceptor of Wnt) inhibits Wnt/beta-catenin signalling. In Xenopus gastrula embryos, Wnt/beta-catenin signalling regulates anterior-posterior patterning, with low-signalling in anterior regions. Inhibition of Krm1/2 induces embryonic head defects. Together with anterior localization of Krms and Dkks, the inhibition of Wnt signalling by Dkk-Krm action seems to allow anterior embryonic development. During mammalian development, krm1 mRNA expression is low in the early stages, but gradually and continuously increases with developmental progression and differentiation. In contrast with the wide, strong expression of krm1 mRNA in mature tissues, expression of krm1 is diminished in a variety of human tumor cells. Since stem cells and undifferentiated cells rely on Wnt/beta-catenin signalling for maintenance in a low differentiation state, the physiological shutdown of Wnt/beta-catenin signalling by Dkk-Krm is likely to set cells on a divergent path toward differentiation. In tumour cells, a deficit of Krm may increase the susceptibility to tumourigenic transformation. Both positive and negative regulation of Wnt/beta-catenin signalling definitively contributes to diverse developmental and physiological processes, including cell-fate determination, tissue patterning and stem cell regulation. Krm is quite significant in these processes as the gatekeeper of the Wnt/beta-catenin signalling pathway.     

Crystallization and Preliminary X-Ray Analysis of Neuropsin, a Serine Protease Expressed in the Limbic System of Mouse Brain

Neuropsin (M_r25 032) is a serine protease expressed in the limbic system of mouse brain. It has been implicated in various neurological processes including formation of memory and may be important as a drug target in the treatment of epilepsy. The recombinant protein was produced using a baculovirus expression system and was purified. Two crystal forms were obtained by a hanging-drop vapor-diffusion method with polyethylene glycol. Preliminary X-ray crystallographic analysis revealed that crystal form I belongs to triclinic space groupP1 with unit cell dimensionsa= 97.16 Å,b= 97.12 Å,c= 46.75 Å and α = 99.17°, β = 99.77°, γ = 117.35°. Self-rotation function analysis of these data of form I indicates the position of a noncrystallographic threefold axis. There are six molecules in the crystallographic asymmetric unit. Crystal form II also belongs to triclinic space groupP1 but has unit cell dimensions ofa= 38.40 Å,b= 55.16 Å,c= 65.37 Å and α = 95.38°, β = 89.98°, γ = 110.46° with two molecules in the crystallographic asymmetric unit. Form II has a noncrystallographic twofold axis. Intensity data to 3.1 Å resolution for form I and to 2.2 Å resolution for form II have been collected.     

Combination of hTERT and bmi-1, E6, or E7 induces prolongation of the life span of bone marrow stromal cells from an elderly donor without affecting their neurogenic potential

Murine bone marrow stromal cells differentiate not only into mesodermal derivatives, such as osteocytes, chondrocytes, adipocytes, skeletal myocytes, and cardiomyocytes, but also into neuroectodermal cells in vitro. Human bone marrow stromal cells are easy to isolate but difficult to study because of their limited life span. To overcome this problem, we attempted to prolong the life span of bone marrow stromal cells and investigated whether bone marrow stromal cells modified with bmi-1, hTERT, E6, and E7 retained their differentiated capability, or multipotency. In this study, we demonstrated that the life span of bone marrow stromal cells derived from a 91-year-old donor could be extended and that the stromal cells with an extended life span differentiated into neuronal cells in vitro. We examined the neuronally differentiated cells morphologically, physiologically, and biologically and compared the gene profiles of undifferentiated and differentiated cells. The neuronally differentiated cells exhibited characteristics similar to those of midbrain neuronal progenitors. Thus, the results of this study support the possible use of autologous-cell graft systems to treat central nervous system diseases in geriatric patients.     

Pyramiding of male-sterile genes in <Emphasis Type="Italic">Cryptomeria japonica</Emphasis> D. Don with the aid of closely linked markers

Gene pyramiding is a breeding method used to combine multiple useful genes. Although several genes have been pyramided in certain crops, gene pyramiding has not previously been applied to forest trees. In this study, we used the markers closely linked to the two male-sterile genes MS1 and MS2 for the effective development of individuals doubly heterozygous for these two genes. This is the first example of gene pyramiding through marker-assisted selection (MAS) in forest trees. The markers gSNP06239, which is closely linked to the MS1 gene, and estSNP00695, which is closely linked to MS2, were used in MAS. On the basis of the linkage phase between the markers and male-sterile loci, we selected five F₁ individuals (S3-64 from Shindai-3 × Kamikiri-31, S3-70 from Shindai-3 × Kamikiri-38, S3-77 from Shindai-3 × Kamikiri-47, S1-22 from Shindai-1 × Nakakubiki-4, and S1-56 from Shindai-1 × Setsugai-20) as parents for artificial crossing. The 268 seedlings obtained from six artificial cross combinations were used in this study. Chi-squared tests showed no significant deviation from the expected Mendelian ratios of genotypes, indicating that MAS using markers closely linked to the male-sterile genes worked very well. Fifteen individuals that showed unexpected genotypes were probably recombinants, because the map distances between the male-sterile locus and the DNA markers were 4.1 cM (gSNP06239 to MS1) and 6.9 cM (estSNP00695 to MS2). Development of markers more closely linked to the male-sterile loci will facilitate precise gene pyramiding in the future.