Soluble branched beta-(1,4)glucans from Acetobacter species show strong activities to induce interleukin-12 in vitro and inhibit T-helper 2 cellular response with immunoglobulin E production in vivo

An extracellular polysaccharide, AC-1, produced by Acetobacter polysaccharogenes is composed of beta-(1,4)glucan with branches of glucosyl residues. We found that AC-1 showed a strong activity to induce production of interleukin-12 P40 and tumor necrosis factor-alpha by macrophage cell lines in vitro. Cellulase treatment completely abolished the activity of AC-1 to induce tumor necrosis factor-alpha production by macrophages, whereas treatment of AC-1 with polymyxin B or proteinase did not affect the activity. Results of experiments using toll-like receptor (TLR) 4-deficient mice and TLR4-transfected human cell line indicated that TLR4 is involved in pattern recognition of AC-1. In vivo administration of AC-1 significantly reduced the serum levels of ovalbumin (OVA)-specific IgE and interleukin-4 production by T cells in response to OVA in mice immunized with OVA. AC-1, a soluble branched beta-(1,4)glucan may be useful in prevention and treatment of allergic disorders With IgE production.     

p57KIP2 modulates stress-activated signaling by inhibiting c-Jun NH2-terminal kinase/stress-activated protein Kinase

p57KIP2, a member of the Cip/Kip family of enzymes that inhibit several cyclin-dependent kinases, plays a role in many biological events including cell proliferation, differentiation, apoptosis, tumorigenesis and developmental changes. The human p57KIP2 gene is located in chromosome 11p15.5, a region implicated in sporadic cancers and Beckwith-Wiedemann syndrome. We here report that p57KIP2 physically interacts with and inhibits c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK). The carboxyl-terminal QT domain of p57KIP2 is crucial for the inhibition of JNK/SAPK. Overexpressed p57KIP2 also suppressed UV- and MEKK1-induced apoptotic cell death. p57KIP2 expression during C2C12 myoblast differentiation resulted in repression of the JNK activity stimulated by UV light. Furthermore, UV-stimulated JNK1 activity was higher in mouse embryonic fibroblasts derived from p57-/- mice than in the cells from wild-type mice. Taken together, these findings suggest that p57KIP2 modulates stress-activated signaling by functioning as an endogenous inhibitor of JNK/SAPK.     

Picomolar analysis of flavins in biological samples by dynamic pH junction-sweeping capillary electrophoresis with laser-induced fluorescence detection

Sensitive capillary electrophoresis (CE) methods are required for emerging areas of biochemical research such as the metabolome. In this report, dynamic pH junction-sweeping CE with laser-induced fluorescence (LIF) detection is applied as a robust single method to analyze trace amounts of three flavin derivatives, riboflavin, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD), from several types of samples including bacterial cell extracts, recombinant protein, and biological fluids. Submicromolar amounts of flavin coenzymes were measured directly from formic acid cell extracts of Bacillus subtilis. Significant differences in flavin concentration were measured in cell extracts derived from either glucose or malate as the carbon source in the culture media. Quantitative assessment of FAD and FMN content from selected flavoenzymes was demonstrated after heat denaturation to release noncovalently bound coenzymes and deproteinization. This method was also applied to the analysis of free flavins in pooled human plasma and urine without the need for laborious off-line sample preconcentration. Picomolar detectability of flavins by CE-LIF detection was realized with on-line preconcentration (up to 15% capillary length used for injection) by dynamic pH junction-sweeping, resulting in a limit of detection (S/N = 3) of about 4.0 pM for FAD and FMN. This represents over a 60-fold improvement in concentration sensitivity compared to those of previous techniques using conventional injections. The method was validated in terms of reproducibility, sensitivity, linearity, and specificity. Flavin analysis by dynamic pH junction-sweeping CE-LIF offers a simple, yet sensitive way to analyze trace levels of flavin metabolites from complex biological samples.     

Kinetic mechanism of Staphylococcus aureus sortase SrtA

Staphylococcus aureus sortase (SrtA) is a thiol transpeptidase. The enzyme catalyzes a cell wall sorting reaction in which a surface protein with a sorting signal containing a LPXTG motif is cleaved between the threonine and glycine residues. The resulting threonine carboxyl end of this protein is covalently attached to a pentaglycine cross-bridge of peptidoglycan. The transpeptidase activity of sortase has been demonstrated in in vitro reactions between a LPETG-containing peptide and triglycine. When a nucleophile is not available, sortase slowly hydrolyzes the LPETG peptide at the same site. In this study, we have analyzed the steady-state kinetics of these two types of reactions catalyzed by sortase. The kinetic results fully support a ping-pong mechanism in which a common acyl-enzyme intermediate is formed in transpeptidation and hydrolysis. However, each reaction has a distinct rate-limiting step: the formation of the acyl-enzyme in transpeptidation and the hydrolysis of the same acyl-enzyme in the hydrolysis reaction. We have also demonstrated in this study that the nucleophile binding site of S. aureus sortase SrtA is specific for diglycine. While S1' and S2' sites of the enzyme both prefer a glycine residue, the S1' site is exclusively selective for glycine. Lengthening of the polyglycine acceptor nucleophile beyond diglycine does not further enhance the binding and catalysis.     

