Utilization of Anhydrous Aluminum Phosphate as a Sole Source of Phosphorous by a Selected Carrot Cell Line

Two variants of carrot (Daucus carota L. cv. MS Yonsun) celllines, which had been selected with Al-phosphate as a sole sourceof phosphorous, were characterized on their mechanisms of phosphate-utilizationfrom Al-phosphate. Both cell lines excreted citrate into themedium. The amount of citrate excretion was highly correlatedwith cell growth in the presence of Al-phosphate. There wasabout a 1 to 1 correlation between solublized-Al and excreted-citratein the medium during cell growth. These results suggest that1) the citrate could chelate with Al, at a 1 to 1 ratio, inAl-phosphate, 2) the citrate-chelated Al remains outside thecells, and 3) solublized phosphate from Al-phosphate is utilizedfor the growth of carrot cells. The characteristics of the selectedcells were very stable, since the rate of citrate-excretionshowed no change after subculturing 25 passages without Al-phosphate. (Received August 7, 1989; Accepted October 19, 1989)     

Expression, purification, and crystallization of endopolygalacturonase from a pathogenic fungus, Stereum purpureum, in Escherichia coli

Endopolygalacturonases (EC 3.2.1.15) catalyze random hydrolysis of the alpha-1,4 glycosidic linkages in polygalacturonic acid, a component of pectin. Previously, we reported crystal structures of endogenously produced Stereum purprureum endopolygalacturonase I (endoPG I), both in its native form and complexed with its product, galacturonate. However, the substrate-binding mechanism of endoPG I is still unclear, because crystals have not yet been obtained with a substrate analog, or with mutant enzymes that can bind substrates. We describe here an expression system using Escherichia coli and a purification method to prepare functionally active endoPG I for such mutation and crystallographic studies. Expression in E. coli strain Origami (DE3) provided a soluble and active enzyme with proper disulfide bond formation, whereas the enzyme expressed in BL21 (DE3) was localized in inclusion bodies. A sufficient amount of recombinant endoPG I produced by Origami (DE3) was purified by a single-step procedure using cation exchange chromatography. The specific activity of recombinant endoPG I was equivalent to that of the enzyme produced by S. purpureum. Recombinant endoPG I was crystallized under the same conditions as those used for the native enzyme produced by S. purpureum. The crystals diffracted beyond 1.0 A resolution with synchrotron radiation.     

Evolutionarily Conserved Interaction between the Phosphoproteins and X Proteins of Bornaviruses from Different Vertebrate Species

Bornavirus, a non-segmented, negative-strand RNA viruses, is currently classified into several genetically distinct genotypes, such as Borna disease virus (BDV) and avian bornaviruses (ABVs). Recent studies revealed that bornavirus genotypes show unique sequence variability in the putative 5′ untranslated region (5′ UTR) of X/P mRNA, a bicistronic mRNA for the X protein and phosphoprotein (P). In this study, to understand the evolutionary relationship among the bornavirus genotypes, we investigated the functional interaction between the X and P proteins of four bornavirus genotypes, BDV, ABV genotype 4 and 5 and reptile bornavirus (RBV), the putative 5′ UTRs of which exhibit variation in the length. Immunofluorescence and immunoprecipitation analyses using mammalian and avian cell lines revealed that the X proteins of bornaviruses conserve the ability to facilitate the export of P from the nucleus to the cytoplasm via interaction with P. Furthermore, we showed that inter-genotypic interactions may occur between X and P among the genotypes, except for X of RBV. In addition, a BDV minireplicon assay demonstrated that the X and P proteins of ABVs, but not RBV, can affect the polymerase activity of BDV. This study demonstrates that bornaviruses may have conserved the fundamental function of a regulatory protein during their evolution, whereas RBV has evolved distinctly from the other bornavirus genotypes.     

High Prevalence of Simian T-Lymphotropic Virus Type L in Wild Ethiopian Baboons

Simian T-cell leukemia viruses (STLVs) are the simian counterparts of human T-cell leukemia viruses (HTLVs). A novel, divergent type of STLV (STLV-L) from captive baboons was reported in 1994, but its natural prevalence remained unclear. We investigated the prevalence of STLV-L in 519 blood samples from wild-living nonhuman primates in Ethiopia. Seropositive monkeys having cross-reactive antibodies against HTLV were found among 22 out of 40 hamadryas baboons, 8 of 96 anubis baboons, 24 of 50 baboons that are hybrids between hamadryas and anubis baboons, and 41 of 177 grivet monkeys, but not in 156 gelada baboons. A Western blotting assay showed that sera obtained from seropositive hamadryas and hybrid baboons exhibited STLV-L-like reactivity. A PCR assay successfully amplified STLV sequences, which were subsequently sequenced and confirmed as being closely related to STLV-L. Surprisingly, further PCR showed that nearly half of the hamadryas (20 out of 40) and hybrid (19 out of 50) baboons had STLV-L DNA sequences. In contrast, most of the seropositive anubis baboons and grivet monkeys carried typical STLV-1 but not STLV-L. These observations demonstrate that STLV-L naturally prevails among hamadryas and hybrid baboons at significantly high rates. STLV-1 and -2, the close relative of STLV-L, are believed to have jumped across simian-human barriers, which resulted in widespread infection of HTLV-1 and -2. Further studies are required to know if STLV-L is spreading into human populations.     

