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.     

The third naturally occurring attractant toward zoospores of phytopathogenic Aphanomyces cochlioides from the Spinacia oleracea host plant

A bioassay-guided survey of spinach leaf constituents resulted in 5,4'-dihydroxy-3,3'-dimethoxy-6,7-methylenedioxyflavone being identified as the third naturally-occurring attractant in the host plant toward the zoospores of its pathogen, Aphanomyces cochlioides. The isolate showed attracting activity around Chromosorb W AW particles (60-80 mesh) coated with a 10(-5) M solution in a zoospore suspension. However, this activity was 1/100-1/1000 less than that of cochliophilin A, an attractant in the roots of spinach. Bioassays with the present isolate and related compounds revealed that 5,3',4'-trihydroxy-3-methoxy-6,7-methylenedioxyflavone did not possess attractant activity, but rather weak antagonistic activity toward the former two attractants from spinach.     

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.     

Crystal structure of the antigen-binding fragment of apoptosis-inducing mouse anti-human Fas monoclonal antibody HFE7A.

Binding of Fas ligand to Fas induces apoptosis. The Fas-Fas ligand system plays important roles in many biological processes, including the elimination of autoreactive lymphoid cells. The mouse anti-human Fas monoclonal antibody HFE7A (m-HFE7A), which induces apoptosis, has been humanized based on a structure predicted by homology modeling. A version of humanized HFE7A is currently under development for the treatment of autoimmune diseases such as rheumatoid arthritis. For a deeper understanding of the protein engineering aspect of antibody humanization, for which information on the three-dimensional structure is essential, we determined the crystal structure of the m-HFE7A antigen-binding fragment (Fab) by X-ray crystallography at 2.5 A resolution. The main-chain conformation of the five loops in the six complementarity-determining regions (CDRs) was correctly predicted with root-mean-square deviations of 0.30-1.04 A based on a comparison of the crystal structure with the predicted structure. The CDR-H3 conformation of the crystal structure, which was not classified as one of the canonical structures, was completely different from that of the predicted structure but adopted the conformation which followed the "H3-rules." The results of charge distribution analysis of the antigen-binding site suggest that electrostatic interactions may be important for its binding to Fas.     

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.     

Resistance to protoporphyrinogen oxidase-inhibiting compound S23142 from overproduction of mitochondrial protoporphyrinogen oxidase by gene amplification in photomixotrophic tobacco cells

Tobacco YZI-IS cells exhibit a 150-fold greater resistance to the protoporphyrinogen oxidase (Protox)-inhibiting compound, S23142, from wild-type tobacco cells. To investigate the mechanism for this S23142 resistance, the protein level, enzymatic activity, and sensitivity to S23142 in two Protox isoenzymes (plastidal and mitochondrial forms) were examined. The level of mitochondrial Protox protein was greater, and its activity 5-times higher, in YZI-IS cells than in wild-type cells. Furthermore, the apparent IC50 value of S23142 was about 20 nM, which is 20-fold higher than that observed in wild-type cells. In contrast, no differences were found in the plastidal Protox protein level, activity or its inhibition by S23142 between YZI-1S and wild-type cells. A southern blot analysis revealed that the mitochondrial Protox gene had been significantly amplified in the YZI-1S cells. These results suggest that the S23142 resistance of YZI-1S cells was due to the overproduction of mitochondrial Protox by gene amplification.