Engineering of polyploid Saccharomyces cerevisiae for secretion of large amounts of fungal glucoamylase

We engineered Saccharomyces cerevisiae cells that produce large amounts of fungal glucoamylase (GAI) from Aspergillus awamori var. kawachi. To do this, we used the delta-sequence-mediated integration vector system and the heat-induced endomitotic diploidization method. delta-Sequence-mediated integration is known to occur mainly in a particular chromosome, and the copy number of the integration is variable. In order to construct transformants carrying the GAI gene on several chromosomes, haploid cells carrying the GAI gene on different chromosomes were crossed with each other. The cells were then allowed to form spores, which was followed by dissection. Haploid cells containing GAI genes on multiple chromosomes were obtained in this way. One such haploid cell contained the GAI gene on five chromosomes and exhibited the highest GAI activity (5.93 U/ml), which was about sixfold higher than the activity of a cell containing one gene on a single chromosome. Furthermore, we performed heat-induced endomitotic diploidization for haploid transformants to obtain polyploid mater cells carrying multiple GAI genes. The copy number of the GAI gene increased in proportion to the ploidy level, and larger amounts of GAI were secreted.     

Effects of orexin on cultured porcine adrenal medullary and cortex cells

New orexigenic peptides called orexins have recently been described in the neurons of the lateral hypothalamus and perifornical area. No orexins have been found in the adipose tissues or visceral organs, including the adrenal gland. However, expression of the orexin receptor (OXR) in the rat adrenal gland has been reported. With regard to the effects of orexins on peripheral organs, we previously reported that orexins suppress catecholamine synthesis and secretion in the rat pheochromocytoma cell line PC12. To further clarify the pharmacological effects of orexins on peripheral organs, we examined the effects of orexin-A on catecholamine, cortisol, and aldosterone secretion, using cultured porcine adrenal glands. We initially confirmed the expression of the orexin receptor (OXR-1) in cultured porcine adrenal medulla and cortex. Orexin-A (1000 nM) significantly increased the release of both epinephrine (E) and norepinephrine (NE) from porcine adrenal medullary cells. Similarly, orexin-A (> or = 100 nM) significantly increased the release of both cortisol and aldosterone from porcine adrenal cortex cells. Orexin-A (100 nM) significantly inhibited basal and the PACAP-induced increase in cAMP levels in adrenal medullary cells. Conversely, orexin-A (>o = 100 nM) significantly increased the cAMP level in adrenal cortex cells. These results indicate that orexin-A induces the release of catecholamine from porcine adrenal medullary cells, and aldosterone and cortisol from the cortex cells and has opposite effects on cAMP levels in adrenal medulla and cortex.     

Microdistribution of alpha particles in pathological sections of tissues from thorotrast patients detected by imaging plate autoradiography

Thorotrast is a colloidal suspension of radioactive (232)ThO(2) that naturally emits alpha particles (90%), beta particles and gamma rays (10%). Thorotrast was used as a radiographic contrast agent in the 1930s-1950s; it caused liver cancer several decades after injection because of its life-long deposition and exposure. Determination of the amount and the distribution of radioactive thorium are essential for assessment of radiation risks. We visualized alpha particles on ordinary archival tissue sections using an imaging plate and a BAS5000 image analyzer. Furthermore, we confirmed that the imaging system is sensitive enough to detect alpha particles and accurate in measuring the total amount of thorium deposited in the organ from a single tissue section. This method revealed that the amount of thorium deposited in tumor tissue is correlated to that in non-tumor tissue. Thorotrast deposition was not associated with DNA damage determined by histochemistry. In combination with histological findings, it is suggested that radioactive thorium always migrates within the deposited organs by macrophages, and that the organs are evenly exposed to alpha particles.     

Single-chain Fv fragments derived from an anti-11-deoxycortisol antibody. Affinity,specificity, and idiotype analysis

Single-chain Fv fragments (scFvs) against a corticosteroid, 11-deoxycortisol (11-DC), have been generated as a template antibody fragment from which a comprehensive mutated antibody library containing various anti-steroid antibodies could be constructed. The cDNAs encoding variable heavy (V(H)) and light (V(L)) domains of a mouse anti-11-DC antibody (CET-M8), were amplified by RT-PCR, combined via a common linker to construct the sequence of 5'-V(H)-(Gly(4)Ser)(3)-V(L)-3', and cloned into a phagemid vector, pEXmide 5. The phage clones exhibiting binding activity to 11-DC were isolated after single panning against a hapten-immobilizing immunotube. The scFv gene in one of these clones was reamplified to introduce the ochre codons, and then expressed in the bacterial periplasm as the soluble antibody fragment. Two different scFvs (#6 and #12) were cloned, whose binding characteristics were examined by a radioimmunoassay using a tritium-labeled 11-DC. Both of them showed high affinity (K(a)=1.3x10(10)M(-1)) and practical specificity (cross-reactivity: cortisol, <0.2%; cortisone, <0.3%) to 11-DC, and furthermore, strong reactivity with an anti-idiotype antibody which recognizes the paratope of CET-M8. These results suggest that the present scFvs retain the three-dimensional structure of the paratope of the original monoclonal antibody.     

Dual action of isoprenols from herbal medicines on both PPARgamma and PPARalpha in 3T3-L1 adipocytes and HepG2 hepatocytes

Several herbal medicines improve hyperlipidemia, diabetes and cardiovascular diseases. However, the molecular mechanism underlying this improvement has not yet been clarified. In this study, we found that several isoprenols, common components of herbal plants, activate human peroxisome proliferator-activated receptors (PPARs) as determined using the novel GAL4 ligand-binding domain chimera assay system with coactivator coexpression. Farnesol and geranylgeraniol that are typical isoprenols in herbs and fruits activated not only PPARgamma but also PPARalpha as determined using the chimera assay system. These compounds also activated full-length human PPARgamma and PPARalpha in CV1 cells. Moreover, these isoprenols upregulated the expression of some lipid metabolic target genes of PPARgamma and PPARalpha in 3T3-L1 adipocytes and HepG2 hepatocytes, respectively. These results suggest that herbal medicines containing isoprenols with dual action on both PPARgamma and PPARalpha can be of interest for the amelioration of lipid metabolic disorders associated with diabetes.     

Linear and Conformation-Dependent Antigenic Sites on the Nucleoprotein of Rabies Virus

A set of 29 monoclonal antibodies (MAbs) specific for the rabies virus nucleoprotein (N protein) was prepared and used to analyze the topography of antigenic sites. At least four partially overlapping antigenic sites were delineated on the N protein of rabies virus by competitive binding assays. Indirect immunofluorescent antibody tests using MAbs with a series of rabies and rabies-related viruses showed that epitopes shared by various fixed and street strains of rabies virus were mainly localized at antigenic sites II and III, while epitopes representing the genus-specific antigen of Lyssavirus were widely presented at sites I, III and IV. All but one of seven MAbs specific for antigenic sites I, IV and bridge site (I and II) reacted with the antigen that had been denatured by sodium dodecyl sulfate or 2-mercaptoethanol, as well as with the denatured N protein in Western blotting assays. However, none of the MAbs against antigenic sites II and III reacted with the denatured antigen. These data indicate that antigenic sites I and IV, and sites II and III on the N protein of rabies virus are composed of linear and conformation-dependent epitopes, respectively.