Protein folding and unfolding are coupled to a range of biological phenomena, from the regulation of cellular activity to the onset of neurodegenerative diseases. Defining the nature of the conformations sampled in nonnative proteins is crucial for understanding the origins of such phenomena. We have used a combination of nuclear magnetic resonance (NMR) spectroscopy and site-directed mutagenesis to study unfolded states of the protein lysozyme. Extensive clusters of hydrophobic structure exist within the wild-type protein even under strongly denaturing conditions. These clusters involve distinct regions of the sequence but are all disrupted by a single point mutation that replaced residue Trp62 with Gly located at the interface of the two major structural domains in the native state. Thus, nativelike structure in the denatured protein is stabilized by the involvement of Trp62 in nonnative and long-range interactions.

To demonstrate the extent of phylogenetic diversity of diazotrophic bacteria associated with rice roots, we characterized phylogenetically 23 nifH gene sequences obtained by PCR amplification of mixed organism DNA extracted directly from rice roots without culturing the organisms. The analyses document the presence of eight novel NifH types, which appear to be a variety of significant components of the diazotrophic community, dominated mainly by proteobacteria.The method developed by Pace et al. to determine species diversity and composition, using rRNA (or ribosomal DNA) isolated directly from nature has opened a window into the world of unculturable bacteria, although this method has the disadvantage that organisms whose genes have been isolated by the method cannot be studied for any other trait (11,16). This report extends the method by applying it to a functionally important gene, nifH, to determine the importance and diversity of nitrogen-fixing bacteria associated with rice roots. The nitrogenase iron protein gene nifH is one of the oldest existing and functioning genes in the history of gene evolution, and the outline of the NifH tree is reported to be largely consistent with the 16S rRNA phylogeny (21,22). These features prompted us to study the genetic diversity of N 2 -fixing bacteria by the molecular evolutionary analysis of nifH sequences amplified directly from rice root DNA, because the rice root DNA contains not only plant DNA but also microbial DNA in the roots. In the rice root zone, N 2 fixation is associated with the activity of N 2 -fixing heterotrophic bacteria (4,20). It has been reported that a large percentage of the total aerobic heterotrophic population in the root is diazotrophic (1, 10, 17). However, studies on the rhizospheric N 2 -fixing microflora have until now suffered from the use of selective media for counts and isolations, because it is widely believed that only a small percentage of natural prokaryotes may actually be culturable (18,19). Here we report a study of N 2 -fixing bacterial diversity by analysis of nifH gene sequences without a cultivation technique.DNA extraction. Rice, Oryza sativa L. cv. nihonnbare, was raised under flooded conditions in the Kyushu University Farm. Rice plants taken at the heading stage in September were dug out from a wetland rice field, and the roots were washed to remove the attached soil. The washed roots were cut into segments, frozen with liquid nitrogen, and ground to a fine powder in a mortar and pestle. The fine powder was suspended in extraction buffer (100 mM Tris, 100 mM EDTA, 250 mM NaCl, 100 g of proteinase K per ml) supplemented with Sarkosyl (1% final concentration) and lysed by incubation at 55ЊC for 1 h. Treatment of the lysate with RNase A was followed by chloroform extraction and isopropanol precipitation. Crude DNA was purified by phenol extraction, chloroform extraction, and isopropanol precipitation.PCR amplification of nifH genes. The primers for PCR amplification were chosen by careful inspection of the 37 published ni...

