Selection and proliferation of rapid growing cell lines from embryo derived cell cultures of yew tree (Taxus cuspidata Sieb. et Zucc)

Calli were induced from 300,000 embryos isolated from immature to mature stage of seeds collected on late September from 14 elite trees. When the embryos were cultured onto plastic Petri-dish containing 20 mL of modified B5 basal medium supplemented with 3% (w/v) sucrose, 500 mg/L casein hydrolysate, 250 mg/L myo-inositol, 0.5% (w/v) polyvinyl polypyrrolidon (PVPP), 2×MS vitamins, 0.5 mg/L gibberellic acid, and 10 mg/L 2,4-D after 2 weeks of culture, yellowish-white calli were immediately formed on the surfaces of embryos, and subcultured for 4 weeks in same culture medium. Because most of calli maintained for more than 3 months were revealed differences in their colors, surface texture, and growth rate, visual selection was made for first round screening. When the size of visually selected calli larger than 19 mm in their diameter were inoculated, persistent proliferation was observed. Among the plating methods tested for the selection of rapid growing cell lines at single cell and/or small cell aggregate level, 2-layer spread plating revealed as the best for single cell cloning. To enhance cell growth and maintain high rate of viability for long-term culture of yew cells in bioreactor, final cell volume less than 50% in SCV seemed to be the best. Time course study revealed that 30% of inoculum density was suitable for fed batch culture. Among the tested conditional media, the rate of 1∶2 (old medium: fresh medium) was recorded at the best for cell growth.     

Probing the substrate specificity of the intracellular brain platelet-activating factor acetylhydrolase.

Platelet-activating factor acetylhydrolases (PAF-AHs) are unique PLA2s which hydrolyze the sn-2 ester linkage in PAF-like phospholipids with a marked preference for very short acyl chains, typically acetyl. The recent solution of the crystal structure of the alpha(1) catalytic subunit of isoform Ib of bovine brain intracellular PAF-AH at 1.7 A resolution paved the way for a detailed examination of the molecular basis of substrate specificity in this enzyme. The crystal structure suggests that the side chains of Thr103, Leu48 and Leu194 are involved in substrate recognition. Three single site mutants (L48A, T103S and L194A) were overexpressed and their structures were solved to 2.3 A resolution or better by X-ray diffraction methods. Enzyme kinetics showed that, compared with wild-type protein, all three mutants have higher relative activity against phospholipids with sn-2 acyl chains longer than an acetyl. However, for each of the mutants we observed an unexpected and substantial reduction in the V(max) of the reaction. These results are consistent with the model in which residues Leu48, Thr103 and Leu194 indeed contribute to substrate specificity and in addition suggest that the integrity of the specificity pocket is critical for the expression of full catalytic function, thus conferring very high substrate selectivity on the enzyme.     

Morphological Parameters Associated with Ruptured Posterior Communicating Aneurysms

The rupture risk of unruptured intracranial aneurysms is known to be dependent on the size of the aneurysm. However, the association of morphological characteristics with ruptured aneurysms has not been established in a systematic and location specific manner for the most common aneurysm locations. We evaluated posterior communicating artery (PCoA) aneurysms for morphological parameters associated with aneurysm rupture in that location. CT angiograms were evaluated to generate 3-D models of the aneurysms and surrounding vasculature. Univariate and multivariate analyses were performed to evaluate morphological parameters including aneurysm volume, aspect ratio, size ratio, distance to ICA bifurcation, aneurysm angle, vessel angles, flow angles, and vessel-to-vessel angles. From 2005–2012, 148 PCoA aneurysms were treated in a single institution. Preoperative CTAs from 63 patients (40 ruptured, 23 unruptured) were available and analyzed. Multivariate logistic regression revealed that smaller volume (p = 0.011), larger aneurysm neck diameter (0.048), and shorter ICA bifurcation to aneurysm distance (p = 0.005) were the most strongly associated with aneurysm rupture after adjusting for all other clinical and morphological variables. Multivariate subgroup analysis for patients with visualized PCoA demonstrated that larger neck diameter (p = 0.018) and shorter ICA bifurcation to aneurysm distance (p = 0.011) were significantly associated with rupture. Intracerebral hemorrhage was associated with smaller volume, larger maximum height, and smaller aneurysm angle, in addition to lateral projection, male sex, and lack of hypertension. We found that shorter ICA bifurcation to aneurysm distance is significantly associated with PCoA aneurysm rupture. This is a new physically intuitive parameter that can be measured easily and therefore be readily applied in clinical practice to aid in the evaluation of patients with PCoA aneurysms.     

