Origin of Triploids of Elatostema suzukii (Urticaceae) on Okinawa Island, the Ryukyus

Elatostema suzukii , an endemic species occurring only along streams in the Yanbaru area in the north of Okinawa Island, the Ryukyus, is known to comprise di-, tri-, and tetraploids with x=13. Molecular analysis of chloroplast DNA sequences has shown that two haplotypes, AT- and NI-types, characterized by the insertion or deletion of four nucleotides “ATAT”, exist in E. suzukii. The AT-type was found in some diploids and triploids only at Genkagawa, whereas the NI-type is widespread, occurring in both diploids and triploids of the Genkagawa site as well as at other localities. The triploids share both types with the diploids, indicating that the triploids have at least two independent origins. Because of its restricted distribution, the AT-type triploid may have arisen within the Genkagawa area. Received 16 October 2000/ Accepted in revised form 18 June 2001     

Highly stable and efficient mRNA templates for mRNA–protein fusions and C-terminally labeled proteins

For high-throughput in vitro protein selection using genotype (mRNA)–phenotype (protein) fusion formation and C-terminal protein labeling as a post-selection analysis, it is important to improve the stability and efficiency of mRNA templates for both technologies. Here we describe an efficient single-strand ligation (90% of the input mRNAs) using a fluorescein-conjugated polyethylene glycol puromycin (Fluor-PEG Puro) spacer. This ligation provides a stable c-jun mRNA with a flexible Fluor-PEG Puro spacer for efficient fusion formation (70% of the input mRNA with the PEG spacer) in a cell-free wheat germ translation system. When using a 5′ untranslated region including SP6 promoter and Ω29 enhancer (a part of tobacco mosaic virus Ω), an A₈ sequence (eight consecutive adenylate residues) at the 3′ end is suitable for fusion formation, while an XA₈ sequence (XhoI and the A₈ sequence) is suitable for C-terminal protein labeling. Further, we report that Fluor-PEG N-t-butyloxycarbonylpuromycin [Puro(Boc)] spacer enhances the stability and efficiency of c-jun mRNA template for C-terminal protein labeling. These mRNA templates should be useful for puromycin-based technologies (fusion formation and C-terminal protein labeling) to facilitate high-throughput in vitro protein selection for not only evolutionary protein engineering, but also proteome exploration.     

Mechanism of asymmetric decarboxylation of α-aryl-α-methylmalonate catalyzed by arylmalonate decarboxylase originated from Alcaligenes bronchisepticus

Arylmalonate decarboxylase (EC. 4.1.1.76, originated from Alcaligenes bronchisepticus KU 1201) is an enzyme which catalyzes asymmetric decarboxylation of arylmalonate. We have once proposed the intermediary formation of a thiol ester between the substrate and the enzyme based on the inhibition studies with α-bromophenylacetate. We misinterpreted the binding mode of this acid as formation of thiol ester and estimated that the substrate also bound to the enzyme in the same manner. However, reinvestigation indicated that the mode of inhibition by this acid is irreversible, different from the previous conclusion. Accordingly the above mechanism became very unlikely. Instead, we would like to propose that Cys 188 is working as a proton donor on the basis of following evidence. The pH-rate of reaction profiles of the native and C188S mutant enzyme greatly differed in alkaline region. This is estimated to come from the difference in pK_a values of Cys and Ser, and suggested that Cys 188 is a proton donor. Homology alignment showed that this enzyme has some homology with glutamete racemase and some other isomerases. The presence of Cys 188 is conserved to all these enzymes as well as to AMDase. The role of this amino acid residue in glutamate racemase has been established to interchange a proton between the substrate. This fact also supports that Cys 188 of AMDase is working as a proton donor to form the asymmetric center of the product.     

G-electrode-loading method for isoelectric focusing, enabling separation of low-abundance and high-molecular-mass proteins

The G-electrode-loading method (GELM) is a technique enabling a large number of proteins from rat liver to enter an immobilized pH gradient (IPG) gel strip for isoelectric focusing (IEF). In this method, three slips containing the sample solution are placed on the cathodic edge of an IPG gel strip and a slip containing Chaps solution, a filtration membrane, and an electrode slip are placed on top. Finally, a G-electrode is placed on these slips. The Chaps solution (an amphoteric compound) is supplied gently to the sample solution during IEF and helps the proteins in the sample solution to enter the IPG gel strips with a high solubilization capacity. This method was compared with traditional slip-loading and in-gel rehydration, and it showed the best results for protein separation, including high-molecular-mass proteins.     

