Features of bacterial cellulose synthesis in a mutant generated by disruption of the diguanylate cyclase 1 gene of <Emphasis Type="Italic">Acetobacter xylinum</Emphasis> BPR 2001

The diguanylate cyclase 1 (DGC1) (dgc1) gene in Acetobacter xylinum BPR 2001—a bacterial cellulose (BC) producer—was cloned and sequenced, and a DGC1 gene-disrupted mutant, strain DD, was constructed. The production and structural characteristics of the BC formed by DD were compared with those of the parental strain BPR 2001. BC production by DD was almost the same as that by BPR 2001 in static cultivation and in shake flask cultivation. However, in a jar fermentor DD produced about 36% more BC than the parental strain. DD produced suspended particle materials that cannot aggregate owing to their random structural characteristics in static cultivation; more uniformly dispersed BC pellicles and smaller BC pellets are produced on average in a jar fermentor, as reflected by the higher BC production by DD than by the parental strain in a jar fermentor. Micrographs of BC produced by DD revealed that the width of cellulose ribbons assemblies decreased as a result of differences in the ultrastructure and mechanism of formation of BC between the two strains. These results reveal that disruption of the dgc1 gene, which catalyzes synthesis of c-di-GMP (an effector of BC synthase), is not fatal for BC synthesis, although it affects BC structure.     

A distinctive class of spermidine synthase is involved in chilling response in rice

A cDNA for a putative 42 kD spermidine synthase (OsSPDS2) was cloned from rice. The deduced OsSPDS2 sequence showed highest similarity with Arabidopsis AtSPDS3. Phylogenetic analysis revealed that OsSPDS2 and AtSPDS3 form a distinctive subclass in the spermidine synthase family in plants. OsSPDS2 mRNA accumulated in roots during long term exposure to chilling temperature (12 degrees C). In contrast, no such induction of the paralogous OsSPDS1 was observed during the chilling treatment. ABA treatment up-regulated OsSPDS2, whereas salt stress did not change OsSPDS2 levels significantly. Data suggested a distinct function of OsSPDS2 in chilling response in rice.     

Thienopyridine and benzofuran derivatives as potent anti-tumor agents possessing different structure-activity relationships

(3-Amino-6-thiophen-2-yl-thieno[2,3-b]pyridin-2-yl)phenylmethanone (3) was discovered as a new type of cytotoxic agent selective against a tumorigenic cell line. The molecular structure of a previously reported compound, (4-hydroxy-3-methyl-6-phenylbenzofuran-2-yl)phenylmethanone (2), had remarkably similar bioisosteric substructures to that of compound 3. Although the relationship between the molecular structure and biological activity of each derivative synthesized from these two hit compounds (2 and 3) were studied, unexpectedly no correlation was observed. However, after further synthetic study from 3, one of the most potent derivative (10k) having a different SAR profile from 2, was discovered.     

FORTE: a profile-profile comparison tool for protein fold recognition

We present FORTE, a profile-profile comparison tool for protein fold recognition. Users can submit a protein sequence to explore the possibilities of structural similarity existing in known structures. Results are reported via email in the form of pairwise alignments.     

Artificial metallo-DNA: a bio-inspired approach to metal array programming

The structure of DNA is such that the multi-array of functionalized units with desired numbers and sequences within the DNA is possible. In particular, to replace DNA bases, which are biologically important elements for gene expression, by alternative bases would provide powerful tools for programming molecular arrays in a pre-designed manner. This review focuses on recent chemical approaches to self-assembled metal arrays within DNA with metal-mediated base pairing.     

Conserved protein kinases encoded by herpesviruses and cellular protein kinase cdc2 target the same phosphorylation site in eukaryotic elongation factor 1delta

