Green tea (-)-epigallocatechin-3-gallate inhibits HGF-induced progression in oral cavity cancer through suppression of HGF/c-Met

Hepatocyte growth factor (HGF) and c-Met have recently attracted a great deal of attention as prognostic indicators of patient outcome, and they are important in the control of tumor growth and invasion. Epigallocatechin-3-gallate (EGCG) has been shown to modulate multiple signal pathways in a manner that controls the unwanted proliferation and invasion of cells, thereby imparting cancer chemopreventive and therapeutic effects. In this study, we investigated the effects of EGCG in inhibiting HGF-induced tumor growth and invasion of oral cancer in vitro and in vivo. We examined the effects of EGCG on HGF-induced cell proliferation, migration, invasion, induction of apoptosis and modulation of HGF/c-Met signaling pathway in the KB oral cancer cell line. We investigated the antitumor effect and inhibition of c-Met expression by EGCG in a syngeneic mouse model (C3H/HeJ mice, SCC VII/SF cell line). HGF promoted cell proliferation, migration, invasion and induction of MMP (matrix metalloproteinase)-2 and MMP-9 in KB cells. EGCG significantly inhibited HGF-induced phosphorylation of Met and cell growth, invasion and expression of MMP-2 and MMP-9. EGCG blocked HGF-induced phosphorylation of c-Met and that of the downstream kinases AKT and ERK, and inhibition of p-AKT and p-ERK by EGCG was associated with marked increases in the phosphorylation of p38, JNK, cleaved caspase-3 and poly-ADP-ribose polymerase. In C3H/HeJ syngeneic mice, as an in vivo model, tumor growth was suppressed and apoptosis was increased by EGCG. Our results suggest that EGCG may be a potential therapeutic agent to inhibit HGF-induced tumor growth and invasion in oral cancer.     

A one-step electrochemical method for DNA detection that utilizes a peroxidase-mimicking DNAzyme amplified through PCR of target DNA

A novel one-step electrochemical method for DNA detection is described. The procedure utilizes a reaction catalyzed by a peroxidase-mimicking DNAzyme to produce a product, which forms an insoluble precipitation layer on the surface of an electrode. A rationally designed forward primer, conjugated with a peroxidase DNAzyme complementary sequence at its 5′-end, is used for PCR amplification of target DNA. As a result, the DNAzyme sequence is produced by amplification only when the target DNA is present in the sample. The PCR product is then subjected to the precipitation reaction on the electrode surface using an electrolyte assay buffer containing 4-chloronaphthol, hydrogen peroxide, ferrocenemethanol, hemin, and 5′-lambdaexonuclease. Finally, analysis is carried out using Faradaic impedance spectroscopy. The impedance value was found to greatly increase when target DNA is present owing to the formation of a precipitation layer on the electrode surface caused by the catalytic action of the DNAzyme. In contrast, no impedance increase is observed when a control sample not containing target DNA is utilized. By employing this strategy, target DNA from Chlamydia trachomatis was reliably detected within a 10 min period following precipitation without the need for complicated secondary procedures. This effort has led to the development of a highly convenient electrochemical one-step method for DNA detection that utilizes a peroxidase-mimicking DNAzyme, which is specifically designed to undergo amplification during PCR of target DNA.     

Electrical immunosensor based on a submicron-gap interdigitated electrode and gold enhancement

We demonstrated that the detection of human interleukin 5 (IL5) with a higher sensitivity than the enzyme-linked immunosorbent assay (ELISA) was possible using mass-producible submicron-gap interdigitated electrodes (IDEs) combined with signal amplification by a gold nanoparticle (AuNP) and gold enhancement. IDEs, facing comb-shape electrodes, can act as simple and miniaturized devices for immunoassay. An IDE with a gap size of 400nm was fabricated by a stepper photolithography process and was applied for the immunoassay of human IL5. A biotinylated anti-human IL5 was immobilized on the streptavidin-modified IDE, and biotin-bovine serum albumin (BSA) and BSA were added sequentially to reduce non-specific binding between the streptavidin-immobilized IDE surface and other proteins. The immunoassay procedure included three main steps: the reaction of human IL5 to form antigen-antibody complexes, the binding of AuNP conjugation with an antibody against human IL5 for the sandwich immunoassay, and gold enhancement for electrical signal amplification. The measurement of electrical current at each step showed that the gold enhancement step was very critical in detection of the concentration of human IL5. Analysis by scanning electron microscope (SEM) showed that close to 1μm particles were formed from 10nm AuNP by the gold enhancement reaction using gold ions and hydroxylamine. Under optimized conditions, human IL5 could be analyzed at 1pgmL(-1) with a wide dynamic range (from 10(-3) to 100ngmL(-1) concentrations).     

