The leukocyte common antigen (CD45) of the Pacific hagfish,Eptatretus stoutii: implications for the primordial function of CD45

CD45, originally known as the leukocyte common antigen, is a prototypical transmembrane protein tyrosine phosphatase that plays a critical role in signal transduction through T-cell and B-cell receptors, as well as in T-cell and B-cell development. In the present study, we show that the Pacific hagfish, widely believed to lack the adaptive immune system, has CD45. The presence of CD45 in jawless fish is consistent with the recent discovery that CD45 also plays a crucial role in innate immunity via the regulation of signaling through type I and type II cytokine receptors. It is likely that CD45 was recruited to activate lymphocytes through antigen receptors encoded by rearranging genes in jawed vertebrates.     

Activation of Ca2+/calmodulin-dependent protein kinase I in cultured rat hippocampal neurons

We have focused on activation mechanisms of calcium/calmodulin-dependent protein kinase (CaM) kinase I in the hippocampal neurons and compared them with that of CaM kinase IV. Increased activation of CaM kinase I occurred by stimulation with glutamate and depolarization in cultured rat hippocampal neurons. Similar to CaM kinases II and IV, CaM kinase I was essentially activated by stimulation with the NMDA receptor. Although both CaM kinases I and IV seem to be activated by CaM kinase kinase, the activation of CaM kinase I was persistent during stimulation with glutamate in contrast to a transient activation of CaM kinase IV. In addition, CaM kinase I was activated in a lower concentration of glutamate than that of CaM kinase IV. Depolarization-induced activation of CaM kinase I was also evident in the cultured neurons and was largely blocked by nifedipine. In the experiment with 32P-labeled cells, phosphorylation of CaM kinase I was stimulated by glutamate treatment and depolarization. The glutamate- and depolarization-induced phosphorylation was inhibited by the NMDA receptor antagonist and nifedipine, respectively. These results suggest that, although CaM kinases I and IV are activated by the NMDA receptor and depolarization stimulation, these kinase activities are differently regulated in the hippocampal neurons.     

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.     

Gene expression profile analysis of the mouse liver during bacteria-induced fulminant hepatitis by a cDNA microarray system

Fulminant hepatic failure (FHF) is a disease characterized by sudden and severe impairment of liver function. To elucidate the mechanism involved in FHF, we adopted a murine model of FHF by administrating mice with heat-killed Propionibacterium acnes (P. acnes), followed by a low dose of lipopolysaccharide (LPS), and analyzed the dynamic change of gene expression profile of the murine liver using an in-house cDNA microarray system which contained most of the cDNAs encoding chemokines/cytokines and their receptors (33 chemokines/21 chemokine receptors, 28 cytokines/35 cytokine receptors) as well as 230 liver related proteins mostly selected by serial analysis of gene expression (SAGE). Among them, 335 genes were found to differ by more than 2-fold in at least one time point comparing with normal liver. Hierarchical cluster analysis revealed that except for a few genes, such as heme oxygenase (HO)-1 and nicotinamide N-methyltransferase (NNMT) of which expression increased, the expression of most of the genes encoding drug metabolizing enzymes decreased with the progress of the disease. The expression of the genes encoding chemokines/cytokines was dramatically changed, such as Mig, IP-10, RANTES, TNF-alpha, and IFN-gamma. In addition, the expression of those that were not previously linked to this murine model was also identified to be changed. These include endogenous IL-18 binding protein (IL-18BP), CXCL16 (the ligand of Bonzo, CXCR6) as well as ESTs. Taken together this study has shown the systemic and comprehensive gene expression profile during FHF and may contribute to better understanding of the mechanism of FHF.     

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.     

Identification of the mouse killer immunoglobulin-like receptor-like (<Emphasis Type="Italic">Kirl</Emphasis>) gene family mapping to Chromosome X

Natural killer (NK) inhibitory receptors, which recognize major histocompatability complex (MHC) proteins in humans, are known as killer Ig-like receptors (KIRs) and are encoded by a multi-gene immunoglobulin (Ig) superfamily. In a screen for genes differentially expressed in the mouse thymus, we discovered the first close rodent homologue of the NK receptor KIR family, which we named KIR- Like (Kirl). KIRL1 shares 40% amino acid identity with primate KIR family members, with the majority of the homology contained within the Ig-like ectodomains. KIRL1 is more similar to the KIRs than to any other known member of the Ig domain-containing leukocyte receptor superfamily. This highly significant homology suggests that the KIR family did not arise independently in primates, as has been previously suggested, but rather evolved from a primordial gene already present in the common rodent/primate ancestor. KIRL1 lacks the cytoplasmic protein motifs that mediate inhibition in KIRs (immunoregulatory tyrosine inhibiting motif, ITIM); KIRL1 also lacks the transmembrane activation signature (a conserved K residue involved in association with the immunoregulatory tyrosine activating motif-containing DAP12 molecule) found in some KIRs. Nevertheless, we hypothesize that Kirl1 is functional, for the following reasons: (1) Kirl1 mRNA is expressed at high levels in immature thymocytes; (2) Kirl1 is regulated during thymocyte development; (3) KIRL1 protein is detected in thymus. We also show that the mouse genome contains a closely related, transcribed gene, which we name Kirl2. Kirl2 encodes a KIR-like molecule with three Ig-like domains and also lacks tyrosine-based immunoregulatory motifs in its cytoplasmic region.     

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+.