Facile determination of DNA-binding nuclear factor-kappaB by chemiluminescence detection

A simple, rapid, and sensitive method for the assay of a sequence-specific DNA-binding protein, nuclear factor-kappaB (NF-kappaB), has been developed by using a DNA-detectable chemiluminogenic reagent and a centrifugal filter that distinguishes different molecular sizes. After the formation of a complex between NF-kappaB and DNA, the unbound DNA is separated from the complex by the centrifugal filter. The amount of the bound NF-kappaB is estimated by chemiluminescence detection of the bound DNA. This detection is performed within 2 min at room temperature by the use of a chemiluminogenic reagent, 3',4',5'-trimethoxyphenylglyoxal, which selectively recognizes guanine moiety in oligonucleotides or DNAs. This method does not require any labeled probes or antibodies and can determine a concentration as low as 5 nM of DNA-binding NF-kappaB. The sensitivity is nearly the same as that of other methods such as gel shift assay using fluorescence-labeled probes and enzyme-linked immunosorbent assay. Therefore, the current method provides a convenient tool for surveying various DNA-binding proteins.     

Exercise causes tissue-specific enhancement of endothelin-1 mRNA expression in internal organs

Endothelin-1 (ET-1) is a potent vasoconstrictorpeptide, which also potentiates contractions to norepinephrine in humaninternal mammary and coronary vessels. Exercise causes a redistribution of blood flow, i.e., the increase in working muscles that is partly attributable to a decrease in visceral blood flow. We hypothesized thatexercise causes a tissue-specific increase in ET-1 expression ininternal organs. We studied whether exercise affects expression ofpreproET-1 mRNA in the kidneys and lungs. The rats performed treadmillrunning (0% grade) for 45 min at a speed of 25 m/min. The plasmaconcentrations of ET-1, epinephrine, and norepinephrine were greater inthe exercise rats than in the sedentary control rats. The expression ofpreproET-1 mRNA in the kidneys was markedly higher in the exercise ratsthan in the sedentary control rats, whereas that in the lungs did notdiffer between the two groups. Therefore, the present study provides apossibility that the exercise-induced increase in production of ET-1 inthe kidneys causes vasoconstriction and hence decreases blood flow inthe kidneys through its direct vasoconstrictive action and/orits indirect effect of enhancing vasoconstrictions to norepinephrine.

     

Feedback controls restrain the initiation of Escherichia coli chromosomal replication

In Escherichia coli, initiation of chromosomal replication is activated by a nucleoprotein complex formed primarily between the DnaA protein and oriC (replication origin) DNA. After replicational initiation, this complex has to be inactivated in order to repress the appearance of initiation events until the next scheduled round of initiation. Studies of the mechanisms responsible for this repression have recently revealed direct coupling between these mechanisms and key elements of the replication process, suggesting that feedback-type regulatory loops exist between the factors implicated in initiation and the elements yielded by the replication process. The loading of the ring-shaped beta-subunit of DNA polymerase III onto DNA plays a key role in the inactivation of the DnaA protein. Duplication of oriC DNA results in hemimethylated DNA, which is inert for reinitiation. Titration of large amounts of DnaA protein to a non-oriC locus can repress untimely initiations, and timely duplication of this locus is required for this repression in rapidly growing cells. All these systems functionally complement one another to ensure the maintenance of the interinitiation interval between two normal DNA replication cycles. The mechanisms that link the replication cycle to the progression of the cell cycle are also discussed.     

cDNA sequence and expression of a phosphoenolpyruvate carboxylase gene from soybean

A full-length cDNA encoding a subunit of phosphoenolpyruvate carboxylase (PEPC) was isolated from a developing seed expression library of the C₃ plant Glycine max. The corresponding mRNA is present at similar levels in leaf, stem, root and developing seed. Two potential start codons exist, and the activity of protein initiated from the first such codon could be subject to regulation by protein kinase. Sequence comparison shows a similar upstream start codon in the case of the Ppc2 gene from Mesembryanthemum crystallinum, previously assumed to lack the sequences necessary for phosphorylation. The soybean encoded protein tends to resemble other C₃-type PEPC proteins more closely than those implicated in C₄ or crassulacean acid metabolism.     

Cutting edge: a potent adjuvant effect of ligand to receptor activator of NF-kappa B gene for inducing antigen-specific CD8+ T cell response by DNA and viral vector vaccination

The ligand to receptor activator of NF-kappaB (RANK-L)/RANK interaction has been implicated in CD40 ligand/CD40-independent T cell priming by dendritic cells. In this report, we show that the coadministration of the RANK-L gene with a Trypanosoma cruzi gene markedly enhances the induction of Trypanosoma Ag-specific CD8(+) T cells and improves the DNA vaccine efficacy. A similarly potent adjuvant effect of the RANK-L gene on the induction of Ag-specific CD8(+) T cells was also observed when recombinant influenza virus expressing murine malaria Ag was used as an immunogen. In contrast, the coadministration of the CD40L gene was not effective in these systems. Our results demonstrated, for the first time, the potent immunostimulatory effect of the RANK-L gene to improve the CD8(+) T cell-mediated immunity against infectious agents.     