Thermotropic and barotropic phase transitions of dilauroylphosphatidylcholine bilayer

The bilayer phase transitions of dilauroylphosphatidylcholine (DLPC), containing two linear acyl chains with 12 carbon atoms, were observed by means of differential scanning calorimetry (DSC) under ambient pressure and light transmittance under high pressure. When the heating scan for the DLPC bilayer in 50 wt.% aqueous ethylene glycol (EG) solution began at -30 degrees C after cold storage, the DSC thermogram showed two endothermic peaks at 1.7 and 4.5 degrees C, which correspond to the transition from the lamellar crystalline (Lc) phase to the intermediate liquid crystalline (Lx) phase and the transition from the Lx phase to the liquid crystalline (L) phase, respectively. Extremely large enthalpy change (32.9 kJ mol(-1)) is characteristic of the Lc/Lx phase transition. The DSC thermogram for the heating scan beginning from -10 degrees C showed a single endothermic peak with 9.2 kJ mol(-1) at -0.4 degrees C, which was assigned as the so-called main transition between the metastable ripple gel (P'(beta)) and metastable Lalpha phases. The DLPC bilayer under high pressure underwent three kinds of transitions in EG solution, whereas only one transition was observed in water under high pressure. The middle-temperature transition in EG solution could be assigned to the main transition because of its consistency with the main transition in water. The lower-temperature transition is probably assigned as transition from the Lc phase to the P'(beta) phase. Since the slope (dT/dp) of the Lc/P'(beta) phase boundary is smaller than that for the main transition, the Lc/P'(beta) phase boundary and the main transition curves crossed each other at 40 MPa on the temperature-pressure phase diagram. The higher-temperature transition in EG solution refers to the transition from the Lx phase to the Lalpha phase. The Lx phase disappeared at about 180 MPa, and the direct transition from the P'(beta) phase to the Lalpha phase was observed at high pressures above 180 MPa.     

Serological evidence for hepatitis e virus infection in laboratory monkeys and pigs in animal facilities in Japan

In laboratory animal facilities, monkeys and pigs are used for animal experiments, but the details of hepatitis E virus (HEV) infection in these animals are unknown. The risk of infection from laboratory animals to humans has become a concern; therefore, much attention should be paid to the handling of these animals during their care and use, including surgical procedures performed on infected animals. In this connection, serum samples collected from 916 monkeys and 77 pigs kept in 23 animal facilities belonging to the Japanese Association of Laboratory Animal Facilities of National University Corporations (JALAN) and the Japanese Association of Laboratory Animal Facilities of Public and Private Universities (JALAP) in Japan were examined for the purpose of detecting antibodies to HEV and HEV RNA by using ELISA and RT-PCR, respectively. One hundred and seven serum samples of 916 (11.7%) monkeys were positive for anti-HEV IgG, and 7 and 17 serum samples of 916 (0.8% and 5.3%) monkeys were positive for anti-HEV IgM and IgA, respectively. Thirty-six samples from 62 (58.1%) farm pigs were positive for anti-HEV IgG, whereas all samples tested from miniature pigs were negative (0/15, 0%). Seven samples from 62 (9.1%) farm pigs and 7 samples from 916 (0.8%) monkeys were positive for IgM antibody, but these HEV-IgM antibody positive serum samples were HEV-RNA negative by RT-PCR. The IgM antibody positive rate (9.1%) of farm pigs was much higher than that of monkeys (0.8%). These results suggest the relative levels of risk of HEV infection from these animals to animal handlers and researchers who work with them in laboratory animal facilities.     

Identification and expression of human epiglycanin/MUC21: a novel transmembrane mucin

The gene for the human orthologue of mouse epiglycanin, a mucin expressed on mammary carcinoma TA3-Ha cells but not TA3-St cells, was identified by homology search to a mouse epiglycanin cDNA fragment identified by representational difference analysis between TA3-Ha and TA3-St cells. The open reading frame of this gene was cloned from human cervical carcinoma ME-180 cells. It consists of a mucin domain with 28 nonidentical tandem repeats of 45 nucleotides each corresponding to a threonine/serine-rich peptide, a stem domain, a transmembrane domain, and a cytoplasmic tail. The cloned cDNA with a FLAG sequence was expressed in K562 cells. A combination of immunoprecipitation with a polyclonal antibody specific for the cytoplasmic tail and Western blotting analysis with an anti-FLAG antibody and lectins revealed a mucin-like component as the gene product. Analysis by the use of tissue cDNA libraries indicated that the gene is expressed in lung, large intestine, thymus, and testis among 16 normal tissues tested. The polyclonal antibody specific for a synthetic peptide from the cytoplasmic tail, when tested for its reactivity with normal lung tissues, reacted with epithelia of bronchi and bronchioli but not with alveoli. All of 24 lung adenocarcinomas specimens tested were reactive with the antibody, whereas reactivity was observed with only 2 out of 24 squamous and none out of 24 small cell lung carcinomas. This is a novel transmembrane mucin and designated as MUC21.     

