Complete nucleotide sequence of the prophage VT2-Sakai carrying the verotoxin 2 genes of the enterohemorrhagic Escherichia coli O157:H7 derived from the Sakai outbreak

The enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain RIMD 0509952, derived from an outbreak in Sakai city, Japan, in 1996, produces two kinds of verotoxins, VT1 and VT2, encoded by the stx1 and stx2 genes. In the EHEC strains, as well as in other VT-producing E. coli strains, the toxins are encoded by lysogenic bacteriophages. The EHEC O157:H7 strain RIMD 0509952 did not produce plaque-forming phage particles upon inducing treatments. We have determined the complete nucleotide sequence of a prophage, VT2-Sakai, carrying the stx2A and stx2B genes on the chromosome, and presumed the putative functions of the encoded proteins and the cis-acting DNA elements based on sequence homology data. To our surprise, the sequences in the regions of VT2-Sakai corresponding to the early gene regulators and replication proteins, and the DNA sequences recognized by the regulators share very limited homology to those of the VT2-encoding 933W phage carried by the EHEC O157:H7 strain EDL933 reported by Plunkett et al. (J. Bacteriol., p1767-1778, 181, 1999), although the sequences corresponding to the structural components are almost identical. These data suggest that these two phages were derived from a common ancestral phage and that either or both of them underwent multiple genetic rearrangements. An IS629 insertion was found downstream of the stx2B gene and upstream of the lysis gene S, and this might be responsible for the absence of plaque-forming activity in the lysate obtained after inducing treatments.     

Structure of rat gamma-tubulin and its binding to HP33.

Gamma-tubulin is localized at the microtubule organizing center and is thought to participate in the organizing of the microtubule network. In this study, we isolated a cDNA of rat gamma-tubulin. The rat gamma-tubulin cDNA encoded 451 amino acids, the same number as that of its counterpart in other vertebrates, and its structure was found to be highly conserved in vertebrates. In a previous work, we identified HP33 (hepatocarcinogenesis- and hepatocellular proliferation-related 33-kDa protein) that was localized at the centrosome of hepatic cells and that exhibited MAP-like activity. In vitro GST pull-down assay using highly purified recombinant HP33 and bacterially expressed gamma-tubulin demonstrated that HP33 bound to gamma-tubulin directly. These results suggest that HP33 is localized at the centrosome via association with both the microtubule and its minus end-specific component, gamma-tubulin.     

EPR characterization of axial bond in metal center of native and cobalt-substituted guanylate cyclase

The nature of the metal-proximal base bond of soluble guanylate cyclase from bovine lung was examined by EPR spectroscopy. When the ferrous enzyme was mixed with NO, a new species was transiently produced and rapidly converted to a five-coordinate ferrous NO complex. The new species exhibited the EPR signal of six-coordinate ferrous NO complex with a feature of histidine-ligated heme. The histidine ligation was further examined by using the cobalt protoporphyrin IX-substituted enzyme. The Co2+-substituted enzyme exhibited EPR signals of a broad g perpendicular;1 component and a g;1 component with a poorly resolved triplet of 14N superhyperfine splittings, which was indicative of the histidine ligation. These EPR features were analogous to those of alpha-subunits of Co2+-hemoglobin in tense state, showing a tension on the iron-histidine bond of the enzyme. The binding of NO to the Co2+-enzyme markedly stimulated the cGMP production by forming the five-coordinate NO complex. We found that N3- elicited the activation of the ferric enzyme by yielding five-coordinate high spin N3- heme. These results indicated that the activation of the enzymes was initiated by NO binding to the metals and proceeded via breaking of the metal-histidine bonds, and suggested that the iron-histidine bond in the ferric enzyme heme was broken by N3- binding.     

A new sugar chain of the proteinase inhibitor from latex of Carica papaya

The structure of a sugar chain of the proteinase inhibitor from the latex of Carica papaya was studied. Sugar chains liberated on hydrazinolysis were N-acetylated, and their reducing-end residues were tagged with 2-aminopyridine. One major sugar chain was detected on size-fractionation and reversed-phase HPLC analyses. The structure of the PA-sugar chain was determined by two-dimensional sugar mapping combined with sequential exoglycosidase digestion and partial acid hydrolysis, and by 750 MHz 1H-NMR spectroscopy. The structure found was Manalpha1-6(Manalpha1-3)Manalpha1-6(Manalpha1-3) (Xylbeta1-2)Manbeta1- 4GlcNAcbeta1-4(Fucalpha1-3)GlcNAc. This sugar chain represents a new plant-type sugar chain with five mannose residues.     

