Design of multifunctional peptides expressing both antimicrobial activity and shiga toxin neutralization activity

We have designed novel short peptides expressing both antimicrobial and Shiga-toxin (Stx) neutralization activities by combining nuclear localization signal (NLS) peptides (RIRKKLR, PKKKRKV, and PRRRK) tandemly with globotriaoside (Gb3) mimic peptide (WHWTWL). These fusion peptides exhibited excellent antimicrobial activity against both gram-positive and gram-negative bacteria. A peptide WHWTWLRIRKKLR (Trp-His-Trp-Thr-Trp-Leu-Arg-Ile-Arg-Lys-Lys-Leu-Arg), especially, exhibited about 100 times higher activity than the original NLS peptide. SPR analysis demonstrated that the binding of this peptide to both Stxs was strong: K(d) = 6.6 x 10(-6) to Stx-1 and 6.8 x 10(-6) to Stx-2. The in vitro assay against Stx-1 using HeLa cells showed that this peptide increased the survival rate of HeLa cells against the infection of Stx-1. The peptide has been found to maintain high antimicrobial activity, Stx neutralization activity, and no cytotoxicity at its concentration of 7.8-31.3 microg/mL (4.2-16.7 microM). The present peptide design has a prospect of developing potent multifunctional drugs to destroy proteinaceous toxin-producing bacteria and to simultaneously neutralize the toxins released by bacteriolysis.     

First systematic chiral syntheses of two pairs of enantiomers with 3,5-dihydroxyheptenoic acid chain, associated with a potent synthetic statin NK-104

First systematic chiral syntheses of two pairs of enantiomers with 3,5-dihydroxyheptenoic acid chain, associated with a potent synthetic statin NK-104 are reported. A pair of syn diol isomers (NK-104 and its enantiomer) was obtained efficiently by diastereomeric resolution. The synthesis of a pair of anti diol isomers (3-epimer and 5-epimer) was accomplished effectively by the asymmetric aldol reaction followed by anti stereoselective reduction as key steps. Their purity determinations were effected by chiral HPLC analysis.     

Involvement of auxin distribution in root nodule development of <Emphasis Type="Italic">Lotus japonicus</Emphasis>

The symbiosis between legume plants and rhizobia causes the development of new organs, nodules which function as an apparatus for nitrogen fixation. In this study, the roles of auxin in nodule development in Lotus japonicus have been demonstrated using molecular genetic tools and auxin inhibitors. The expression of an auxin-reporter GH3 fused to β-glucuronidase (GUS) was analyzed in L. japonicus roots, and showed a strong signal in the central cylinder of the root, whereas upon rhizobium infection, generation of GUS signal was observed at the dividing outer cortical cells during the first nodule cell divisions. When nodules were developed to maturity, strong GUS staining was detected in vascular tissues of nodules, suggesting distinct auxin involvement in the determinate nodule development. Numbers and the development of nodules were affected by auxin transport inhibitors (1-naphthylphthalamic acid, NPA and triindobenzoic acid, TIBA), and by a newly synthesized auxin antagonist, α-(phenyl ethyl-2-one)-indole-3-acetic acid (PEO-IAA). The common phenotypical alteration by these auxin inhibitors was the inhibition in forming lenticel which is normally developed on the nodule surface from the root outer cortex. The inhibition of lenticel formation was correlated with the inhibition of nodule vascular bundle development. These results indicate that auxin is required for the normal development of determinate nodules in a multidirectional manner.     

Caecal environment of rats fed far East Asian-modelled diets

To clarify the effect of type of foods on the intestinal environment, Far East Asian- (FEA; rich in rice starch, soy protein and soy oil) and Far East Asian marine- (FEAM; rich in rice starch, fish meal, fish oil and brown alga) modelled diets and sucrose, casein and beef tallow-rich (SCB) diet were prepared. After the 2-week administration of diets in rats, caecal organic acids and putrefactive compounds (ammonia, indole, phenol and H₂S, which are regarded as putative risk factors for tumours) were determined. The caecal microbiota was also analyzed using denaturing gradient gel electrophoresis and pyrosequencing with bar-coded primers targeting the bacterial 16S rRNA gene. Levels of n-butyrate, acetate, indole and phenol were high in rats fed FEA. On the other hand, H₂S was clearly suppressed by both FEA and FEAM comparing with SCB. These results suggest that FEAM is preferable to FEA for optimal intestinal environment and host health. Both microbial analyses showed that the diversity of microbiota in the FEAM group was lower than in the other diet groups. Ratio of Firmicutes, Bacteroidetes and Proteobacteria in the SCB group was about 5:4:1. Firmicutes, particularly Lachnospiraceae, was promoted by FEA and FEAM.     

CD166/ALCAM Expression Is Characteristic of Tumorigenicity and Invasive and Migratory Activities of Pancreatic Cancer Cells

Background

CD166, also known as activated leukocyte cell adhesion molecule (ALCAM), is expressed by various cells in several tissues including cancer. However, the role of CD166 in malignant tumors is controversial, especially in pancreatic cancer. This study aimed to clarify the role and significance of CD166 expression in pancreatic cancer.