Molecular signatures of lineage‐specific adaptive evolution in a unique sea basin: the example of an anadromous goby Leucopsarion petersii

Climate changes on various time scales often shape genetic novelty and adaptive variation in many biotas. We explored molecular signatures of directional selection in populations of the ice goby Leucopsarion petersii inhabiting a unique sea basin, the Sea of Japan, where a wide variety of environments existed in the Pleistocene in relation to shifts in sea level by repeated glaciations. This species consisted of two historically allopatric lineages, the Japan Sea (JS) and Pacific Ocean (PO) lineages, and these have lived under contrasting marine environments that are expected to have imposed different selection regimes caused by past climatic and current oceanographic factors. We applied a limited genome‐scan approach using seven candidate genes for phenotypic differences between two lineages in combination with 100 anonymous microsatellite loci. Neuropeptide Y (NPY) gene, which is an important regulator of food intake and potent orexigenic agent, and three anonymous microsatellites were identified as robust outliers, that is, candidate loci potentially under directional selection, by multiple divergence‐ and diversity‐based outlier tests in comparisons focused on multiple populations of the JS vs. PO lineages. For these outlier loci, populations of the JS lineage had putative signals of selective sweeps. Additionally, real‐time quantitative PCR analysis using fish reared in a common environment showed a higher expression level for NPY gene in the JS lineage. Thus, this study succeeded in identifying candidate genomic regions under selection across populations of the JS lineage and provided evidence for lineage‐specific adaptive evolution in this unique sea basin.     

Effects of gestational exposure to 1.95‐GHz W‐CDMA signals for IMT‐2000 cellular phones: Lack of embryotoxicity and teratogenicity in rats

The present study was designed to evaluate whether gestational exposure to an EMF targeting the head region, similar to that from cellular phones, might affect embryogenesis in rats. A 1.95‐GHz wide‐band code division multiple access (W‐CDMA) signal, which is one applied for the International Mobile Telecommunication 2000 (IMT‐2000) system and used for the freedom of mobile multimedia access (FOMA), was employed for exposure to the heads of four groups of pregnant CD(SD) IGS rats (20 per group) for gestational days 7–17. The exposure was performed for 90 min/day in the morning. The spatial average specific absorption rate (SAR) for individual brains was designed to be 0.67 and 2.0 W/kg with peak brain SARs of 3.1 and 7.0 W/kg for low (group 3) and high (group 4) exposures, respectively, and a whole‐body average SAR less than 0.4 W/kg so as not to cause thermal effects due to temperature elevation. Control and sham exposure groups were also included. At gestational day 20, all dams were killed and fetuses were taken out by cesarean section. There were no differences in maternal body weight gain. No adverse effects of EMF exposure were observed on any reproductive and embryotoxic parameters such as number of live (243–271 fetuses), dead or resorbed embryos, placental weights, sex ratios, weights or external, visceral or skeletal abnormalities of live fetuses. Bioelectromagnetics 30:205–212, 2009. © 2008 Wiley‐Liss, Inc.     

Intravesical treatment of bladder cancer with recombinant human interferon-β

Summary In order to examine its clinical efficacy, recombinant human interferon- (rIFN-) was instilled intravesically into 51 patients with superficial bladder cancer. Ten patients, who received intermittent intravesical instillation at a dose of (3–36) × 10⁶ U rIFN- on days 1–3 every week, showed no response. Thirty-two patients received intravesical instillation at a dose of (3–36) × 10⁶ U every day for 10–20 days. Eight patients showed partial response, indicating an efficacy rate of 25%. Nine patients received divided doses of 18 × 10⁶ U twice a day every day for 10–20 days. Six patients showed partial response, indicating an efficacy rate of 67%. This value was significantly higher than that obtained by administering divided doses. The response to intravesical instillation therapy with rIFN- varies with treatment protocol. Frequent and longer exposure to rIFN- may induce better regression of superficial bladder cancer. Six incidences of side-effects were found in five cases (9.8%): pollakiuria in one, pain on micturition in two, fever in two, and eruption in one case. All of these side-effects were slight and reversible after drug withdrawal. Laboratory tests showed only a few changes with low severity. Thus, rIFN- is potentially a new drug for instillation therapy of superficial bladder cancer, in view of the absence of adverse effects.     

A Pichia pastoris single-cell biosensor for detection of enzymatically produced methanol

Applied Microbiology and Biotechnology - We conducted single-cell analyses of the methylotrophic yeast Pichia pastoris to develop a biosensor for the detection of methanol produced by heterologous...