Design,synthesis, and evaluation of curcumin analogues as potential inhibitors of bacterial sialidase

Sialidases are key virulence factors that remove sialic acid from the host cell surface glycan, unmasking receptors that facilitate bacterial adherence and colonisation. In this study, we developed potential agents for treating bacterial infections caused by Streptococcus pneumoniae Nan A that inhibit bacterial sialidase using Turmeric and curcumin analogues. Design, synthesis, and structure analysis relationship (SAR) studies have been also described. Evaluation of the synthesised derivatives demonstrated that compound 5e was the most potent inhibitor of S. pneumoniae sialidase (IC₅₀?=?0.2?±?0.1?µM). This compound exhibited a 3.0-fold improvement in inhibitory activity over that of curcumin and displayed competitive inhibition. These results warrant further studies confirming the antipneumococcal activity 5e and indicated that curcumin derivatives could be potentially used to treat sepsis by bacterial infections.     

Effectiveness of Losartan-Loaded Hyaluronic Acid (HA) Micelles for the Reduction of Advanced Hepatic Fibrosis in C3H/HeN Mice Model

Advanced hepatic fibrosis therapy using drug-delivering nanoparticles is a relatively unexplored area. Angiotensin type 1 (AT1) receptor blockers such as losartan can be delivered to hepatic stellate cells (HSC), blocking their activation and thereby reducing fibrosis progression in the liver. In our study, we analyzed the possibility of utilizing drug-loaded vehicles such as hyaluronic acid (HA) micelles carrying losartan to attenuate HSC activation. Losartan, which exhibits inherent lipophilicity, was loaded into the hydrophobic core of HA micelles with a 19.5% drug loading efficiency. An advanced liver fibrosis model was developed using C3H/HeN mice subjected to 20 weeks of prolonged TAA/ethanol weight-adapted treatment. The cytocompatibility and cell uptake profile of losartan-HA micelles were studied in murine fibroblast cells (NIH3T3), human hepatic stellate cells (hHSC) and FL83B cells (hepatocyte cell line). The ability of these nanoparticles to attenuate HSC activation was studied in activated HSC cells based on alpha smooth muscle actin (α-sma) expression. Mice treated with oral losartan or losartan-HA micelles were analyzed for serum enzyme levels (ALT/AST, CK and LDH) and collagen deposition (hydroxyproline levels) in the liver. The accumulation of HA micelles was observed in fibrotic livers, which suggests increased delivery of losartan compared to normal livers and specific uptake by HSC. Active reduction of α-sma was observed in hHSC and the liver sections of losartan-HA micelle-treated mice. The serum enzyme levels and collagen deposition of losartan-HA micelle-treated mice was reduced significantly compared to the oral losartan group. Losartan-HA micelles demonstrated significant attenuation of hepatic fibrosis via an HSC-targeting mechanism in our in vitro and in vivo studies. These nanoparticles can be considered as an alternative therapy for liver fibrosis.     

Modulation of Intracellular Calcium Levels by Calcium Lactate Affects Colon Cancer Cell Motility through Calcium-Dependent Calpain

Cancer cell motility is a key phenomenon regulating invasion and metastasis. Focal adhesion kinase (FAK) plays a major role in cellular adhesion and metastasis of various cancers. The relationship between dietary supplementation of calcium and colon cancer has been extensively investigated. However, the effect of calcium (Ca²⁺) supplementation on calpain-FAK-motility is not clearly understood. We sought to identify the mechanism of FAK cleavage through Ca²⁺ bound lactate (CaLa), its downstream signaling and role in the motility of human colon cancer cells. We found that treating HCT116 and HT-29 cells with CaLa immediately increased the intracellular Ca²⁺ (iCa²⁺) levels for a prolonged period of time. Ca²⁺ influx induced cleavage of FAK into an N-terminal FAK (FERM domain) in a dose-dependent manner. Phosphorylated FAK (p-FAK) was also cleaved in to its p-N-terminal FAK. CaLa increased colon cancer cells motility. Calpeptin, a calpain inhibitor, reversed the effects of CaLa on FAK and pFAK cleavage in both cancer cell lines. The cleaved FAK translocates into the nucleus and modulates p53 stability through MDM2-associated ubiquitination. CaLa-induced Ca²⁺ influx increased the motility of colon cancer cells was mediated by calpain activity through FAK and pFAK protein destabilization. In conclusion, these results suggest that careful consideration may be given in deciding dietary Ca²⁺ supplementation to patient undergoing treatment for metastatic cancer.     