Phenotypic differences in PFIC2 and BRIC2 correlate with protein stability of mutant Bsep and impaired taurocholate secretion in MDCK II cells

Progressive familial cholestasis (PFIC) 2 and benign recurrent intrahepatic cholestasis (BRIC) 2 are caused by mutations in the bile salt export pump (BSEP, ABCB11) gene; however, their prognosis differs. PFIC2 progresses to cirrhosis and requires liver transplantation, whereas BRIC2 is clinically benign. To identify the molecular mechanism(s) responsible for the phenotypic differences, eight PFIC2 and two BRIC2 mutations were introduced in rat Bsep, which was transfected in MDCK II cells. Taurocholate transport activity, protein expression, and subcellular distribution of these mutant proteins were studied in a polarized MDCK II monolayer. The taurocholate transport activity was approximately half of the wild-type (WT) in BRIC2 mutants (A570T and R1050C), was substantially less in two PFIC2 mutants (D482G and E297G), and was almost abolished in six other PFIC2 mutants (K461E, G982R, R1153C, R1268Q, 3767-3768insC, and R1057X). Bsep protein expression levels correlated closely with transport activity, except for R1057X. The half-life of the D482G mutant was shorter than that of the WT (1.35 h vs. 3.49 h in the mature form). BRIC2 mutants and three PFIC mutants (D482G, E297G, and R1057X) were predominantly distributed in the apical membrane. The other PFIC2 mutants remained intracellular. The R1057X mutant protein was stably expressed and trafficked to the apical membrane, suggesting that the COOH-terminal tail is required for transport activity but not for correct targeting. In conclusion, taurocholate transport function was impaired in proportion to rapid degradation of Bsep protein in the mutants, which were aligned in the following order: A570T and R1050C > D482G > E297G > K461E, G982R, R1153C, R1268Q, 3767-3768insC, and R1057X. These results may explain the phenotypic difference between BRIC2 and PFIC2.     

Shootin1 interacts with actin retrograde flow and L1-CAM to promote axon outgrowth

Actin polymerizes near the leading edge of nerve growth cones, and actin filaments show retrograde movement in filopodia and lamellipodia. Linkage between actin filament retrograde flow and cell adhesion molecules (CAMs) in growth cones is thought to be one of the mechanisms for axon outgrowth and guidance. However, the molecular basis for this linkage remains elusive. Here, we show that shootin1 interacts with both actin filament retrograde flow and L1-CAM in axonal growth cones of cultured rat hippocampal neurons, thereby mediating the linkage between them. Impairing this linkage, either by shootin1 RNA interference or disturbing the interaction between shootin1 and actin filament flow, inhibited L1-dependent axon outgrowth, whereas enhancing the linkage by shootin1 overexpression promoted neurite outgrowth. These results strengthen the actin flow-CAM linkage model ("clutch" model) for axon outgrowth and suggest that shootin1 is a key molecule involved in this mechanism.     

Discovery of mitocryptide-1, a neutrophil-activating cryptide from healthy porcine heart

Although neutrophils are known to migrate in response to various chemokines and complement factors, the substances involved in the early stages of their transmigration and activation have been poorly characterized to date. Here we report the discovery of a peptide isolated from healthy porcine hearts that activated neutrophils. Its primary structure is H-Leu-Ser-Phe-Leu-Ile-Pro-Ala-Gly-Trp-Val-Leu-Ser-His-Leu-Asp-His-Tyr-Lys-Arg-Ser-Ser-Ala-Ala-OH, and it was indicated to originate from mitochondrial cytochrome c oxidase subunit VIII. This peptide caused chemotaxis at concentrations lower than that inducing beta-hexosaminidase release. Such responses were observed in neutrophilic/granulocytic differentiated HL-60 cells but not in undifferentiated cells, and G(i2)-type G proteins were suggested to be involved in the peptide signaling. Moreover the peptide activated human neutrophils to induce beta-hexosaminidase secretion. A number of other amphipathic neutrophil-activating peptides presumably originating from mitochondrial proteins were also found. The present results suggest that neutrophils monitor such amphipathic peptides including the identified peptide as an initiation signal for inflammation at injury sites.