Characterization of the mouse gene for the U-box-type ubiquitin ligase UFD2a

UFD2a is a mammalian homolog of Saccharomyces cerevisiae Ufd2, originally described as an E4 ubiquitination factor. UFD2a belongs to the U-box family of ubiquitin ligases (E3s) and likely functions as both an E3 and E4. We have isolated and characterized the mouse gene (Ube4b) for UFD2a. A full-length (approximately 5700 bp) Ube4b cDNA was isolated and the corresponding gene spans >100 kb, comprising 27 exons. Luciferase reporter gene analysis of the 5(') flanking region of Ube4b revealed that nucleotides -1018 to -943 (relative to the translation initiation site) possess promoter activity. This functional sequence contains two putative Sp1 binding sites but not a TATA box. Immunoblot and immunohistochemical analyses revealed that UFD2a is expressed predominantly in the neuronal tissues. We also show that UFD2a interacts with VCP (a AAA-family ATPase) that is thought to mediate protein folding. These data implicate UFD2a in the degradation of neuronal proteins by the ubiquitin-proteasome pathway.     

Positive and negative roles of homologous recombination in the maintenance of genome stability in Saccharomyces cerevisiae

In previous studies of the loss of heterozygosity (LOH), we analyzed a hemizygous URA3 marker on chromosome III in S. cerevisiae and showed that homologous recombination is involved in processes that lead to LOH in multiple ways, including allelic recombination, chromosome size alterations, and chromosome loss. To investigate the role of homologous recombination more precisely, we examined LOH events in rad50 Delta, rad51 Delta, rad52 Delta, rad50 Delta rad52 Delta, and rad51 Delta rad52 Delta mutants. As compared to Rad(+) cells, the frequency of LOH was significantly increased in all mutants, and most events were chromosome loss. Other LOH events were differentially affected in each mutant: the frequencies of all types of recombination were decreased in rad52 mutants and enhanced in rad50 mutants. The rad51 mutation increased the frequency of ectopic but not allelic recombination. Both the rad52 and rad51 mutations increased the frequency of intragenic point mutations approximately 25-fold, suggesting that alternative mutagenic pathways partially substitute for homologous recombination. Overall, these results indicate that all of the genes are required for chromosome maintenance and that they most likely function in homologous recombination between sister chromatids. In contrast, other recombination pathways can occur at a substantial level even in the absence of one of the genes and contribute to generating various chromosome rearrangements.     

Effects of recombinant cholera toxin B subunit on IL-1beta production by macrophages in vitro

Recombinant cholera toxin B subunit (rCTB) is a safe and potent mucosal adjuvant. As a clue to the mechanism of the adjuvant effect of rCTB, the profile of cytokines secreted in vitro by the mouse peritoneal macrophage (Mphi) treated with rCTB was examined. IL-1beta secretion, intracellular production, and expression of its mRNA of LPS-stimulated Mphi was greatly enhanced by treatment with rCTB. IL-1beta production in response to other microbial stimulators, such as Pansorbin, Sansorbin, insoluble peptidoglycan, and Taxol, was also potentiated by rCTB. Mphi pretreated with rCTB before 24 hr could maintain the ability to produce a high level of IL-1beta, suggesting that this ability may be involved in the adjuvant activity of rCTB on Mphi stimulation. The possibility of close association between rCTB and signal transduction of a Toll-like receptor family in Mphi is discussed.     

Role of internalization in the pathogenicity of Shiga toxin-producing Escherichia coli infection in a gnotobiotic murine model

We investigated the role of bacterial internalization in the killing caused by Shiga toxin-producing Escherichia coli (STEC) infection using a gnotobiotic murine model. A high number of internalized STEC was found in the colonic epithelial cells of STEC-infected mice by both an ex vivo assay and transmission electron microscopy. Most of these mice were killed within 10 days after infection. However, the implantation of lactic acid bacteria in such mice before infection markedly decreased the number of internalized STECs and also completely protected these hosts from killing by a STEC infection. The inhibition of such internalization by immunoglobulin also prevented the hosts from being killed. The Shiga toxin levels in these hosts indicated an inhibition of the penetration of Shiga toxins produced in the colon to the underlying tissue. These results suggested that the internalization plays an important role in the pathogenicity caused by STEC infection in a gnotobiotic murine model.