Earlier studies have shown that translation elongation factor 1delta (EF-1delta) is hyperphosphorylated in various mammalian cells infected with representative alpha-, beta-, and gammaherpesviruses and that the modification is mediated by conserved viral protein kinases encoded by herpesviruses, including UL13 of herpes simplex virus type 1 (HSV-1), UL97 of human cytomegalovirus, and BGLF4 of Epstein-Barr virus (EBV). In the present study, we attempted to identify the site in EF-1delta associated with the hyperphosphorylation by the herpesvirus protein kinases. Our results are as follows: (i) not only in infected cells but also in uninfected cells, replacement of the serine residue at position 133 (Ser-133) of EF-1delta by alanine precluded the posttranslational processing of EF-1delta, which corresponds to the hyperphosphorylation. (ii) A purified chimeric protein consisting of maltose binding protein (MBP) fused to a domain of EF-1delta containing Ser-133 (MBP-EFWt) is specifically phosphorylated in in vitro kinase assays by purified recombinant UL13 fused to glutathione S-transferase (GST) expressed in the baculovirus system. In contrast, the level of phosphorylation by the recombinant UL13 of MBP-EFWt carrying an alanine replacement of Ser-133 (MBP-EFS133A) was greatly impaired. (iii) MBP-EFWt is also specifically phosphorylated in vitro by purified recombinant BGLF4 fused to GST expressed in the baculovirus system, and the level of phosphorylation of MBP-EFS133A by the recombinant BGLF4 was greatly reduced. (iv) The sequence flanking Ser-133 of EF-1delta completely matches the consensus phosphorylation site for a cellular protein kinase, cdc2, and in vitro kinase assays revealed that purified cdc2 phosphorylates Ser-133 of EF-1delta. (v) As observed with EF-1delta, the casein kinase II beta subunit (CKIIbeta) was specifically phosphorylated by UL13 in vitro, while the level of phosphorylation of CKIIbeta by UL13 was greatly diminished when a serine residue at position 209, which has been reported to be phosphorylated by cdc2, was replaced with alanine. These results indicate that the conserved protein kinases encoded by herpesviruses and a cellular protein kinase, cdc2, have the ability to target the same amino acid residues for phosphorylation. Our results raise the possibility that the viral protein kinases mimic cdc2 in infected cells.     

Na+/Mg2+ transporter acts as a Mg2+ buffering mechanism in PC12 cells

Mg(2+) buffering mechanisms in PC12 cells were demonstrated with particular focus on the role of the Na(+)/Mg(2+) transporter by using a newly developed Mg(2+) indicator, KMG-20, and also a Na(+) indicator, Sodium Green. Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), a protonophore, induced a transient increase in the intracellular Mg(2+) concentration ([Mg(2+)](i)). The rate of decrease of [Mg(2+)](i) was slower in a Na(+)-free extracellular medium, suggesting the coupling of Na(+) influx and Mg(2+) efflux. Na(+) influxes were different for normal and imipramine- (a putative inhibitor of the Na(+)/Mg(2+) transporter) containing solutions. FCCP induced a rapid increase in [Na(+)](i) in the normal solution, while the increase was gradual in the imipramine-containing solution. The rate of decrease of [Mg(2+)](i) in the imipramine-containing solution was also slower than that in the normal solution. From these results, we show that the main buffering mechanism for excess Mg(2+) depends on the Na(+)/Mg(2+) transporter in PC12 cells.     

Remarkable stabilization of neutrophil NADPH oxidase using RacQ61L and a p67phox-p47phox fusion protein

Activation of the phagocyte NADPH oxidase occurs via assembly of cytosolic p47(phox), p67(phox), and Rac with the membrane-bound flavocytochrome b(558). Recently, we have found that p67(phox)-(1-210) (p67N) fused with p47(phox)-(1-286) (p47N) or with Rac efficiently stabilizes the oxidase in a cell-free reconstitution system. In an attempt to further stabilize the oxidase, we herein used a constitutively active Rac, RacQ61L, and examined its effect on the oxidase stability. The half-life (t(1/2)) of the activity reconstituted with wild-type Rac was 12 min at 37 degrees C, which was extended 6-fold by RacQ61L. Also, the stability of the oxidase without p47(phox) increased 8-fold using RacQ61L. RacQ61L had a higher affinity for the complex than wild-type Rac and increased the affinity of p67N for the complex. Far-western blotting showed an enhanced binding between RacQ61L and p67N. The oxidase was stabilized by nanomolar FAD, and RacQ61L lowered the FAD concentration required. The combination of RacQ61L and a fusion protein consisting of p67N and p47N produced an extremely stable enzyme (t(1/2) = 184 min at 37 degrees C). The effectiveness of RacQ61L and fusion proteins on stabilization was in the following order: p67N-Rac < p67N + RacQ61L < or = p67N-RacQ61L < p67N-p47N + RacQ61L. These results indicate that a tightly bound ternary complex of p67(phox), Rac, and p47(phox) is very effective in maintaining the oxidase and confirm that the longevity of the activated state requires continuous association of these components. This simple and efficient method of stabilization may provide a useful tool to elucidate the nature of the activated oxidase.