A novel way to express proline-selectively labeled proteins with a wheat germ cell-free protein synthesis system

For high-throughput protein structural analyses, it is essential to develop a reliable protein overexpression system. Although many protein overexpression systems, such as ones involving Escherichia coli cells, have been developed, the number of overexpressed proteins exhibiting the same biological activities as those of the native ones is limited. A novel wheat germ cell-free protein synthesis system was developed recently, and most of the synthesized proteins that should function in solution were found to be in soluble forms. This suggests the applicability of this protein synthesis method to determination of the functional structures of soluble proteins. In our previous work, we developed a selective labeling technique for amino acids having amide functional groups (other than proline residues) involving the use of several inhibitors for transaminases. This paper in turn describes a proline-selective labeling technique. Based on our results, we have succeeded in constructing a complete amino acid selective labeling technique for the wheat germ cell-free protein synthesis system.     

Coupling of GABAB receptor GABAB2 subunit to G proteins: evidence from Xenopus oocyte and baby hamster kidney cell expression system

Coupling of functional GABA_B receptors (GABA_BR) to G proteins was investigated with an expression system of baby hamster kidney (BHK) cells and Xenopus oocytes. Fluorescence resonance energy transfer (FRET) analysis of BHK cells coexpressing GABA_B1a receptor (GB_1aR) fused to Cerulean, a brighter variant of cyan fluorescent protein, and GABA_B2 receptor (GB₂R) fused to Venus, a brighter variant of yellow fluorescent protein, revealed that GB_1aR-Cerulean and GB₂R-Venus form a heterodimer. The GABA_BR agonists baclofen and 3-aminopropylphosphonic acid (3-APPA) elicited inward-rectifying K⁺ currents in a concentration-dependent manner in oocytes expressing GB_1aR and GB₂R, or GB_1aR-Cerulean and GB₂R-Venus, together with G protein-activated inward-rectifying K⁺ channels (GIRKs), but not in oocytes expressing GB_1aR alone or GB₂R alone together with GIRKs. Oocytes coexpressing GB_1aR + G_i2-fused GB₂R (GB₂R-G_i2) caused faster K⁺ currents in response to baclofen. Furthermore, oocytes coexpressing GB_1aR + GB₂R fused to G_qi5 (a chimeric G_q protein that activates PLC pathways) caused PLC-mediated Ca²⁺-activated Cl^– currents in response to baclofen. In contrast, these responses to baclofen were not observed in oocytes coexpressing GB_1aR-G_i2 or GB_1aR-G_qi5 together with GB₂R. BHK cells and Xenopus oocytes coexpressing GB_1aR-Cerulean + a triplet tandem of GB₂R-Venus-G_qi5 caused FRET and Ca²⁺-activated Cl^– currents, respectively, with a similar potency in BHK cells coexpressing GB_1aR-Cerulean + GB₂R-Venus and in oocytes coexpressing GB_1aR + GB₂R-G_qi5. Our results indicate that functional GABA_BR forms a heterodimer composed of GB₁R and GB₂R and that the signal transducing G proteins are directly coupled to GB₂R but not to GB₁R. fluorescence resonance energy transfer     

Isolation and analysis of genes specifically expressed during fruiting body development in the basidiomycete Flammulina velutipes by fluorescence differential display

Using fluorescence differential display, cDNAs specifically expressed at the primordial stage of fruiting body development were isolated from the basidiomycete, Flammulina velutipes. Seventy-five cDNAs were sequenced and compared with the amino-acid sequences of proteins in the database by BLASTX search. Significant similarity was found for 29 cDNAs coding for proteins with known function, GTP-binding protein, growth factor, ubiquitin-proteasome, cytochrome P450 and hydrophobin, all of which would be associated with fruiting body development. Seventeen cDNAs were not similar to proteins in the database and may represent unique genes that play specific roles in the process of fruiting in F. velutipes.     

Comparison of PCR-RFLP and PFGE for determining the clonality of enterohemorrhagic Escherichia coli strains

We report here on a comparative evaluation of PCR-restriction fragment length polymorphism (PCR-RFLP) and pulsed-field gel electrophoresis (PFGE) assays, and ascertain the clonal relationship between 13 enterohemorrhagic Escherichia coli O157 : H7 strains isolated from fecal samples collected from three cows over a period of 2 months. PCR-RFLP analysis was carried out with either BglI or EcoRV digested LA-PCR amplicons, generated by targeting region V of the Stx-phage. While PCR-RFLP analysis placed these 13 strains into a single clonal type, pulsotyping analysis, as reported earlier, grouped these strains into four different PFGE subtypes of which three were closely related, while the other appeared to be different. The comparative analysis was extended further using two clonally different wild-type (3-0 and Sakai 215) strains and 17 derivative strains which had passed through an animal's gastrointestinal tract. The PCR-RFLP assay, which was not only able to differentiate the wild-type strains, but also placed the passaged derivative strains into their respective parental group, although PFGE patterns of the same set of strains resulted from different PFGE subtypes. These data indicate that PCR-RFLP is the more reliable and useful assay for a molecular epidemiological survey of enterohemorrhagic E. coli strains.     

