Emodin Induces Apoptotic Death in Murine Myelomonocytic Leukemia WEHI-3 Cells In Vitro

Emodin is one of major compounds in rhubarb (Rheum palmatum L.), a plant used as herbal medicine in Chinese population. Although many reports have shown that emodin exhibits anticancer activity in many tumor cell types, there is no available information addressing emodin-affected apoptotic responses in the murine leukemia cell line (WEHI-3) and modulation of the immune response in leukemia mice. We investigated that emodin induced cytotoxic effects in vitro and affected WEHI-3 cells in vivo. This study showed that emodin decreased viability and induced DNA fragmentation in WEHI-3 cells. Cells after exposure to emodin for 24 h have shown chromatin condensation and DNA damage. Emodin stimulated the productions of ROS and Ca2+ and reduced the level of ΔΨm by flow cytometry. Our results from Western blotting suggest that emodin triggered apoptosis of WEHI-3 cells through the endoplasmic reticulum (ER) stress, caspase cascade-dependent and -independent mitochondrial pathways. In in vivo study, emodin enhanced the levels of B cells and monocytes, and it also reduced the weights of liver and spleen compared with leukemia mice. Emodin promoted phagocytic activity by monocytes and macrophages in comparison to the leukemia mice group. In conclusions, emodin induced apoptotic death in murine leukemia WEHI-3 cells and enhanced phagocytosis in the leukemia animal model

Ciprofloxacin-resistant Salmonella enterica Typhimurium and Choleraesuis from Pigs to Humans, Taiwan

We evaluated the disk susceptibility data of 671 nontyphoid Salmonella isolates collected from different parts of Taiwan from March 2001 to August 2001 and 1,261 nontyphoid Salmonella isolates from the National Taiwan University Hospital from 1996 to 2001. Overall, ciprofloxacn resistance was found in 2.7% (18/671) of all nontyphoid Salmonella isolates, in 1.4% (5/347) of Salmonella enterica serotype Typhimurium and in 7.5% (8/107) in S. enterica serotype Choleraesuis nationwide. MICs of six newer fluoroquinolones were determined for the following isolates: 37 isolates of ciprofloxacin-resistant (human) S. enterica Typhimurium (N = 26) and Choleraesuis (N = 11), 10 isolates of ciprofloxacin-susceptible (MIC <1 μg/mL) (human) isolates of these two serotypes, and 15 swine isolates from S. enterica Choleraesuis (N = 13) and Typhmurium (N = 2) with reduced susceptibility to ciprofloxacin (MIC >0.12 μg/mL). Sequence analysis of the gryA, gyrB, parC, parE, and acrR genes, ciprofloxacin accumulation; and genotypes generated by pulsed-field gel electrophoresis with three restriction enzymes (SpeI, XbaI, and BlnI) were performed. All 26 S. enterica Typhimurium isolates from humans and pigs belonged to genotype I. For S. enterica Choleraesuis isolates, 91% (10/11) of human isolates and 54% (7/13) of swine isolates belonged to genotype B. These two genotypes isolates from humans all exhibited a high-level of resistance to ciprofloxacin (MIC 16–64 μg/mL). They had two-base substitutions in the gyrA gene at codons 83 (Ser83Phe) and 87 (Asp87Gly or Asp87Asn) and in the parC gene at codon 80 (Ser80Arg, Ser80Ile, or Ser84Lys). Our investigation documented that not only did these two S. enterica isolates have a high prevalence of ciprofloxacin resistance nationwide but also that some closely related ciprofloxacin-resistant strains are disseminated from pigs to humans

