5-Halogenated thymidine analogues induce a senescence-like phenomenon in HeLa cells

We tested various thymidine analogues for induction of a senescence-like phenomenon in HeLa cells. CldU, BrdU, and IdU similarly induced the morphology of senescent cells and typical senescence markers. Thymidine analogues other than 5-halogenated forms caused only cell death. BrdU efficiently killed the cells in cooperation with irradiation with light and a brief treatment with Hoechst 33258, but CldU did not at all. 5-Halogenated thymidine analogues were thus shown to be specific inducers of cellular senescence in mammalian cells.     

The synthetic rate of dipeptide catalyzed by organic solvent-stable protease from Pseudomonas aeruginosa PST-01 in the presence of water-soluble organic solvents

The initial synthetic rates of peptide Cbz-Arg-Leu-NH(2) from Cbz-Arg and Leu-NH(2) using PST-01 protease in the presence and absence of organic solvents were investigated under various conditions. The synthetic rates of Cbz-Arg-Leu-NH(2) in the presence of 50% (v/v) methanol, 50% (v/v) N,N-dimethylformamide (DMF) and 60% (v/v) dimethyl sulfoxide (DMSO) were 1.6-, 2.4-, and 5.1-times higher than that in the absence of organic solvent, respectively. The PST-01 protease was not only stable in the presence of organic solvents but also exhibited high reaction rates in the presence of methanol, DMF, and DMSO. When the Cbz-Arg concentration was lower than 60mM or the Leu-NH(2) concentration was lower than 400mM, the initial rates increased lineally with increase in their concentrations. However, the rates did not increase when the Leu-NH(2) concentration was more than 500mM. The optimum temperature and pH of the reaction were 40 degrees C and 7.0, respectively.     

Suppression of GD1alpha ganglioside-mediated tumor metastasis by liposomalized WHW-peptide

GD1alpha ganglioside-replica peptides were recently isolated from a phage-displayed random pentadecapeptide library by assaying for inhibition of adhesion of RAW117-H10 lymphosarcoma cells to hepatic sinusoidal microvessel endothelial (HSE) cells. We show here that the Trp-His-Trp (WHW) peptide was identified as a minimal sequence of the GD1alpha-replica peptide WHWRHRIPLQLAAGR. The addition of WHW peptide-attached liposomes displayed efficient inhibition of liver metastasis of RAW117-H10 cells as well as of GD1alpha-mediated adhesion of RAW117-H10 cells to HSE cells in vitro. These results suggest that engineered liposomes for peptide delivery are applicable to treatment for metastasis.     

Pathogenicity of Hantaan virus in newborn mice: genetic reassortant study demonstrating that a single amino acid change in glycoprotein G1 is related to virulence

Two Hantaan virus strains, clone 1 (cl-1), which is virulent in newborn mice, and its attenuated mutant (mu11E10), were used to examine the pathogenesis of Hantaan virus infection in a mouse model and identify virus factors relating to virulence. After subcutaneous inoculation of newborn BALB/c mice, cl-1 caused fatal disease with high viral multiplication in peripheral organs, but mu11E10 produced nonfatal infection with a low level of virus multiplication. Intracerebral inoculation of either strain caused fatal disease. Histopathological changes in the dead animals were prominent in the brain, indicating that the brain is the target organ and produces the fatal outcome. These results indicate that mu11E10 has a generally less virulent phenotype, and because of decreased multiplication in peripheral tissues, neuroinvasiveness is also decreased. An experiment with genetic reassortant viruses showed that in newborn mice the M segment is the most related to virulence and the L segment is partly related. Sequence comparison detected a single deduced amino acid change (cl-1 Ile to mu11E10 Thr) at amino acid number 515 in glycoprotein G1. One nucleotide change, but no amino acid substitution, was observed in the noncoding region of the L segment. In mouse brain microvascular endothelial cells in vitro, viruses possessing a cl-1-derived M segment grew more rapidly than viruses containing a mu11E10-derived M segment. These results suggest that the single amino acid change in the glycoprotein alters peripheral growth, which affects invasion of the central nervous system in mice.     

Clustering of Human Chromosome Fragments on the Mouse Genome by Chromosome-Mediated Gene Transfer

Pieces of metaphase chromosomes prepared from mouse cells containingneo-tagged human chromosome 7 were transferred to mouse cellswith calcium phosphate to isolate G418-resistant clones. FISHanalysis revealed that the majority of them contained humanDNA at a single site on their genome. These transformants containedSTS markers mapped to various regions of chromosome 7. It isthus suggested that pieces of human chromosomes tend to assembleand integrate on the mouse genome.     

