Tetrahydrouridine inhibits cell proliferation through cell cycle regulation regardless of cytidine deaminase expression levels

Tetrahydrouridine (THU) is a well characterized and potent inhibitor of cytidine deaminase (CDA). Highly expressed CDA catalyzes and inactivates cytidine analogues, ultimately contributing to increased gemcitabine resistance. Therefore, a combination therapy of THU and gemcitabine is considered to be a potential and promising treatment for tumors with highly expressed CDA. In this study, we found that THU has an alternative mechanism for inhibiting cell growth which is independent of CDA expression. Three different carcinoma cell lines (MIAPaCa-2, H441, and H1299) exhibited decreased cell proliferation after sole administration of THU, while being unaffected by knocking down CDA. To investigate the mechanism of THU-induced cell growth inhibition, cell cycle analysis using flow cytometry was performed. This analysis revealed that THU caused an increased rate of G1-phase occurrence while S-phase occurrence was diminished. Similarly, Ki-67 staining further supported that THU reduces cell proliferation. We also found that THU regulates cell cycle progression at the G1/S checkpoint by suppressing E2F1. As a result, a combination regimen of THU and gemcitabine might be a more effective therapy than previously believed for pancreatic carcinoma since THU works as a CDA inhibitor, as well as an inhibitor of cell growth in some types of pancreatic carcinoma cells.     

HIV-1 Suppressive Sequences Are Modulated by Rev Transport of Unspliced RNA and Are Required for Efficient HIV-1 Production

The unspliced human immunodeficiency virus type 1 (HIV-1) RNAs are translated as Gag and Gag-Pol polyproteins or packaged as genomes into viral particles. Efficient translation is necessary before the transition to produce infective virions. The viral protein Rev exports all intron-containing viral RNAs; however, it also appears to enhance translation. Cellular microRNAs target cellular and viral mRNAs to silence their translation and enrich them at discrete cytoplasmic loci that overlap with the putative interim site of Gag and the genome. Here, we analyzed how Rev-mediated transport and the splicing status of the mRNA influenced the silencing status imposed by microRNA. Through identification and mutational analysis of the silencing sites in the HIV-1 genome, we elucidated the effect of silencing on virus production. Renilla luciferase mRNA, which contains a let-7 targeting site in its 3′ untranslated region, was mediated when it was transported by Rev and not spliced, but it was either not mediated when it was spliced even in a partial way or it was Rev-independent. The silencing sites in the pol and env-nef regions of the HIV-1 genome, which were repressed in T cells and other cell lines, were Drosha-dependent and could also be modulated by Rev in an unspliced state. Mutant viruses that contained genomic mutations that reflect alterations to show more derepressive effects in the 3′ untranslated region of the Renilla luciferase gene replicated more slowly than wild-type virus. These findings yield insights into the HIV-1 silencing sites that might allow the genome to avoid translational machinery and that might be utilized in coordinating virus production during initial virus replication. However, the function of Rev to modulate the silencing sites of unspliced RNAs would be advantageous for the efficient translation that is required to support protein production prior to viral packaging and particle production.     

Investigation of oral opportunistic pathogens in independent living elderly Japanese

doi: 10.1111/j.1741‐2358.2010.00449.x
Investigation of oral opportunistic pathogens in independent living elderly Japanese Objective: Pneumonia is reported to be associated with high morbidity in elderly and compromised individuals, with poor oral health demonstrated to be a significant risk factor for pneumonia. Several opportunistic pathogens, such as Streptococcus pneumoniae, Klebsiella pneumoniae, Escherichia coli and Staphylococcus aureus, have been detected in patients with pneumonia. We investigated the prevalence of opportunistic pathogens in the oral cavity of healthy independent living Japanese elderly subjects and analysed factors related to harbouring those pathogens. Materials and methods: We studied 265 subjects, each of whom received a dental examination, during which specimens were collected with a tongue swab and examined for the presence of 10 oral opportunistic pathogens using single or multiple selective media. Furthermore, the presence of occult blood in saliva was examined using test paper strips. Results: Oral opportunistic pathogens were detected in 13.6% of the subjects. Those positive for occult blood in saliva had a significantly higher rate of harbouring the pathogens (p < 0.05). In addition, age was a significant factor for the presence of pathogenic microbes in the oral cavity (p = 0.033). Conclusion: Positive findings of occult blood in saliva and older age are suggested to be significant factors for harbouring opportunistic pathogens in the oral cavity.     