Immunological effects of recombinant feline interferon-omega (KT-80) administration in the dog

The immunological effects of recombinant feline interferon-omega (rFeIFN-omega ; KT-80, Toray) were examined on administration to healthy dogs. The activities of whole blood cells, macrophages, and natural killer cells were enhanced. Moreover, the whole blood activity was examined when KT-80 was administered to dogs which had been diagnosed as having natural canine parvovirus (CPV) infection. Only some cases in which the activity increased until 3 hr post-administration survived. These results suggest that rFeIFN-omega (KT-80) treatment enhanced the cellular immunity of normal dogs, and could exert significant therapeutic effects on only natural CPV infected dogs with induced continuous immunoenhancement.     

Effect of family 22 carbohydrate-binding module on the thermostability of Xyn10B catalytic module from Clostridium stercorarium

A family 22 carbohydrate-binding module (CBM22) from Clostridium stercorarium Xylanase10B raised the optimum temperature of the xylanase, but in the remaining activity of heating test, apparently the catalytic module alone showed higher remaining activity. Differential scanning calorimetry showed that CBM22 conferred resistance to thermal unfolding of the enzyme and prevented the enzyme from refolding after thermal unfolding.     

Effects of sequential exposure to lipopolysaccharide and heat stress on dental pulp cells

In the present study, we examined the effects of sequential exposure to bacterial lipopolysaccharide (LPS) and heat stress on dental pulp cells. LPS induced the proliferation of pulp cells through the activation of p38 MAPK. HSP27 was expressed in cells with or without LPS during the entire period of heat stress, while transiently phosphorylated by short-term heat stress. In LPS-treated cells, short-term heat stress also induced the phosphorylation of HSF1. The immediate phosphorylation of HSF1 and HSP27 in LPS-treated cells by short-term heat stress occurred dependent on the activation of p38 MAPK. However, with long-term heat stress, the activation of HSF1 and induction of HSP27 occurred independent of p38 MAPK. Further, full activation of Akt in LPS-treated cells was immediately induced by short-term heat stress and lasted during the entire period of heat stress. IkappaB alpha was induced and phosphorylated throughout sequential exposure to LPS and heat stress. These results suggest that LPS has the unique effects on the cytoprotection and the cell death of pulp cells during heat stress through the modification and the activation of heat stress responsive molecules, HSF1 and HSP27, and cell survival molecules, Akt and NF-kappaB/IkappaB alpha.     

Unique quadruplex structure and interaction of an RNA aptamer against bovine prion protein

RNA aptamers against bovine prion protein (bPrP) were obtained, most of the obtained aptamers being found to contain the r(GGAGGAGGAGGA) (R12) sequence. Then, it was revealed that R12 binds to both bPrP and its β-isoform with high affinity. Here, we present the structure of R12. This is the first report on the structure of an RNA aptamer against prion protein. R12 forms an intramolecular parallel quadruplex. The quadruplex contains G:G:G:G tetrad and G(:A):G:G(:A):G hexad planes. Two quadruplexes form a dimer through intermolecular hexad–hexad stacking. Two lysine clusters of bPrP have been identified as binding sites for R12. The electrostatic interaction between the uniquely arranged phosphate groups of R12 and the lysine clusters is suggested to be responsible for the affinity of R12 to bPrP. The stacking interaction between the G:G:G:G tetrad planes and tryptophan residues may also contribute to the affinity. One R12 dimer molecule is supposed to simultaneously bind the two lysine clusters of one bPrP molecule, resulting in even higher affinity. The atomic coordinates of R12 would be useful for the development of R12 as a therapeutic agent against prion diseases and Alzheimer''s disease.     