Investigation of size–dependent cell adhesion on nanostructured interfaces

BACKGROUND: Cells explore the surfaces of materials through membrane-bound receptors, such as the integrins, and use them to interact with extracellular matrix molecules adsorbed on the substrate surfaces, resulting in the formation of focal adhesions. With recent advances in nanotechnology, biosensors and bioelectronics are being fabricated with ever decreasing feature sizes. The performances of these devices depend on how cells interact with nanostructures on the device surfaces. However, the behavior of cells on nanostructures is not yet fully understood. Here we present a systematic study of cell-nanostructure interaction using polymeric nanopillars with various diameters. RESULTS: We first checked the viability of cells grown on nanopillars with diameters ranging from 200 nm to 700 nm. It was observed that when cells were cultured on the nanopillars, the apoptosis rate slightly increased as the size of the nanopillar decreased. We then calculated the average size of the focal adhesions and the cell-spreading area for focal adhesions using confocal microscopy. The size of focal adhesions formed on the nanopillars was found to decrease as the size of the nanopillars decreased, resembling the formations of nascent focal complexes. However, when the size of nanopillars decreased to 200 nm, the size of the focal adhesions increased. Further study revealed that cells interacted very strongly with the nanopillars with a diameter of 200 nm and exerted sufficient forces to bend the nanopillars together, resulting in the formation of larger focal adhesions. CONCLUSIONS: We have developed a simple approach to systematically study cell-substrate interactions on physically well-defined substrates using size-tunable polymeric nanopillars. From this study, we conclude that cells can survive on nanostructures with a slight increase in apoptosis rate and that cells interact very strongly with smaller nanostructures. In contrast to previous observations on flat substrates that cells interacted weakly with softer substrates, we observed strong cell-substrate interactions on the softer nanopillars with smaller diameters. Our results indicate that in addition to substrate rigidity, nanostructure dimensions are additional important physical parameters that can be used to regulate behaviour of cells

Bufalin-Inhibited Migration and Invasion in Human Osteosarcoma U-2 OS Cells Is Carried Out by Suppression of the Matrix Metalloproteinase-2, ERK, and JNK Signaling Pathways

[[abstract]]Bufalin has been shown to exhibit multiple pharmacological activities, including induction of apoptosis in many types of cancer cell lines. Osteosarcoma is a type of cancer which is difficult to treat and the purpose of this study was to investigate the effects of bufalin on the migration and invasion of human osteosarcoma U-2 OS cells. The wound healing assay and Boyden chamber transwell assay were used for examining the migration of U-2 OS cells. Western blotting and gelatin zymography assays were used for theexpression and activities of metalloproteinase (MMP)-2, MMP-7 or MMP-9 levels. Western blotting analysis also was used for measuring the levels of growth factor receptor-bound protein 2 (GRB2), son of sevenless homolog 1 (SOS1), c-Jun N-terminal kinases 1/2 (JNK1/2), extracellular signal-regulated kinase 1/2 (ERK1/2), and p38 in bufalin-treated U-2 OS cells. Bufalin inhibited the cell migration and invasion of U-2 OS cells in vitro. Moreover, bufalin reduced MMP-2 and MMP-9 enzyme activities of U-2 OS cells. Bufalin also suppressed the protein level of MMP-2 and reduced the levels of mitogen-activated protein kinases (MAPKs) such as JNK1/2 and ERK1/2 signals in U-2 OS cells. Our results suggest that signaling pathways for bufalin-inhibited migration and invasion of U-2 OS cells might be mediated through blocking MAPK signaling and resulting in the inhibition of MMP-2. Bufalin could be a useful agent to develop as a novel antitumor agent by virtue of its ability to inhibit tumor cell migration and invasion