Stability of the dimerization domain effects the cooperative DNA binding of short peptides

The basic region peptide derived from the basic leucine zipper protein GCN4 bound specifically to the native GCN4 binding sequences in a dimeric form when the beta-cyclodextrin/adamantane dimerization domain was introduced at the C-terminus of the GCN4 basic region peptide. We describe here how the structure and stability of the dimerization domain affect the cooperative formation of the peptide dimer-DNA complex. The basic region peptides with five different guest molecules were synthesized, and their equilibrium dissociation constants with a peptide possessing beta-cyclodextrin were determined. These values, ranging from 1.3 to 15 microM, were used to estimate the stability of the complexes between the dimers with various guest/cyclodextrin dimerization domains and GCN4 target sequences. An efficient cooperative formation of the dimer complexes at the GCN4 binding sequence was observed when the adamantyl group was replaced with the norbornyl or noradamantyl group, but not with the cyclohexyl group that formed a beta-cyclodextrin complex with a stability that was 1 order of magnitude lower than that of the adamantyl group. Thus, cooperative formation of the stable dimer-DNA complex appeared to be effected by the stability of the dimerization domain. For the peptides that cooperatively formed dimer-DNA complexes, there was no linear correlation between the stability of the inclusion complex and that of the dimer-DNA complex. With the beta-cyclodextrin/adamantane dimerization domain, the basic region peptide dimer preferred to bind to a palindromic 5'-ATGACGTCAT-3' sequence over the sequence lacking the central G.C base pair and that with an additional G.C base pair in the middle. Changing the adamantyl group into a norbornyl group did not alter the preferential binding of the peptide dimers to the palindromic sequence, but slightly affected the selectivity of the dimer for other nonpalindromic sequences. The helical contents of the peptides in the DNA-bound dimer with the adamantyl group were decreased by reducing the stability of the dimer-DNA complex, which was possibly caused by deformation of the helical structure proximal to the dimerization domain.     

Structure and stability of the consecutive stereoregulated chiral phosphorothioate DNA duplex

The duplex structures of the stereoregulated phosphorothioate DNAs, [R(p),R(p)]- and [S(p),S(p)]-[d(GC(ps)T(ps)ACG)] (ps, phosphorothioate; PS-DNA), with their complementary RNA have been investigated by combined use of (1)H NMR and restrained molecular dynamics calculation. Compared to those obtained for the unmodified duplex structures (PO-DNA.RNA), the NOE cross-peak intensities are virtually identical for the PS-DNA.RNA hybrid duplexes. The structural analysis on the basis of the NOE restraints reveals that all of the three DNA.RNA duplexes take a A-form conformation and that there is no significant difference in the base stacking for the DNA.RNA hybrid duplexes. On the other hand, the NOE cross-peak intensities of the protons around the central T(ps)A step of the PS-DNA.DNA duplexes are apparently different from those of PO-DNA. DNA. The chemical shifts of H8/6 and H1' at the T(ps)A step are also largely different among PS-DNA.DNAs and PO-DNA.DNA, suggesting that the DNA.DNA structure is readily changed by the introduction of the phosphorothioate groups to the central T(p)A step. The structure calculations indicate that all of these DNA.DNA duplexes are B-form although there exist some small differences in helical parameters between the [R(p),R(p)]- and [S(p),S(p)]PS-DNA.DNA duplexes. The melting temperatures (T(m)) were determined for all of the duplexes by plotting the chemical shift change of isolated peaks as a function of temperature. For the PS-DNA.RNA hybrid duplexes, the [S(p),S(p)] isomer is less stable than the [R(p),R(p)] isomer while this trend is reversed for the PS-DNA.DNA duplexes. Consequently, although the PS-DNA.RNA duplexes take the similar A-form structure, the duplex stability is different between PS-DNA.RNA duplexes. The stability of the DNA.RNA duplexes may not be governed by the A-form structure itself but by some other factors such as the hydration around the phosphorothioate backbone, although the T(m) difference of the DNA.DNA duplexes could be explained by the structural factor.     

Local delivery of soluble TNF-alpha receptor 1 gene reduces infarct size following ischemia/reperfusion injury in rats

Apoptosis in the myocardium is linked to ischemia/reperfusion injury, and TNF-alpha induces apoptosis in cardiomyocytes. A significant amount of TNF-alpha is detected after ischemia and reperfusion. Soluble TNF-alpha receptor 1 (sTNFR1) is an extracellular domain of TNF-alpha receptor 1 and is an antagonist to TNF-alpha. In the present study, we examined the effects of sTNFR1 on infarct size in acute myocardial infarction (AMI) following ischemia/reperfusion. Male Wistar rats were subjected to left coronary artery (LCA) ligation. After 30 min of LCA occlusion, the temporary ligature on the LCA was released and blood flow was restored. Immediately after reperfusion, a total of 200 microg of sTNFR1 or LacZ plasmid was injected into three different sites of the left ventricular wall. At 6 h, 1 and 2 days after reperfusion, the TNF-alpha bioactivity in the myocardium was significantly higher in rats receiving LacZ plasmid than in sham-operated rats, whereas sTNFR1 plasmid significantly suppressed the increase in the TNF-alpha bioactivity. The sTNFR1 plasmid significantly reduced DNA fragmentation and caspase activity compared to the LacZ plasmid. Finally, the sTNFR1 expression-plasmid treatment significantly reduced the area of myocardial infarction at 2 days after ischemia/reperfusion compared to LacZ plasmid. In conclusion, the TNF-alpha bioactivity in the heart increased from the early stage of ischemia/reperfusion, and this increase was thought to contribute in part to the increased area of myocardial infarction. Suppression of TNF-alpha bioactivity with the sTNFR1 plasmid reduced the infarct size in AMI following ischemia and reperfusion.