The Direct Binding of Mrc1, a Checkpoint Mediator,to Mcm6, a Replication Helicase,Is Essential for the Replication Checkpoint against Methyl Methanesulfonate-Induced Stress

Mrc1 plays a role in mediating the DNA replication checkpoint. We surveyed replication elongation proteins that interact directly with Mrc1 and identified a replicative helicase, Mcm6, as a specific Mrc1-binding protein. The central portion of Mrc1, containing a conserved coiled-coil region, was found to be essential for interaction with the 168-amino-acid C-terminal region of Mcm6, and introduction of two amino acid substitutions in this C-terminal region abolished the interaction with Mrc1 in vivo. An mcm6 mutant bearing these substitutions showed a severe defect in DNA replication checkpoint activation in response to stress caused by methyl methanesulfonate. Interestingly, the mutant did not show any defect in DNA replication checkpoint activation in response to hydroxyurea treatment. The phenotype of the mcm6 mutant was suppressed when the mutant protein was physically fused with Mrc1. These results strongly suggest for the first time that an Mcm helicase acts as a checkpoint sensor for methyl methanesulfonate-induced DNA damage through direct binding to the replication checkpoint mediator Mrc1.Progression of the DNA replication machinery along chromosomes is a complex process. Replication forks pause occasionally when they encounter genomic regions that are difficult to replicate, such as highly transcribed regions, tRNA genes, and regions with specialized chromatin structure, like centromeric and heterochromatic regions (17). Replication forks also stall when treated with chemicals like methyl methanesulfonate (MMS), which causes DNA damage, or hydroxyurea (HU), which limits the cellular concentration of the deoxynucleoside triphosphate pool (17). Because de novo assembly and programming of the replisome do not occur after the onset of S phase (18), DNA replication forks must be protected from replicative stresses. The DNA replication checkpoint constitutes a surveillance mechanism for S-phase progression that safeguards replication forks from various replicative stresses (22, 38, 40), and malfunction of this checkpoint leads to chromosome instability and cancer development in higher organisms (4, 9).The Saccharomyces cerevisiae DNA replication checkpoint mediator Mrc1 is functionally conserved and is involved directly in DNA replication as a component of the replisome (1, 8, 16, 19, 29, 30). Mrc1, together with Tof1 and Csm3, is required for forming a replication pausing complex when the fork is exposed to replicative stress by HU (16). The pausing complex subsequently triggers events leading to DNA replication checkpoint activation and hence stable replicative arrest. A sensor kinase complex, Mec1-Ddc2 (ATR-ATRIP homolog of higher eukaryotes), is then recruited to the complex (14, 16). Mec1-Ddc2-mediated phosphorylation of Mrc1 activates the pausing complex, and phosphorylated Mrc1 likely recruits Rad53 (a putative homolog of CHK2 of higher eukaryotes), which is then activated via phosphorylation by Mec1-Ddc2 (1, 16, 20, 30). Activated Rad53 subsequently elicits a stress responses, i.e., stabilization of replication forks, induction of repair genes, and suppression of late-firing origins (24). It remains unclear, however, whether DNA replication checkpoint activation is induced in response to DNA damage by MMS, a reagent commonly used to study the DNA replication stress response. Several lines of evidence have suggested that MMS-induced damage is also sensed directly by the replication machinery (38, 40).Although biochemical and genetic interaction data have placed Mrc1 at the center of the replication checkpoint signal transduction cascade, its molecular function remains largely unknown. The proteins Mrc1, Tof1, and Csm3 associate with the Mcm complex (8, 27), a heterohexameric DNA helicase consisting of Mcm2 to Mcm7 proteins which unwinds the parental DNA duplex to allow replisome progression (3, 12, 18, 31, 32, 35). The Mcm complex associates with a specific set of regulatory proteins at forks to form replisome progression complexes (8). In addition to Mcm, Tof1, Csm3, and Mrc1, replisome progression complexes include factors such as Cdc45 and the GINS complex that are also required for fork progression (13, 26, 31, 32, 39). Claspin, a putative Xenopus laevis homolog of Mrc1, is also reported to associate with Cdc45, DNA polymerase ɛ (Polɛ), replication protein A, and two of the replication factor C complexes in aphidicolin-treated Xenopus egg extracts (19). Recently, Mrc1 was reported to interact directly with Polɛ (23).The aim of this study was to provide mechanistic insight into Mrc1 function in the DNA replication checkpoint. For this purpose, it was essential to identify, among all the essential proteins in the replication machinery, a specific protein that interacts with Mrc1 and to examine the role of this interaction in the DNA replication checkpoint. We found that Mrc1 interacts with Mcm6 directly and specifically. When the interaction between Mrc1 and Mcm6 was impaired, cells no longer activated the DNA replication checkpoint in response to MMS-induced replicative stress. Interestingly and unexpectedly, this interaction was not required for DNA replication checkpoint activation in response to HU-induced replicative stress. Our results provide the first mechanistic evidence that cells use separate mechanisms to transmit replicative stresses caused by MMS and HU for DNA replication checkpoint activation.