Cancer Cell Analyses at the Single Cell-Level Using Electroactive Microwell Array Device

Circulating tumor cells (CTCs), shed from primary tumors and disseminated into peripheral blood, are playing a major role in metastasis. Even after isolation of CTCs from blood, the target cells are mixed with a population of other cell types. Here, we propose a new method for analyses of cell mixture at the single-cell level using a microfluidic device that contains arrayed electroactive microwells. Dielectrophoretic (DEP) force, induced by the electrodes patterned on the bottom surface of the microwells, allows efficient trapping and stable positioning of single cells for high-throughput biochemical analyses. We demonstrated that various on-chip analyses including immunostaining, viability/apoptosis assay and fluorescent in situ hybridization (FISH) at the single-cell level could be conducted just by applying specific reagents for each assay. Our simple method should greatly help discrimination and analysis of rare cancer cells among a population of blood cells.     

Localized reversible frameshift mutation in an adhesin gene confers a phase-variable adherence phenotype in mycoplasma

The variable adherence-associated (Vaa) antigen of Mycoplasma hominis is an abundant surface lipoprotein adhesin that may mediate important interactions of this wall-less prokaryotic pathogen with the human host. Extensive mutational variation of Vaa size, as well as sequence and antigenic divergence, has been described previously. Using a series of clonal isolates representing an isogenic lineage of variants oscillating in Vaa expression, Vaa is further shown in this study to undergo high-frequency phase variation in expression, which correlated precisely with the ability of M . hominis to adhere to cultured human cells. Although no DNA rearrangements or sequence differences in the 5' regions flanking vaa alleles were detected between Vaa⁺ and Vaa⁻ variants, intragenic vaa sequences from this lineage revealed an oscillating mutation involving a single nucleotide deletion/insertion in a short tract of adenine residues near the 5' end of the mature Vaa coding sequence, which created a translational frameshift resulting in either a complete Vaa ORF or an in-frame UAG stop codon immediately downstream of the poly-A tract. Evidence for the occurrence of this high-frequency frameshift mutation in vivo was obtained from analysis of PCR-generated vaa sequences amplified from the joint synovial fluid of a patient with M . hominis -associated arthritis, which indicated that Vaa phase variation occurs during M . hominis infection in the natural host. These results identify a distinctive frameshift mutator element in the vaa gene that governs M . hominis adherence and highlight the importance of mutational alteration of primary gene products on the mycoplasma surface as a means of generating and maintaining functional diversity in the host.     

In vivo antiplasmodial activity of 11(13)-dehydroivaxillin from Carpesium ceruum

The whole plants of Carpesium genus are used in traditional medicine as anti-pyretic, analgesic and vermifugic, including a topical application for sores and inflammation. A previous study on Carpesium genus suggested that the antiplasmodial activity against Plasmodium falciparum was due to the existence of 11(13)- dehydroivaxillin (DDV) from EtOAc extracts of C. ceruum (Compositae). Here, the antimalarial activity of DDV was evaluated against Plasmodium berghei in mice. The LD₅₀ of the compound was determined as 51.2 mg/kg, while doses of 124 mg/kg and above were found to be lethal to mice. DDV (2, 5, 10 mg/kg/day) exhibited a significant blood schizontocidal activity in 4-day early infection, repository evaluation and in an established infection with a significant mean survival time comparable to that of the standard drug, chloroquine, 5 mg/kg/day. DDV possesses a promising antiplasmodial activity, which can be exploited in malaria therapy.     

Genetic diversity and population structure of the gizzard shad,Konosirus punctatus (Clupeidae,Pisces), in Korean waters based on mitochondrial DNA control region sequences

In this study, we clarified the population structure of the gizzard shad, Konosirus punctatus, in Korean waters. We analyzed 896 base pairs of the mitochondrial DNA control region in 182 individuals, which were sampled from eight localities between the East Sea and the Yellow Sea. The haplotype diversity (h) was very high (0.9662–1.0000) but the nucleotide diversity (π) was very low (0.0061–0.0434). A neighbor-joining tree showed that the population clustered into two reciprocal monophyletic groups, lineages A and B. Lineage A is distributed on all coasts of Korea, from the Yellow Sea to the East Sea, declining to the east, whereas lineage B is distributed in the East Sea and Korea Strait, disappearing completely from middle Korea Strait to the west. Analysis of molecular variance showed strong structuring (F _ST = 0.856; P < 0.0001) between the two lineages. Neutrality tests and mismatch distribution analyses showed that a recent rapid expansion event occurred only in lineage A. Our results suggest that the management unit of the Korean gizzard shad may be divided in two, lineage A and lineage B.