Isolation of a New Xanthylium-Related Pigment from Adzuki Beans Vigna angularis

A new type of flavonoid was isolated from an adzuki bean-water extract by various chromatographic techniques. The chemical structure was determined by ultraviolet-visible (UV-Vis) spectroscopy, mass spectrometry (MS), and various nuclear magnetic resonance (NMR) experiments, and it was found to be unique in that the xanthylium skeleton was present in this molecule.     

Assembly of synthetic locked phycocyanobilin derivatives with phytochrome in vitro and in vivo in Ceratodon purpureus and Arabidopsis

Phytochromes are photoreceptors with a bilin chromophore in which light triggers the conversion between the red light-absorbing form, Pr, and the far-red-light-absorbing form, Pfr. Here we performed in vitro and in vivo studies using locked phycocyanobilin derivatives, termed 15 Z anti phycocyanobilin (15ZaPCB) and 15 E anti PCB (15EaPCB). Recombinant bacterial and plant phytochromes incorporated either chromophore in a noncovalent or covalent manner. All adducts were photoinactive. The absorption spectra of the 15ZaPCB and 15EaPCB adducts were comparable with those of the Pr and Pfr form, respectively. Feeding of 15EaPCB, but not 15ZaPCB, to protonemal filaments of the moss Ceratodon purpureus resulted in increased chlorophyll accumulation, modulation of gravitropism, and induction of side branches in darkness. The effect of locked chromophores on phytochrome responses, such as induction of seed germination, inhibition of hypocotyl elongation, induction of cotyledon opening, randomization of gravitropism, and gene regulation, were investigated in wild-type Arabidopsis thaliana and the phytochrome-chromophore-deficient long hypocotyl mutant hy1. All phytochrome responses were induced in darkness by 15EaPCB, not only in the mutant but also in the wild type. These studies show that the 15Ea stereochemistry of the chromophore results in the formation of active Pfr-like phytochrome in the cell. Locked chromophores might be used to investigate phytochrome responses in many other organisms without the need to isolate mutants. The induction of phytochrome responses in the hy1 mutant by 15EaPCB were however less efficient than by red light irradiation given to biliverdin-rescued seeds or seedlings.     

Effects of interleukin-8 on estradiol and progesterone production by bovine granulosa cells from large follicles and progesterone production by luteinizing granulosa cells in culture

Interleukin 8 (IL-8) is a chemoattractant involved in the recruitment and activation of neutrophils and is associated with the ovulate process. We examined the possible role of IL-8 in steroid production by bovine granulosa cells before and after ovulation. The concentration of IL-8 in the follicular fluid of estrogen-active dominant (EAD) and pre-ovulatory follicles (POF) was higher than that of small follicles (SF). CXCR1 mRNA expression was higher in the granulosa cells of EAD and POF than that of SF. In contrast, CXCR2 mRNA expression was lower in granulosa cells of EAD and POF than in SF. IL-8 inhibited estradiol (E2) production in follicle-stimulating hormone (FSH)-treated granulosa cells at 48 h of culture. IL-8 also suppressed CYP19A1 mRNA expression in FSH-treated granulosa cells. IL-8 stimulated progesterone (P4) production in luteinizing hormone (LH)-treated granulosa cells at 48 h of culture. Although IL-8 did not alter the expression of genes associated with P4 production, it induced StAR protein expression in LH-treated granulosa cells. The expression of CXCR1 mRNA in corpus luteum (CL) did not change during the luteal phase. In contrast, the expression of CXCR2 mRNA in middle CL was significantly higher than in early and regression CL during the luteal phase. In luteinizing granulosa cells, an in vitro model of granulosa cell luteinization, CXCR2 mRNA expression was downregulated, whereas CXCR1 mRNA expression was unchanged. IL-8 also stimulated P4 production in luteinizing granulosa cells. These data provide evidence that IL-8 functions not only as a chemokine, but also act as a regulator of steroid synthesis in granulosa cells to promote luteinization after ovulation.     