Processed and Unprocessed Red Meat and Risk of Colorectal Cancer: Analysis by Tumor Location and Modification by Time

Although the association between red meat consumption and colorectal cancer (CRC) is well established, the association across subsites of the colon and rectum remains uncertain, as does time of consumption in relation to cancer development. As these relationships are key for understanding the pathogenesis of CRC, they were examined in two large cohorts with repeated dietary measures over time, the Nurses’ Health Study (n = 87,108 women, 1980–2010) and Health Professionals Follow-up Study (n = 47,389 men, 1986–2010). Cox proportional hazards regression models generated hazard ratios (HRs) and 95% confidence intervals (CIs), which were pooled by random-effects meta-analysis. In combined cohorts, there were 2,731 CRC cases (1,151 proximal colon, 816 distal colon, and 589 rectum). In pooled analyses, processed red meat was positively associated with CRC risk (per 1 serving/day increase: HR = 1.15, 95% CI: 1.01–1.32; P for trend 0.03) and particularly with distal colon cancer (per 1 serving/day increase; HR = 1.36; 95% CI: 1.09–1.69; P for trend 0.006). Recent consumption of processed meat (within the past 4 years) was not associated with distal cancer. Unprocessed red meat was inversely associated with risk of distal colon cancer and a weak non-significant positive association between unprocessed red meat and proximal cancer was observed (per 1 serving/day increase: distal HR = 0.75; 95% CI: 0.68–0.82; P for trend <0.001; proximal HR = 1.14, 95% CI: 0.92–1.40; P for trend 0.22). Thus, in these two large cohorts of US health professionals, processed meat intake was positively associated with risk of CRC, particularly distal cancer, with little evidence that higher intake of unprocessed red meat substantially increased risk of CRC. Future studies, particularly those with sufficient sample size to assess associations by subsites across the colon are needed to confirm these findings and elucidate potentially distinct mechanisms underlying the relationship between processed meat and subtypes of unprocessed red meat with CRC.     

Display of both N- and C-terminal target fusion proteins on the Aspergillus oryzae cell surface using a chitin-binding module

A novel cell surface display system in Aspergillus oryzae was established by using a chitin-binding module (CBM) from Saccharomyces cerevisiae as an anchor protein. CBM was fused to the N or C terminus of green fluorescent protein (GFP) and the fusion proteins (GFP-CBM and CBM-GFP) were expressed using A. oryzae as a host. Western blotting and fluorescence microscopy analysis showed that both GFP-CBM and CBM-GFP were successfully expressed on the cell surface. In addition, cell surface display of triacylglycerol lipase from A. oryzae (tglA), while retaining its activity, was also successfully demonstrated using CBM as an anchor protein. The activity of tglA was significantly higher when tglA was fused to the C terminus than N terminus of CBM. Together, these results show that CBM used as a first anchor protein enables the fusion of both the N and/or C terminus of a target protein.     

Selection of DNA aptamers using atomic force microscopy

Atomic force microscopy (AFM) can detect the adhesion or affinity force between a sample surface and cantilever, dynamically. This feature is useful as a method for the selection of aptamers that bind to their targets with very high affinity. Therefore, we propose the Systematic Evolution of Ligands by an EXponential enrichment (SELEX) method using AFM to obtain aptamers that have a strong affinity for target molecules. In this study, thrombin was chosen as the target molecule, and an ‘AFM-SELEX’ cycle was performed. As a result, selected cycles were completed with only three rounds, and many of the obtained aptamers had a higher affinity to thrombin than the conventional thrombin aptamer. Moreover, one type of obtained aptamer had a high affinity to thrombin as well as the anti-thrombin antibody. AFM-SELEX is, therefore, considered to be an available method for the selection of DNA aptamers that have a high affinity for their target molecules.     

Protein–protein interactions and selection: yeast-based approaches that exploit guanine nucleotide-binding protein signaling

For elucidating protein–protein interactions, many methodologies have been developed during the past two decades. For investigation of interactions inside cells under physiological conditions, yeast is an attractive organism with which to quickly screen for hopeful candidates using versatile genetic technologies, and various types of approaches are now available.Among them, a variety of unique systems using the guanine nucleotide-binding protein (G-protein) signaling pathway in yeast have been established to investigate the interactions of proteins for biological study and pharmaceutical research. G-proteins involved in various cellular processes are mainly divided into two groups: small monomeric G-proteins,and heterotrimeric G-proteins. In this minireview, we summarize the basic principles and applications of yeast-based screening systems, using these two types of G-protein, which are typically used for elucidating biological protein interactions but are differentiated from traditional yeast two-hybrid systems.     

Ethanol production from cellulosic materials using cellulase-expressing yeast

We demonstrate direct ethanol fermentation from amorphous cellulose using cellulase-co-expressing yeast. Endoglucanases (EG) and cellobiohydrolases (CBH) from Trichoderma reesei, and β-glucosidases (BGL) from Aspergillus aculeatus were integrated into genomes of the yeast strain Saccharomyces cerevisiae MT8-1. BGL was displayed on the yeast cell surface and both EG and CBH were secreted or displayed on the cell surface. All enzymes were successfully expressed on the cell surface or in culture supernatants in their active forms, and cellulose degradation was increased 3- to 5-fold by co-expressing EG and CBH. Direct ethanol fermentation from 10 g/L phosphoric acid swollen cellulose (PASC) was also carried out using EG-, CBH-, and BGL-co-expressing yeast. The ethanol yield was 2.1 g/L for EG-, CBH-, and BGL-displaying yeast, which was higher than that of EG- and CBH-secreting yeast (1.6 g/L ethanol). Our results show that cell surface display is more suitable for direct ethanol fermentation from cellulose.