Proteome analysis of programmed cell death and defense signaling using the rice lesion mimic mutant cdr2

We have previously identified three lesion-mimic mutants, cell death and resistance (cdr), in rice. These mutants induce a series of defense responses, including expression of defense-related genes and high accumulation of phytoalexins, indicating that the cdr mutants are useful materials to study programmed cell death and defense signaling in rice. Here, we carried out a proteome analysis of the cdr2 mutant. Total proteins prepared from the wild type and the cdr2 mutant at three different stages of lesion formation were compared using two-dimensional electrophoresis. We found a total of 37 proteins that were differentially expressed between cdr2 and wild type. Among them, 28 spots were up-regulated and nine were down-regulated in the cdr2 mutant. All the protein spots were identified by mass spectrometric analysis. These differentially regulated proteins included defense-related proteins. In addition, 27 proteins were classified as metabolic enzymes, suggesting that the programmed cell death that occurs in the cdr2 mutant is associated with active metabolic changes. Our study shows that proteome analysis is a useful approach to study programmed cell death and defense signaling in plants.     

A synthesis of 3′,4-́dideoxykanamycin B

3′,4′-Dideoxykanamycin B, the kanamycin B derivative that is active against resistant bacteria, was prepared from kanamycin B viaN-tosylation, 3′,4′-O-sulphonylation, 3′,4′-unsaturation, and hydrogenation. The unsaturated intermediate was obtained from the 3′,4′-di-O-sulphonyl derivatives by the action of sodium iodide in N,N-dimethylformamide; if zinc dust was added in this reaction, aziridine derivatives were formed, Removal of the tosyl group was successfully performed by using sodium in ammonia-ethylamine.     

Repression of sigK intervening (skin) element gene expression by the CI-like protein SknR and effect of SknR depletion on growth of Bacillus subtilis cells

The Bacillus subtilis phage DNA-like sigK intervening (skin) element (48 kb) is excised from the chromosome by DNA rearrangement, and a composite gene, sigK (spoIIIC and spoIVCB), is created on the chromosome during sporulation. In this study, we first focused on the role of sknR (skin repressor), which has homology with the gene encoding the Xre repressor of defective phage PBSX. The depletion of SknR caused overexpression of the region between yqaF and yqaN (the yqaF-yqaN operon) and a growth defect in B. subtilis. Point mutation analysis and an electrophoretic mobility shift assay (EMSA) suggested that SknR functions as a negative regulator of gene expression in the yqaF-yqaN operon of the skin element through direct interaction with operators of 2-fold symmetry located in the intergenic region between sknR and yqaF. Deletion analysis revealed that the lethal effect of depletion of SknR was related to overexpression of yqaH and yqaM, whose products were previously reported to associate with DnaA and DnaC, respectively. Furthermore, overexpression of either yqaH or yqaM caused cell filamentation and abnormal chromosome segregation, which suggested that overproduction of these proteins inhibits DNA replication. Moreover, overexpression of yqaM inhibited the initiation of replication. Taken together, these data demonstrate that the B. subtilis skin element carries lethal genes, which are induced by the depletion of sknR.     

Improvement of isoflavone aglycones production using β-glucosidase secretory produced in recombinant Aspergillus oryzae

β-Glucosidase (BGL1) from Aspergillus oryzae was efficiently produced in recombinant A. oryzae using sodM promoter-mediated expression system. The yield of BGL1 was 960 mg/l in liquid culture, which is 20-fold higher than the yield of BGL1 produced using the yeast Saccharomyces cerevisiae. Recombinant BGL1 converted isoflavone glycosides into isoflavone aglycones more efficiently than β-glucosidase from almond. In addition, BGL1 produced isoflavone aglycones even in the presence of the insoluble form of isoflavone glycosides.     

Colorectal Advanced Neoplasms Occur through Dual Carcinogenesis Pathways in Individuals with Coexisting Serrated Polyps

Background

Individuals with serrated polyps (SP) are at higher risk for synchronous colorectal advanced neoplasms (AN) and cancers. However, it remains unclear whether there is a unique involvement of the serrated pathway and/or the classical adenoma-carcinoma sequence in this setting.

Methods

Colorectal ANs, which include tubular adenomas ≥10 mm, adenomas with villous histology, high-grade intraepithelial neoplasms, and cancers, were collected retrospectively. The groups included ANs with (AN+SP) or without (AN-only) coexisting SPs. Clinicopathological findings were compared between groups. BRAF and KRAS mutations in ANs and SPs, and methylation levels at long interspersed element-1 (LINE-1) in adjacent mucosa were determined by pyrosequencing.

Results

Seventy-five ANs from 40 patients in the AN+SP group, and 179 ANs from 119 patients in the AN-only group were analyzed. There were no significant differences in clinicopathological findings between the two groups, except that intraepithelial neoplasia in the AN+SP group was more likely to be located in the right colon (P = 0.018). BRAF mutations were significantly more frequent in the AN+SP group (P = 0.003), while KRAS mutations showed no significant differences between groups (P = 0.142). The majority of high-grade intraepithelial neoplasms in both groups showed a contiguous component of conventional adenoma. Individuals with large and right-sided SPs had significantly more conventional adenomas compared to those without such SPs (P = 0.027 and P = 0.031, respectively). Adjacent mucosa from individuals with multiple and large SPs showed significantly lower methylation levels at LINE-1 compared to individuals without such associated SPs (P = 0.049 and P = 0.015, respectively).

Conclusion

Our data suggest that both the adenoma-carcinoma sequence and the serrated pathway are operational in individuals with coexisting ANs and SPs. The reduced methylation levels at LINE-1 in the background mucosa suggest the possibility of an underlying ‘field defect’.