Chromosomal replication initiation requires the regulated formation of dynamic higher order complexes. Escherichia coli ATP-DnaA forms a specific multimer on oriC, resulting in DNA unwinding and DnaB helicase loading. DiaA, a DnaA-binding protein, directly stimulates the formation of ATP-DnaA multimers on oriC and ensures timely replication initiation. In this study, DnaA Phe-46 was identified as the crucial DiaA-binding site required for DiaA-stimulated ATP-DnaA assembly on oriC. Moreover, we show that DiaA stimulation requires only a subgroup of DnaA molecules binding to oriC, that DnaA Phe-46 is also important in the loading of DnaB helicase onto the oriCDnaA complexes, and that this process also requires only a subgroup of DnaA molecules. Despite the use of only a DnaA subgroup, DiaA inhibited DnaB loading on oriC-DnaA complexes, suggesting that DiaA and DnaB bind to a common DnaA subgroup. A cellular factor can relieve the DiaA inhibition, allowing DnaB loading. Consistently, DnaA F46A caused retarded initiations in vivo in a DiaA-independent manner. It is therefore likely that DiaA dynamics are crucial in the regulated sequential progress of DnaA assembly and DnaB loading. We accordingly propose a model for dynamic structural changes of initial oriC complexes loading DiaA or DnaB helicase.In many cellular organisms, multiple proteins form dynamic complexes on the chromosomal origin for the initiation of DNA replication. In Escherichia coli, ATP-DnaA forms a specific multimeric complex on the origin (oriC), resulting in an initiation complex that is competent in the replicational initiation (1-3). ATP-DnaA complexes, but not ADP-DnaA complexes, unwind the DNA duplex within the oriC DNA unwinding element (DUE) 2 with the aid of superhelicity of oriC DNA and heat energy, resulting in the formation of open complexes (4, 5). At the unwound region, the loading of a DnaB replicative helicase is mediated by a DnaC helicase loader, resulting in the formation of the prepriming complex (6, 7). DnaG primase then complexes with DnaB loaded on the single-stranded (ss) region, which leads to primer synthesis and the loading of DNA polymerase III holoenzyme (8). The cellular ATP-DnaA level fluctuates during the replication cycle with a peak around the time of initiation (9). At the post-initiation stage, DnaA-ATP is hydrolyzed in a manner depending on ADP-Hda protein and the DNA-loaded form of the ␤-clamp subunit of the polymerase III holoenzyme, yielding inactive ADP-DnaA (10 -13). This DnaA inactivation system is called RIDA (regulatory inactivation of DnaA). Hda consists of a short N-terminal region bearing a clamp-binding motif and a C-terminal AAA ϩ domain. This protein is activated by ADP binding, which allows interaction with ATP-DnaA in a DNA-loaded ␤-clamp-dependent manner. RIDA decreases the level of cellular ATP-DnaA in a replication-coordinated manner and represses extra initiation events (9 -11).The timing of chromosomal replication initiation is strictly regulated and needs to be linked to the regulation o...

BACKGROUND To elucidate the pathophysiological significance of endothelin in pulmonary hypertension associated with congenital heart defects, we measured plasma endothelin-like immunoreactivity (ET-LI) concentrations by using radioimmunoassay in 18 patients with pulmonary hypertension (PH group; age, 6 months to 12 years) in comparison with 27 patients without pulmonary hypertension (non-PH group; age, 6 months to 12 years). METHODS AND RESULTS Blood samples were obtained from the vena cava, right atrium, right ventricle, left or right pulmonary artery, and pulmonary vein or the pulmonary arterial wedge position (pulmonary venous blood) during cardiac catheterization. Plasma ET-LI concentrations in the PH group were significantly higher than those in the non-PH group at all sampling sites. In the PH group, plasma ET-LI concentration showed a significant increase between the right ventricle and pulmonary artery and between the pulmonary artery and pulmonary vein. The increment of plasma ET-LI concentrations from the right ventricle to the pulmonary vein was significantly larger in the PH group than in the non-PH group and was significantly correlated with pulmonary artery pressure. CONCLUSIONS Plasma ET-LI concentrations were elevated in patients with pulmonary hypertension; the elevation was due to the increased production of ET-LI in pulmonary circulation, indicating the possible involvement of endothelin in the pathophysiology of pulmonary hypertension.

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)...

Summary We report here the phylogenetic characterization of small subunit rRNA gene sequences obtained by polymerase chain reaction (PCR) amplification of mixed population DNA extracted directly from soil in a soybean field without culturing the organisms. The phylogenetic analysis of 17 soil clones by the neighbour‐joining method shows that the soil sample contained broadly diverse prokaryotes; a clone related to archaea, a clone to gram‐positive bacteria with high G+C contents, two clones to green sulphur bacteria, four clones to proteobacteria, and nine clones were not in clusters of any previously reported bacterial groups, which suggests they belong to members of novel groups in Bacteria. In addition, the archaeal sequence, FIE16, is phylogenetically similar to ANTARCTIC 12, a clone obtained from surface waters of Antarctica by PCR. Their occurrence in both the ocean and soil suggests a global distribution of this archaeal group. In conclusion, rRNA gene sequences recovered from soil biomass document the occurrence of many more bacterial lingeages than have been recognized previously through cultivation‐based techniques.

