The novel function of a short region K253 XRXXXD259 conserved in the exonuclease domain of hyperthermostable DNA polymerase I from Pyrococcus horikoshii

The DNA polymerase gene of the hyperthermophile Pyrococcus horikoshii was successfully overexpressed after removing an intein. The importance of an amino acid sequence around a highly conserved Asp was studied by site-directed mutagenesis. The results indicated that Lys253, Arg255, and Asp259 form a novel functional motif, K253xRxxxD259 (outside known motifs Exo I, II, and III), that is important not only for exonuclease activity but also for polymerizing activity, confirming functional interdependence between the polymerase and exonuclease domains. The short loop region, K253G254R255, probably contributes to binding to DNA substrates. Moreover, the negative charge and the side-chain length of D259 might play a supporting role in coordinating the conserved Mg2+ to the correct position at the active center in the exonuclease domain.     

Detergent-resistant erythrocyte membrane rafts are modified by a Plasmodium falciparum infection

Detergent resistant membranes (DRMs) have been implicated in numerous cellular processes including signal transduction, membrane trafficking, and molecular sorting. Flotillins-1 and -2 have recently been shown to be large components of erythrocyte DRMs. In this study, we show that a Plasmodium falciparum infection disrupts the association of flotillins with erythrocyte DRMs. Flotillins are probably released from erythrocyte DRMs through the reduction of cholesterol and sphingomyelin levels during the course of a P. falciparum-infection. Although it is well known that a P. falciparum infection can modify the host erythrocyte membrane, this is the first report that P. falciparum can alter the DRM components of erythrocyte membranes.     

Two-dimensional electrophoresis/phage panning (2D-PP): a novel technology for direct antibody selection on 2-D blots

We describe a novel method, two-dimensional electrophoresis/phage panning (2D-PP), for the generation of antibodies against proteins in crude biochemical samples, such as cellular membrane fractions. These sources have traditionally presented problems as to the development of antibodies by conventional techniques. 2D-PP involves two-dimensional resolution of proteins, blotting of the proteins onto a nitrocellulose membrane, and screening of a phage antibody library and isolation of corresponding antibodies. By 2D-PP with detergent-insoluble "lipid rafts" as a target protein complex, we obtained specific phage pools against eight antigen spots (from a total of 39 spots). These antibodies were functional in Western blotting, enzyme-linked immunosorbent assaying (ELISA), and immunoscreening of a cDNA expression library. Propagation of anti-nitrocellulose phages was the major problem in 2D-PP, but was overcome by the use of the soluble anti-nitrocellulose antibody fragment. 2D-PP constitutes a key tool for functional analysis of proteins in complex fractions.     

5-Halogenated thymidine analogues induce a senescence-like phenomenon in HeLa cells

We tested various thymidine analogues for induction of a senescence-like phenomenon in HeLa cells. CldU, BrdU, and IdU similarly induced the morphology of senescent cells and typical senescence markers. Thymidine analogues other than 5-halogenated forms caused only cell death. BrdU efficiently killed the cells in cooperation with irradiation with light and a brief treatment with Hoechst 33258, but CldU did not at all. 5-Halogenated thymidine analogues were thus shown to be specific inducers of cellular senescence in mammalian cells.     

Zinc-coordination of aspartic acid-76 in Sulfolobus ferredoxin is not required for thermal stability of the molecule

The highly thermostable 7Fe-ferredoxin from Sulfolobus sp. strain 7 has tightly bound zinc at the interface between the N-terminal extra domain and the C-terminal core. The zinc is tetrahedrally ligated by His-16, His-19, His-34, and Asp-76. Previous studies on truncated mutants have shown that the zinc and certain parts, i.e. not all, of the N-terminal extra stretch are responsible for the thermal stabilization of the molecule. To study the role of Asp-76, a series of mutants were constructed with Asp-76 replaced by Glu (D76E), Asn (D76N), or Ala (D76A). All the mutants, as well as wild type ferredoxin, bound 1 mol zinc/mol protein, and showed similar kinetics for 2-oxoacid:ferredoxin oxidoreductase. The stability of the protein was examined by thermal degradation of the clusters. In the absence of guanidium thiocyanate, the T(m), defined as the mid-point temperature of the thermal transition from the native to the denatured state, for every mutant was above 100 degrees C. The T(m) values in the presence of 1 M guanidium thiocyanate were determined to be 90.8, 90.2, 87.1, 84.4, and 72.9 degrees C for the natural, recombinant, D76N-, D76A-, and D76E-ferredoxins, respectively. These results indicate that the interaction between zinc and the carboxyl oxygen of Asp-76 has subtle effects on both the zinc-ligation and stability, although the native zinc center is liganded with high symmetry, suggesting that the three His residues are more important for zinc-binding.     