Methods

We performed immunohistochemistry and flow cytometry to analyze the expression of CD166 in surgical pancreatic tissues and pancreatic cancer cell lines. The differences between isolated CD166+ and CD166- pancreatic cancer cells were analyzed by invasion and migration assays, and in mouse xenograft models. We also performed quantitative RT-PCR and microarray analyses to evaluate the expression levels of CD166 and related genes in cultured cells.

Results

Immunohistochemistry revealed high expression of CD166 in pancreatic cancer tissues (12.2%; 12/98) compared with that in normal pancreas controls (0%; 0/17) (p = 0.0435). Flow cytometry indicated that CD166 was expressed in 33.8–70.2% of cells in surgical pancreatic tissues and 0–99.5% of pancreatic cancer cell lines. Invasion and migration assays demonstrated that CD166- pancreatic cancer cells showed stronger invasive and migratory activities than those of CD166+ cancer cells (p<0.05). On the other hand, CD166+ Panc-1 cells showed a significantly stronger colony formation activity than that of CD166- Panc-1 cells (p<0.05). In vivo analysis revealed that CD166+ cells elicited significantly greater tumor growth than that of CD166- cells (p<0.05) in both subcutaneous and orthotopic mouse tumor models. mRNA expression of the epithelial-mesenchymal transition activator Zeb1 was over-expressed in CD166- cells (p<0.001). Microarray analysis showed that TSPAN8 and BST2 were over-expressed in CD166+ cells, while BMP7 and Col6A1 were over-expressed in CD166- cells.

Conclusions

CD166+ pancreatic cancer cells are strongly tumorigenic, while CD166- pancreatic cancer cells exhibit comparatively stronger invasive and migratory activities. These findings suggest that CD166 expression is related to different functions in pancreatic cancer cells.     

Biphasic activation of two mitogen-activated protein kinases during the cell cycle in mammalian cells.

We studied mitogen-activated protein kinase (MAPK) activities during the cell cycle of Chinese hamster ovary (CHO) cells using site-specific antibodies against extracellular signal-regulated kinase-1, a 44-kDa MAPK (Boulton, T.G., Yancopoulos, G.D., Gregory, J.S., Slauer, C., Moomaw, C., Hsu, J., and Cobb, M.H. (1990) Science 249, 64-67). These antibodies detected two distinct MAPKs (44- and 42-kDa MAPKs) in CHO cells. CHO cells were arrested at metaphase in the M phase by treatment with nocodazole, and activities of MAPKs were analyzed at specific time points after release from arrest. Immune complex kinase assay and renaturation and phosphorylation assay in substrate-containing gel revealed that both 44- and 42-kDa MAPKs had activities in the G1 through S and G2/M phases and were activated biphasically, in the G1 phase and around the M phase. MAPKs were inactivated in metaphase-arrested cells. The amount of MAPKs did not change significantly in the cell cycle. In the G1, S, and G2/M phases, MAPKs were phosphorylated on both tyrosine and threonine residues and dephosphorylated in metaphase-arrested cells. Our data suggest that MAPKs may play some role in the cell cycle other than G0/G1 transition.     

Replacement of intracellular C-terminal domain of GLUT1 glucose transporter with that of GLUT2 increases Vmax and Km of transport activity.

The intracellular C-terminal domain is diverse in size and amino acid sequence among facilitative glucose transporter isoforms. The characteristics of glucose transport are also divergent, and GLUT2 has far higher Km and Vmax values compared with GLUT1. To investigate the role of the intracellular C-terminal domain in glucose transport, we expressed in Chinese hamster ovary cells the mutated GLUT1 protein whose intracellular C-terminal domain was replaced with that of GLUT2 by means of engineering the chimeric cDNA. Cytochalasin B, for which GLUT2 protein has much lower affinity, bound to this chimeric protein in a fashion similar to GLUT1. In contrast, greater transport activity was observed in this chimeric glucose transporter compared with the wild-type GLUT1 at 10 mM 2-deoxy-D-glucose concentration. The kinetic studies on 2-deoxy-D-glucose uptake revealed a 3.8-fold increase in Km and a 4.3-fold increase in Vmax in this chimeric glucose transporter compared with the wild-type GLUT1. Thus, replacement of the intracellular C-terminal domain confers the GLUT2-like property on the glucose transporter. These results strongly suggest that the diversity of intracellular C-terminal domain contributes to the diversity of glucose transport characteristics among isoforms.     

Glucose binding enhances the papain susceptibility of the intracellular loop of the GLUT1 glucose transporter.

Digestion of human GLUT1 protein in erythrocytes with 5 micrograms/ml papain for 5 min yielded several fragments. By using several site-specific antibodies, two of these fragments containing the intracellular loop domain between M6 and M7 were demonstrated to be further digested by a prolonged incubation with papain. The addition of 0.2 M D-glucose enhanced this digestion between M6 and M7 by approximately 3.5-fold, while the addition of 0.2 M D-sorbitol exhibited no effects. These results strongly suggest that D-glucose binding induces the conformational change of the intracellular loop domain between M6 and M7 of GLUT1 protein. Since the homology of the amino acid sequence was low in this intracellular domain among the five facilitative glucose transporter isoforms, this intracellular loop might contribute to the difference in their Km and Vmax values for glucose uptake.