[[alternative]]臺灣龍膽細胞懸浮培養之建立及培養條件之探討

[[abstract]]龍膽（Gentianae Radix）首載於神農本草經，列為上品，為常用之苦味健胃消炎藥。近年來由於龍膽藥材市場需求量遠超出其自然繁殖量，使得野生資源瀕臨枯竭。省產優質生藥的開發，藉由細胞懸浮培養技術，進行藥用植物二次代謝物之生產，除具保育作用外，亦有助於臺灣製藥工業競爭力之提升，本研究乃進行臺灣龍膽[Gentiana davidii var. formosana (Hayata) T. N. Ho] 細胞懸浮培養之建立。試驗結果顯示利用含有0.2 mg/L kinetin 及1.0 mg/L NAA之MS (Murashige ＆ Skoog, 1962）基本鹽類培養基培養臺灣龍膽莖段所誘導之癒合組織，經繼代培養在MS基本鹽類添加0.2 mg/L kinetin 、3% sucrose 及pH 4.2~5.2 之液體培養基，於2.33 μE•m-2•s-1光照、25 ± 1°C恆溫及80~100 rev/min 轉速下培養，可得一分散均勻，生長良好之懸浮細胞。本研究所建立之臺灣龍膽懸浮培養細胞，將有助於其二次代謝物龍膽皂啟（gentiopicroside及當藥皂啟（swertiamarin）之生產及高產細胞系之篩選。 Gentiana davidii var. formosana (Hayata) T. N. Ho (Gentianaceae), commonly known as long-dan in Chinese, is a perennial herb indigenous to Taiwan. It is distributed throughout the island, ranging from low to high elevations. The roots, which contain bitter-tasting secoiridoid glucosides, are used in traditional Chinese medicine. It is mainly used in the treatment of gastrointestinal tract diseases. Continuous collection of plant material from natural habitat has led to the depletion of Gentiana population. The purpose of this study was to establish the cell suspension cultures of Gentiana, which could be used for large-scale production of active principles such as gentiopicroside and swertiamarin. Callus was initiated by culturing stem explants of G. davidii var. formosana on Murashige and Skoog’s (1962) basal medium supplemented with 0.2 mg/L 6-furfurylaminopurine (kinetin) and 1.0 mg/L α-naphthaleneacetic acid (NAA). Fast-growing suspension cell cultures were established by subculturing the callus in MS basal medium (pH 4.2-5.2) supplemented with 0.2 mg/L kinetin and 3% sucrose. The cultures were incubated on an orbital shaker (80-100 rev/min) at 25 ± 1°C and low light intensity (2.33 μE•m-2•s-1)

Quantitative Determination of Secoiridoid Glucoside in In vitro Propagated Plant of Gentiana davidii var. formosana by High performance Liquid Chromatography

[[abstract]]A simple protocol for in vitro mass propagation of Gentiana davidii var. formosana (Gentianaceae) has been developed. Multiple shoot development was achieved by culturing the stem node explants on Murashige and Skoog (MS) medium supplemented with 4.44 muM N-6-benzyladenine (BA). The shoots were multiplied by subculturing on MS medium supplemented with 1.07-10,74 muM alpha -naphthaleneacetic acid (NAA) and 8.88 muM BA. Shoots were Footed on MS basal medium supplemented with various auxins. Shoots Footed on growth regulator-free medium were transferred to peat moss:vermiculite mixture and acclimatized in the growth chamber. The contents of gentiopicroside and swertiamarin, the two important secoiridoid glucosides, in different plant material were determined by high performance liquid chromatography (HPLC). The analysis revealed that the content of gentiopicroside and swertiamarin in the aerial and underground parts of G.davidii var, formosana was higher than the marketed crude drug (underground parts of G.scabra) and varied with the age of the plant

Puerarin Acts Through Brain Serotonergic Mechanisms to Induce Thermal Effects.

[[abstract]]The present study was attempted to investigate the effect of puerarin, an isoflavone compound isolated from Pueraria lobata, on both the basal body temperature and pyrogenic fever in unanesthetized, restrained rats. Intraperitoneal administration of puerarin or crude extracts of Pueraria lobata elicited hypothermia. Direct administration of a small amount of puerarin into the lateral cerebral ventricle produced the same extent of hypothermia. Systemic or central administration of puerarin causes a decrease in both colonic temperature and hypothalamic 5-HT efflux in rats. The puerarin-induced hypothermia and decreased 5-HT efflux in the hypothalamus were attenuated by selective depletion of hypothalamic 5-HT produced by intracerebroventricular injection of 5,7-dihydroxytryptamine. Furthermore, the puerarin-induced hypothermia was almost completely abolished by treatment with a 5-HT2A-receptor agonist (DOI or quipazine) or a 5-HT1A-receptor antagonist [(−)-pindolol]. A 5-HT2A-receptor antagonist (ketanserin) or a 5-HT1A-receptor agonist (8-OH-DPAT) had additive effects with puerarin. Intracerebroventricular administration of interleukin-1 caused an increase in both colonic temperature and hypothalamic 5-HT efflux. The interleukin-1-induced hyperthermia and increased 5-HT efflux in the hypothalamus were attenuated by treatment with systemic administration of puerarin. The data indicate that puerarin exerts its hypothermic and antipyretic effects by activating 5-HT1A receptor and/or antagonizing 5-HT2A receptors in the hypothalamus