The ATP-bound DnaA protein opens duplex DNA at the Escherichia coli origin of replication, leading to a series of initiation reactions in vitro. When loaded on DNA, the DNA polymerase III sliding clamp stimulates hydrolysis of DnaA-bound ATP in the presence of the IdaB/Hda protein, thereby yielding ADP-DnaA, which is inactive for initiation in vitro. This negative feedback regulation of DnaA activity is proposed to play a crucial role in the replication cycle. We here report that the mutant protein DnaA R334A is inert to hydrolysis of bound ATP, although its affinities for ATP and ADP remain unaffected. The ATP-bound DnaA R334A protein, but not the ADP form, initiates minichromosomal replication in vitro at a level similar to that seen for wild-type DnaA. When expressed at moderate levels in vivo, DnaA R334A is predominantly in the ATP-bound form, unlike the wild-type and DnaA E204Q proteins, which in vitro hydrolyze ATP in a sliding clamp-and IdaB/Hda-dependent manner. Furthermore, DnaA R334A, but not the wild-type or the DnaA E204Q proteins, promotes overinitiation of chromosomal replication. These in vivo data support a crucial role for bound nucleotides in regulating the activity of DnaA during replication. Based on a homology modeling analysis, we suggest that the Arg-334 residue closely interacts with bound nucleotides.Chromosomal duplication occurs only once during the cell cycle and is regulated mainly by ingenious controls that act during the initiation of replication (1-3). The Escherichia coli initiator protein, DnaA, binds to the chromosomal origin of replication (oriC) and promotes a series of reactions leading to the formation of a replication fork (4, 5). This protein has high affinity for ATP and ADP, but only the ATP-bound form (ATPDnaA) can initiate replication at oriC. ATP-DnaA (but not ADP-DnaA) causes local unwinding of the oriC DNA duplex, which creates a site of entry for the DnaB helicase. DnaB helicase loaded on the unwound site forms a complex with DnaG primase and expands the single-stranded region. DNA polymerase (pol) 1 III holoenzyme is then loaded on the primed DNA and begins synthesis (6, 7). Several features of both DnaA and the oriC locus are important for the regulation of initiation.Immediately after initiation, re-replication of oriC is restrained temporarily (8). E. coli DNA is modified at the adenine residue of the palindromic sequence GATC by DNA-adenine methyltransferase, but newly replicated DNA remains hemimethylated until acted upon by DNA-adenine methyltransferase (9). The hemimethylated oriC locus is bound by the protein SeqA, which likely inhibits the initiation of replication until full methylation is re-established (10 -12). This eclipse time is speculated to persist for ϳ10 min under conditions in which the cellular doubling time is 30 min (9, 10).Also after initiation, DnaA is likely inactivated by the hydrolysis of bound ATP to yield ADP-DnaA (3). In vitro, ATP hydrolysis is promoted by the pol III ␤ subunit that is loaded onto DNA (the so-called sliding clamp)...

AIMTo determine the possibility that diabetes mellitus promotes pancreatic ductal adenocarcinoma via glyceraldehyde (GA)-derived advanced glycation-end products (GA-AGEs).METHODSPANC-1, a human pancreatic cancer cell line, was treated with 1-4 mmol/L GA for 24 h. The cell viability and intracellular GA-AGEs were measured by WST-8 assay and slot blotting. Moreover, immunostaining of PANC-1 cells with an anti-GA-AGE antibody was performed. Western blotting (WB) was used to analyze the molecular weight of GA-AGEs. Heat shock proteins 90α, 90β, 70, 27 and cleaved caspase-3 were analyzed by WB. In addition, PANC-1 cells were treated with GA-AGEs-bovine serum albumin (GA-AGEs-BSA), as a model of extracellular GA-AGEs, and proliferation of PANC-1 cells was measured.RESULTSIn PANC-1 cells, GA induced the production of GA-AGEs and cell death in a dose-dependent manner. PANC-1 cell viability was approximately 40% with a 2 mmol/L GA treatment and decreased to almost 0% with a 4 mmol/L GA treatment (each significant difference was P < 0.01). Cells treated with 2 and 4 mmol/L GA produced 6.4 and 21.2 μg/mg protein of GA-AGEs, respectively (P < 0.05 and P < 0.01). The dose-dependent production of some high-molecular-weight (HMW) complexes of HSP90β, HSP70, and HSP27 was observed following administration of GA. We considered HMW complexes to be dimers and trimers with GA-AGEs-mediated aggregation. Cleaved caspase-3 could not be detected with WB. Furthermore, 10 and 20 μg/mL GA-AGEs-BSA was 27% and 34% greater than that of control cells, respectively (P < 0.05 and P < 0.01).CONCLUSIONAlthough intracellular GA-AGEs induce pancreatic cancer cell death, their secretion and release may promote the proliferation of other pancreatic cancer cells.