Mitogen-activated protein kinases-dependent induction of hepatocyte growth factor production in human dermal fibroblasts by the antibiotic polymyxin B

Hepatocyte growth factor (HGF) stimulates migration and proliferation of keratinocytes and has been suggested to be involved in wound healing. The cationic antibiotic polymyxin B (PMB) is commonly used as a topical antibiotic for wound care. If PMB possesses an HGF-inducing activity, the antibiotic is potentially beneficial for wound healing in addition to minimizing chances of infection. In this study, we found that PMB markedly induced HGF production from various types of cells including human dermal fibroblasts. Its effect was stronger than the effects of epidermal growth factor and cholera toxin and was comparable to the effect of 8-bromo-cAMP. Among the polymyxin family and polymyxin derivatives, colistin was also effective, whereas colistin methanesulfonate had only a marginal effect and PMB nonapeptide was ineffective. The stimulatory effect of PMB was accompanied by upregulation of HGF gene expression. Increase in phosphorylation of extracellular signal-regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK) was observed from 0.25h to 6h after the addition of PMB, while increase in phosphorylation of p38 mitogen-activated protein kinase (MAPK) was detected from 24h to 60h after PMB addition. The MAPK/ERK kinase inhibitor PD98059, the JNK inhibitor SP600125 and the p38 MAPK inhibitor SB203580 all potently inhibited PMB-induced HGF production. Lastly, proliferation of human dermal fibroblasts was significantly stimulated by PMB. These results indicate that PMB-induced HGF production and proliferation of human dermal fibroblasts and suggest that activation of MAPKs is involved in the induction of HGF production.     

Gene network inference and visualization tools for biologists: application to new human transcriptome datasets

Gene regulatory networks inferred from RNA abundance data have generated significant interest, but despite this, gene network approaches are used infrequently and often require input from bioinformaticians. We have assembled a suite of tools for analysing regulatory networks, and we illustrate their use with microarray datasets generated in human endothelial cells. We infer a range of regulatory networks, and based on this analysis discuss the strengths and limitations of network inference from RNA abundance data. We welcome contact from researchers interested in using our inference and visualization tools to answer biological questions.     

Binding of hairpin pyrrole and imidazole polyamides to DNA: relationship between torsion angle and association rate constants

N-methylpyrrole (Py)-N-methylimidazole (Im) polyamides are small organic molecules that bind to DNA with sequence specificity and can be used as synthetic DNA-binding ligands. In this study, five hairpin eight-ring Py–Im polyamides 1–5 with different number of Im rings were synthesized, and their binding behaviour was investigated with surface plasmon resonance assay. It was found that association rate (k_a) of the Py–Im polyamides with their target DNA decreased with the number of Im in the Py–Im polyamides. The structures of four-ring Py–Im polyamides derived from density functional theory revealed that the dihedral angle of the Py amide carbonyl is 14∼18°, whereas that of the Im is significantly smaller. As the minor groove of DNA has a helical structure, planar Py–Im polyamides need to change their conformation to fit it upon binding to the minor groove. The data explain that an increase in planarity of Py–Im polyamide induced by the incorporation of Im reduces the association rate of Py–Im polyamides. This fundamental knowledge of the binding of Py–Im polyamides to DNA will facilitate the design of hairpin Py–Im polyamides as synthetic DNA-binding modules.