Identification by Suppression Subtractive Hybridization of Frankia Genes Induced under Nitrogen-Fixing Conditions

Frankia is an actinobacterium that fixes nitrogen under both symbiotic and free-living conditions. We identified genes upregulated in free-living nitrogen-fixing cells by using suppression subtractive hybridization. They included genes with predicted functions related to nitrogen fixation, as well as with unknown function. Their upregulation was a novel finding in Frankia.Frankia is a Gram-positive actinobacterium that establishes symbiosis with several angiosperms termed actinorhizal plants and forms nitrogen-fixing nodules on their roots (20). Frankia also fixes nitrogen in free-living culture under nitrogen-free conditions (19). Induction of the nitrogen-fixing ability is accompanied by differentiation of vesicles (19). Vesicles are spherical cells specialized to nitrogen fixation and are surrounded by multilayered lipid envelopes by which nitrogenase is protected from oxygen (3). Frankia plays an important role in the global nitrogen cycle, yet little is known about the genes involved in the induction of nitrogen-fixing activity. Recently, three Frankia genome sequences were determined (15), which facilitates the genetic dissection of Frankia biology. In this study, we identified Frankia genes induced in nitrogen-fixing cells under free-living conditions by using suppression subtractive hybridization (SSH) (4).     

Enzymatic synthesis of unique sialyloligosaccharides using marine bacterial α-(2→3)- and α-(2→6)-sialyltransferases

We investigated the acceptor substrate specificities of marine bacterial α-(2→3)-sialyltransferase cloned from Photobacterium sp. JT-ISH-224 and α-(2→6)-sialyltransferase cloned from Photobacterium damselae JT0160 using several saccharides as acceptor substrates. After purifying the enzymatic reaction products, we confirmed their structure by NMR spectroscopy. The α-(2→3)-sialyltransferase transferred N-acetylneuraminic acid (Neu5Ac) from cytidine 5′-monophospho-N-acetylneuraminic acid (CMP-Neu5Ac) to the β-anomeric hydroxyl groups of mannose (Man) and α-Manp-(1→6)-Manp, and α-(2→6)-sialyltransferase transferred N-acetylneuraminic acid to the 6-OH groups of the non-reducing end galactose residues in β-Galp-(1→3)-GlcpNAc and β-Galp-(1→6)-GlcpNAc.     

Genome mining approach for the discovery of novel cytochrome P450 biocatalysts

Cytochrome P450 enzymes (P450s) are able to regioselectively and stereoselectively introduce oxygen into organic compounds under mild reaction conditions. These monooxygenases in particular easily catalyze the insertion of oxygen into less reactive carbon–hydrogen bonds. Hence, P450s are of considerable interest as oxidation biocatalysts. To date, although several P450s have been discovered through screening of microorganisms and have been further genetically engineered, the substrate range of these biocatalysts is still limited to fulfill the requirements for a large number of oxidation processes. On the other hand, the recent rapid expansion in the number of reported microbial genome sequences has revealed the presence of an unexpectedly vast number of P450 genes. This large pool of naturally evolved P450s has attracted much attention as a resource for new oxidation biocatalysts. In this review, we focus on aspects of the genome mining approach that are relevant for the discovery of novel P450 biocatalysts. This approach opens up possibilities for exploitation of the catalytic potential of P450s for the preparation of a large choice of oxidation biocatalysts with a variety of substrate specificities.     

Efficient soluble protein production on transgenic silkworms expressing cytoplasmic chaperones

Baculovirus expression systems (BES) are widely used for recombinant protein production in lepidopteran cells or larvae. However, even in BES, the insolubility of recombinant proteins sometimes makes their expression difficult. In this study, to improve the solubility and yield of foreign proteins, we constructed transgenic silkworms using silkworm heat-shock proteins, Hsp70 and Hsp40, or Hsc70 and Hsp90 co-chaperone Hop. In these transgenic silkworms, the expression levels of the transgenes were under the control of a UAS·hsp mini-promoter driven by a Gal4NFkBp65 activator. When the transgenic silkworm with HSP70 and 40 (TGS-HSP70/40) was infected with BmNPV carrying mC3d and Gal4NFkBp65 under the control of baculovirus polyhedrin or p10 promoters, respectively, the soluble fraction of the His- or His·GST-tagged mC3d increased significantly. Similarly, the transgenic silkworm with HSC70 and HOP (TGS-HOP7) was effective for the expression of a steroid hormone receptor, USP2. In conclusion, the His-tagged baculovirus expression system featuring the chaperone effect TGS-HSP70/40 and TGS-HOP7 silkworms is effective for increasing the yields of soluble and functional foreign gene products.     

Synthesis and pharmacological evaluation of 1,1,3-substituted urea derivatives as potent TNF-α production inhibitors

A three substituted urea derivative, SA13353 (compound 1a), exhibited potent inhibitory activity against lipopolysaccharide (LPS)-induced TNF-α production. We focused on the 1,1-substituted moiety (R¹ and R²) of SA13353 and investigated substituent effects of this moiety on LPS-induced TNF-α production by oral administration in rats. The synthesis of the urea derivatives was performed rapidly in a one-pot manner using a manual synthesizer. Several compounds containing hydrophobic substituents at this moiety showed more potent inhibitory activities than SA13353.     