Fluorescent europium-modified polymer nanoparticles for rapid and sensitive anthrax sensors

Novel fluorescent polyacrylonitrile nanoparticles were synthesized by microemulsion polymerization and Schiff base modification. By further modification with europium, the polyacrylonitrile nanoparticles could be used as a highly sensitive and rapid sensor for Bacillus anthracis spore detection in aqueous solution. The europium-modified polyacrylonitrile nanoparticles were readily combined with dipicolinic acid as a unique biomarker of B. anthracis, leading to high fluorescence emission. These nanoparticles enabled ratiometric detection without instrument-specific calibration due to the internal fluorescence reference. Additionally, the europium-modified polyacrylonitrile nanoparticle sensors exhibited a remarkable limit of detection (10pM) for dipicolinic acid and outstanding selectivity (160×) over aromatic ligands in aqueous solution. The ultrafine nanoparticle sensor showed a high capability for detecting anthrax due to the increased surface area-to-volume ratio and enhanced dispersibility.     

An ABCG/WBC-type ABC transporter is essential for transport of sporopollenin precursors for exine formation in developing pollen

The exine of the pollen wall shows an intricate pattern, primarily comprising sporopollenin, a polymer of fatty acids and phenolic compounds. A series of enzymes synthesize sporopollenin precursors in tapetal cells, and the precursors are transported from the tapetum to the pollen surface. However, the mechanisms underlying the transport of sporopollenin precursors remain elusive. Here, we provide evidence that strongly suggests that the Arabidopsis ABC transporter ABCG26/WBC27 is involved in the transport of sporopollenin precursors. Two independent mutations at ABCG26 coding region caused drastic decrease in seed production. This defect was complemented by expression of ABCG26 driven by its native promoter. The severely reduced fertility of the abcg26 mutants was caused by a failure to produce mature pollen, observed initially as a defect in pollen-wall development. The reticulate pattern of the exine of wild-type microspores was absent in abcg26 microspores at the vacuolate stage, and the vast majority of the mutant pollen degenerated thereafter. ABCG26 was expressed specifically in tapetal cells at the early vacuolate stage of pollen development. It showed high co-expression with genes encoding enzymes required for sporopollenin precursor synthesis, i.e. CYP704B1, ACOS5, MS2 and CYP703A2. Similar to two other mutants with defects in pollen-wall deposition, abcg26 tapetal cells accumulated numerous vesicles and granules. Taken together, these results suggest that ABCG26 plays a crucial role in the transfer of sporopollenin lipid precursors from tapetal cells to anther locules, facilitating exine formation on the pollen surface.     

Photochemical characterization of phytochrome missense mutants

Phytochromes are photoreceptors that regulate many aspects of plant growth and development in response to red/far-red light signals from the environment. In this study, we analyzed chromophore ligation and photochromism of missense phytochrome mutants in the Per-Arnt-Sim (PAS)-related domain (PRD). Among the 14 mutants analyzed, the Gly768Asp mutant of Avena phytochrome A showed aberrant photochromism and dark reversion, suggesting that amino acid residues in the C-terminal domain affect the photochemical properties of the photosensory N-terminal domain.     

Emerging roles of DNA-PK besides DNA repair

The DNA-dependent protein kinase (DNA-PK) is a DNA-activated serine/threonine protein kinase, and abundantly expressed in almost all mammalian cells. The roles of DNA-PK in DNA-damage repair pathways, including non-homologous end-joining (NHEJ) repair and homologous recombinant (HR) repair, have been studied intensively. However, the high levels of DNA-PK in human cells are somewhat paradoxical in that it does not impart any increased ability to repair DNA damage. If DNA-PK essentially exceeds the demand for DNA damage repair, why do human cells universally express such high levels of this huge complex? DNA-PK has been recently reported to be involved in metabolic gene regulation in response to feeding/insulin stimulation; our studies have also suggested a role of DNA-PK in the regulation of the homeostasis of cell proliferation. These novel findings expand our horizons about the importance of DNA-PK.     

A genetic algorithm for the optimization of the thermoinduction protocol for high-level production of recombinant human-like collagen from Escherichia coli

Production of recombinant human-like collagen (RHLC) by thermoinduction of recombinant Escherichia coli BL 21 during high cell density cultivation was investigated in a 30 L bioreactor. The effects of induction temperature (T), pH, and carbon-to-nitrogen molar ratio of the nutrient medium (C/N) were examined. The optimal thermoinduction protocol for RHLC production was determined by using a model coupling genetic algorithm and artificial neural networks. The optimal operating conditions were as follows: maintenance of induction temperature at 42°C for 3 H and then at 39.4°C until the end, induction pH at 7.03, and C/N at 4.8 (mol/mol). The theoretical maximum concentration of RHLC was 12.5 g/L, whereas the experimental value was 12.1 g/L under the optimal induction conditions.