cDNA cloning and characterization of Drb1, a new member of RRM-type neural RNA-binding protein

Neural RNA recognition motif (RRM)-type RNA-binding proteins play essential roles in neural development. To search for a new member of neural RRM-type RNA-binding protein, we screened rat cerebral expression library with polyclonal antibody against consensus RRM sequences. We have cloned and characterized a rat cDNA that belongs to RRM-type RNA-binding protein family, which we designate as drb1. Orthologs of drb1 exist in human and mouse. The predicted amino acid sequence reveals an open reading frame of 476 residues with a corresponding molecular mass of 53kDa and consists of four RNA-binding domains. drb1 gene is specifically expressed in fetal (E12, E16) rat brain and gradually reduced during development. In situ hybridization demonstrated neuron-specific signals in fetal rat brain. RNA-binding assay indicated that human Drb1 protein possesses binding preference on poly(C)RNA. These results indicate that Drb1 is a new member of neural RNA-binding proteins, which expresses under spatiotemporal control.     

Purification and characterization of beta-N-acetylhexosaminidase from maize seedlings

Enzymatic activity of beta-N-acetyhexosaminidase (EC 3.2.1.52) was analysed in seeds and young seedings of maize (Zea mays) using di-N-acetylchitobiose as a substrate. Substantial activity was detected in dry seeds. Activity increased before germination (48 h) but exclusively in the embryo. In seedlings, most of the activity was found in the scutellum, and lower levels in shoots and roots immediately after germination. An isoform of the enzyme was purified from scutellum (72 h after the start of imbibition) by heat treatment of crude extract and four steps of chromatography. Purified beta-N-acetyl-hexosaminidase showed a single band on SDS-PAGE of around 70 kDa. This was almost the same as the molecular weight estimated by size exclusion chromatography, indicating a monomeric form of the active enzyme. The relative activity of the enzyme for di-N-acetylchitobiose was about 15 times greater than that for p-nitrophenyl-N-acetylglucosaminide or p-nitrophenyl-N-acetylgalactosaminide. Analysis of the reaction with oligo-N-acetylchitooliogsaccharides [(GlcNAc)n] revealed an exotype enzyme producing predominantly (GlcNAc)n-1 and N-acetylglucosamine. The optimum pH, temperature, and isoelectric point (pl) were 4.5, 55 degrees C, and 6.75, respectively. The activity was almost completely inhibited in the presence of 5 mmol/L Ag+, Hg2+, or Fe3+.     

Clustering of Human Chromosome Fragments on the Mouse Genome by Chromosome-Mediated Gene Transfer

Pieces of metaphase chromosomes prepared from mouse cells containingneo-tagged human chromosome 7 were transferred to mouse cellswith calcium phosphate to isolate G418-resistant clones. FISHanalysis revealed that the majority of them contained humanDNA at a single site on their genome. These transformants containedSTS markers mapped to various regions of chromosome 7. It isthus suggested that pieces of human chromosomes tend to assembleand integrate on the mouse genome.     

The Suppression of Maternal–Fetal Leukemia Inhibitory Factor Signal Relay Pathway by Maternal Immune Activation Impairs Brain Development in Mice

Recent studies in rodents suggest that maternal immune activation (MIA) by viral infection is associated with schizophrenia and autism in offspring. Although maternal IL-6 is though t to be a possible mediator relating MIA induced these neuropsychiatric disorders, the mechanism remains to be elucidated. Previously, we reported that the maternal leukemia inhibitory factor (LIF)–placental ACTH–fetal LIF signaling relay pathway (maternal–fetal LIF signal relay) promotes neurogenesis of fetal cerebrum in rats. Here we report that the maternal–fetal LIF signal relay in mice is suppressed by injection of polyriboinosinic-polyribocytidylic acid into dams, which induces MIA at 12.5 days post-coitum. Maternal IL-6 levels and gene expression of placental suppressor of cytokine signaling 3 (Socs3) increased according to the severity of MIA and gene expression of placental Socs3 correlated with maternal IL-6 levels. Furthermore, we show that MIA causes reduction of LIF level in the fetal cerebrospinal fluid, resulting in the decreased neurogenesis in the cerebrum. These findings suggest that maternal IL-6 interferes the maternal–fetal LIF signal relay by inducing SOCS3 in the placenta and leads to decreased neurogenesis.