DnaA forms a homomultimeric complex with the origin of chromosomal replication (oriC) to unwind duplex DNA. The interaction of the DnaA N terminus with the DnaB helicase is crucial for the loading of DnaB onto the unwound region. Here, we determined the DnaA N terminus structure using NMR. This region (residues 1-108) consists of a rigid region (domain I) and a flexible region (domain II). Domain I has an ␣-␣-␤-␤-␣-␤ motif, similar to that of the K homology (KH) domain, and has weak affinity for oriC single-stranded DNA, consistent with KH domain function. A hydrophobic surface carrying Trp-6 most likely forms the interface for domain I dimerization. Glu-21 is located on the opposite surface of domain I from the Trp-6 site and is crucial for DnaB helicase loading. These findings suggest a model for DnaA homomultimer formation and DnaB helicase loading on oriC.In Escherichia coli, DnaA initiates the replication of genomic DNA in a cell cycle-coordinated manner (1, 2). The initiation complex contains a homomultimer of DnaA protein and the replication origin, oriC, of the chromosome. DnaA binds cooperatively to the oriC region in a manner depending on tight binding to the DnaA box sequences within the oriC region. The initiation complex containing ATP-bound DnaA molecules unwinds the duplex of the AT-rich 13-mer repeats within oriC, forming an open complex. This leads to the loading of DnaB helicase onto single-stranded DNA (ssDNA) 4 in the presence of the DnaC helicase loader. In this process, the interaction between DnaA and DnaB is crucial. DnaA creates a specific nucleoprotein structure within which ordered structural DNA changes and protein-protein interactions take place. DnaG primase then complexes with the ssDNA-loaded DnaB, which leads to DNA chain synthesis by the DNA polymerase III holoenzyme. The DNA-loaded ␤-clamp subunit of the holoenzyme complexes with Hda, a DnaA paralogue protein, and this complex promotes DnaA-ATP hydrolysis, yielding inactive ADP-DnaA (3-5).DnaA is a 52-kDa basic protein that has four distinct functional domains (1, 2). NMR and crystal structure analyses have revealed that C-terminal domain IV of E. coli DnaA has a helixturn-helix motif, which binds the DnaA box specifically (6 -8). DnaA domain III contains ATP binding/hydrolysis motifs of the AAAϩ ATPase family (9 -14). Erzberger et al. (9) proposed an oligomeric structure for DnaA based on crystal structures of the ATP-and ADP-bound forms of DnaA domains III-IV from the hyperthermophilic bacterium Aquifex aeolicus. In this model of the initiation complex, the ATP-DnaA molecules assemble in a head-to-tail manner, and the resultant oligomers form a spiral helix, consistent with the known features of the AAAϩ family proteins (11, 15). The structure-function relationships of the N-terminal domains I and II of DnaA remain obscure.The amino acid sequence of domain I (residues 1-86 in E. coli DnaA; Fig. 1A) is highly conserved among DnaA homologs of eubacterial species, unlike that of domain II (residues 87-134 in E. coli DnaA) (2)...

We assessed whether the low sigma-alcohol dehydrogenase (ADH) activity in Japanese (compared with Caucasians) affects the first-pass metabolism of ethanol. ADH isozyme activities were determined in endoscopic biopsies of the gastric corpus from 24 Japanese and 41 Caucasian men by starch gel electrophoresis and by comparing the reduction of m-nitrobenzaldehyde (a preferred substrate of sigma-ADH) with that of acetaldehyde (a preferred substrate of gamma-ADH) and the glutathione-dependent formaldehyde oxidation (a specific reaction of chi-ADH). Alcohol pharmacokinetics was compared in 10 Japanese and 10 Caucasians after administration of ethanol (300 mg/kg of body weight) intravenously or orally, using 5 and 40% oral solutions. Japanese exhibited lower sigma-ADH activity than Caucasians, with no difference in the other gastric isozymes. With 5% ethanol, first-pass metabolism was strikingly lower in Japanese than in Caucasians. Blood alcohol levels were similar because of the high elimination rate in Japanese due to the hepatic beta 2-ADH variant. With 40% ethanol, the first-pass metabolism increased in both groups to comparable levels, suggesting an additional contribution by chi-ADH at high ethanol concentrations. These results indicate that sigma-ADH activity contributes significantly to gastric ethanol oxidation and its lower activity in Japanese is associated with lesser first-pass metabolism.

Thin films of octahedral molybdenum metal clusters (Mo 6) were fabricated on indium tin oxide (ITO) glass slides by the electrophoretic deposition (EPD) technique. The solubility of the Cs 2 Mo 6 Br 14 cluster compounds, which are composed of [Mo 6 Br i 8 Br a 6 ] 2− units associated with two cesium cations, in different solvents was investigated in order to achieve a welldispersed colloidal solution of the Mo 6 cluster units that was stable during the following EPD process. Distilled water, ethanol, 1-propanol, acetone, methyl-ethyl ketone (MEK) and acetyl acetone were selected as the dispersing media of the Cs 2 Mo 6 Br 14 cluster compounds. Homogeneous, transparent Mo 6 cluster films with prominent luminescent properties were obtained by the EPD when dissolving Cs 2 Mo 6 Br 14 into the MEK and acetyl acetone solutions. The obtained Mo 6 cluster films were examined by investigating the thickness (color 3D laser spectroscopy), the morphology (FE-SEM), the Br/Mo ratio (XRF), and the crystallographic parameters variation (XRD). The optical emission properties were characterized by UV-Vis spectroscopy, FL spectroscopy and micro PL spectroscopy. The thickness of the Mo 6 cluster films changed depending on the kind of the solvents. The atomic ratio of the Mo and Br elements in the Mo 6 cluster-based unit were relatively maintained during the dispersion and deposition in the MEK solution. The UV-Vis absorption spectra exhibited a high transparency in the visible light range (500-850 nm) accompanied by many peaks due to light interference in the wavelength range from 400 nm to 2000 nm. The emission intensity of the Mo 6 cluster film deposited in MEK slightly decreased during continuous irradiation of 325 nm laser light for 600s.

Escherichia coli DnaA protein initiates chromosomal replication and is an important regulatory target during the replication cycle. In this study, a suppressor mutation isolated by transposon mutagenesis was found to allow growth of the temperature-sensitive dnaA508 and dnaA167 mutants at 40 °°°° C. The suppressor consists of a transposon insertion in a previously annotated ORF, here termed hspQ , a novel heat shock gene whose promoter is recognized by the major heat shock sigma factor σ σ σ σ 32. Expression of hspQ on a pBR322 derivative inhibits growth of the dnaA508 and dnaA167 mutants at 30 °°°° C, whereas growth of dnaA46 and other dnaA mutants is insensitive to changes in the level of hspQ . Cellular DnaA508 protein is degraded rapidly at elevated temperature, but hspQ disruption impedes this process. In contrast, DnaA46 protein is rapidly degraded in an hspQ -independent manner. Gel-filtration and chemical cross-linking experiments suggest that HspQ forms a stable homodimer in solution and can form homomultimers consisting of about four monomers. Heat-shock induced proteases such as Clp contain homomultimers of subunit proteins. We propose that HspQ is a new factor involved in the quality control of proteins and that it functions by excluding denatured proteins.

This result indicates that EVL is not effective for prophylactic therapy for oesophageal varices in liver cirrhosis.