Sleep Deprivation Influences Diurnal Variation of Human Time Perception with Prefrontal Activity Change: A Functional Near-Infrared Spectroscopy Study

Human short-time perception shows diurnal variation. In general, short-time perception fluctuates in parallel with circadian clock parameters, while diurnal variation seems to be modulated by sleep deprivation per se. Functional imaging studies have reported that short-time perception recruits a neural network that includes subcortical structures, as well as cortical areas involving the prefrontal cortex (PFC). It has also been reported that the PFC is vulnerable to sleep deprivation, which has an influence on various cognitive functions. The present study is aimed at elucidating the influence of PFC vulnerability to sleep deprivation on short-time perception, using the optical imaging technique of functional near-infrared spectroscopy. Eighteen participants performed 10-s time production tasks before (at 21:00) and after (at 09:00) experimental nights both in sleep-controlled and sleep-deprived conditions in a 4-day laboratory-based crossover study. Compared to the sleep-controlled condition, one-night sleep deprivation induced a significant reduction in the produced time simultaneous with an increased hemodynamic response in the left PFC at 09:00. These results suggest that activation of the left PFC, which possibly reflects functional compensation under a sleep-deprived condition, is associated with alteration of short-time perception.     

Mgs1 and Rad18/Rad5/Mms2 are required for survival of Saccharomyces cerevisiae mutants with novel temperature/cold sensitive alleles of the DNA polymerase delta subunit, Pol31

Saccharomyces cerevisiae DNA polymerase delta (Pol delta) is a heterotrimeric enzyme consisting of Pol3 (the catalytic subunit), Pol31 and Pol32. New pol31 alleles were constructed by introducing mutations into conserved amino acid residues in all 10 identified regions of Pol31. Six novel temperature-sensitive (ts) or cold-sensitive (cs) alleles, carrying mutations in regions III, IV, VII, VIII or IX, conferred a range of defects in the response to replication stress or DNA damage. Deletion of SGS1, RAD52, SRS2, MRC1 or RAD24 had a deleterious effect only in combination with those pol31 alleles that had a phenotype as single mutants, suggesting a requirement for recombination and checkpoint functions in processing the DNA lesions or structures that form as a consequence of replication with a defective Pol delta. In contrast, deletion of POL32 negatively affected the growth of almost all pol31 mutants, suggesting an important role for all conserved amino acids of Pol31 in maintaining the integrity of Pol delta complex structurally, at least in the absence of the third subunit. Surprisingly, deletions of RAD18 and MGS1 aggravated the temperature sensitivity conferred by most ts or cs alleles and specifically suppressed the hys2-1 and hys2-1-like mutations of POL31. Deletion of RAD5 or MMS2 had an effect on pol31 ts/cs mutants similar to that of RAD18, whereas deletion of RAD30 or REV3 had no effect. We propose that Rad18/Rad5/Mms2 and Mgs1 are required to promote replication when forks are destabilized or stalled due to defects in Pol delta. These data are consistent with the biochemical activity of the human Mgs1 orthologue, which binds and stimulates Pol deltain vitro. We also demonstrate that Mgs1 interacts physically with Pol31 in vivo. Moreover, regions I and VII of Pol31, which are specifically sensitive to high levels of Mgs1 and PCNA, could be sites of interaction.     

Membrane targeting and remodeling through phosphoinositide-binding domains

In mammals, there are seven inositolphospholipids, collectively called phosphoinositides that serve as versatile molecules not only in receptor-mediated signal transduction but also in a variety of cellular events such as cytoskeletal reorganization, membrane trafficking, cell proliferation and cell death. Recent studies have revealed that the latter functions are mediated by direct interactions between phosphoinositides and proteins. Such proteins contain two types of phosphoinositide-binding regions; basic amino acid stretch and globular structural domain. Furthermore, spatially restricted compartment of phosphoinositides and their concentration are finely regulated by a large number of phosphoinositide kinases and -phosphatases, controlling localization-specific metabolism of this simple lipid whose aberrations cause various diseases such as cancer and diabetes.     

Sodium orthovanadate suppresses DNA damage-induced caspase activation and apoptosis by inactivating p53

We previously reported that p42/SETbeta is a substrate for caspase-7 in irradiated MOLT-4 cells, and that treating the cells with sodium orthovanadate (vanadate) inhibits p42/SETbeta's caspase-mediated cleavage. Here, we initially found that the inhibitory effect of vanadate was due to the suppression of caspase activation but not of caspase activity. Further investigations revealed that vanadate suppressed upstream of apoptotic events, such as the loss of mitochondrial membrane potential, the conformational change of Bax, and p53 transactivation, although the accumulation, total phosphorylation, and phosphorylation of six individual sites of p53 were not affected. Importantly, vanadate suppressed p53-dependent apoptosis, but not p53-independent apoptosis. Finally, gel-shift and chromatin immunoprecipitation assays conclusively demonstrated that vanadate inhibits the DNA-binding activity of p53. Vanadate is conventionally used as an inhibitor of protein tyrosine phosphatases (PTPs); however, we recommend that the influence of vanadate not only on PTPs but also on p53 be considered before using it.