Additions to the cytologically investigated species of <Emphasis Type="Italic">Potentilla</Emphasis> L. (Rosaceae) from India

At present 14 species of Potentilla L. have been cytologically worked out from different geographical areas of Kashmir and Himachal Pradesh in the Western Himalayas. New chromosome numbers in nine species—Potentilla argyrophylla (n = 14), P. atrosanguinea (n = 7, 14), P. desertorum (n = 7), P. gerardiana (n = 14), P. indica (n = 14), P. micropetala (n = 14), P. nepalensis (n = 14), P. sibbaldia (n = 14) and P. thomsonii (n = 7)—have been reported on a worldwide basis for the first time. Additional chromosomal races of polyploid cytotypes for P. argyrophylla (n = 28) and P. desertorum (n = 14) along with a diploid cytotype for P. micropetala (n = 7) plus diploid cytotypes for the five species as P. fulgens (n = 7), P. gelida (n = 7), P. kleiniana (n = 7), P. sibbaldia (n = 7) and P. sundaica (n = 7) as well as a tetraploid cytotype for P. fruticosa (n = 14) all have been cytologically worked out from India for the first time. The course of meiosis varies from normal to abnormal in different populations of the majority of the species, such as P. argyrophylla, P. atrosanguinea, P. desertorum, P. fruticosa, P. fulgens, P. gelida, P. indica, P. nepalensis, P. sibbaldia and P. sundaica, except for normal meiosis observed in P. gerardiana, P. kleiniana, P. micropetala and P. thomsonii. The anomalous taxa are marked with meiotic abnormalities in the form of cytomixis, chromosomal stickiness, unoriented bivalents, formation of laggards and bridges resulting in abnormal microsporogenesis, and production of heterogenous-sized fertile pollen grains along with reduced pollen fertility. All the taxa with normal meiotic courses show nearly one hundred percent pollen fertility.     

Rho kinase inhibitor Y-27632 and Accutase dramatically increase mouse embryonic stem cell derivation

Although it has been 30 yr since the development of derivation methods for mouse embryonic stem (ES) cells, the biology of derivation of ES cells is poorly understood and the efficiency varies dramatically between cell lines. Recently, the Rho kinase inhibitor Y-27632 and the cell dissociation reagent Accutase were reported to significantly inhibit apoptosis of human ES cells during passaging. Therefore, in the current study, C57BL/6×129/Sv mouse blastocysts were used to evaluate the effect of the combination of the two reagents instead of using the conventional 129 line in mouse ES cell derivation. The data presented in this study suggests that the combination of Y-27632 and Accutase significantly increases the efficiency of mouse ES cell derivation; furthermore, no negative side effects were observed with Y-27632 and Accutase treatment. The newly established ES cell lines retain stable karyotype, surface markers expression, formed teratomas, and contributed to viable chimeras and germline transmission by tetraploid complementation assay. In addition, Y-27632 improved embryoid body formation of ES cells. During ES cell microinjection, Y-27632 prevented the formation of dissociation-induced cell blebs and facilitates the selection and the capture of intact cells. The methods presented in this study clearly demonstrate that inhibition of Rho kinase with Y-27632 and Accutase dissociation improve the derivation efficiently and reproducibility of mouse ES cell generation which is essential for reducing variability in the results obtained from different cell lines.     

Extracellular overexpression of recombinant <Emphasis Type="Italic">Thermobifida fusca</Emphasis> cutinase by alpha-hemolysin secretion system in <Emphasis Type="Italic">E. coli</Emphasis> BL21(DE3)

Background  

Extracellular expression of proteins has an absolute advantage in a large-scale industrial production. In our previous study, Thermobifida fusca cutinase, an enzyme mainly utilized in textile industry, was expressed via type II secretory system in Escherichia coli BL21(DE3), and it was found that parts of the expressed protein was accumulated in the periplasmic space. Due to the fact that alpha-hemolysin secretion system can export target proteins directly from cytoplasm across both cell membrane of E. coli to the culture medium, thus in the present study we investigated the expression of cutinase using this alpha-hemolysin secretion system.     

Apolipoprotein A-I mimetic peptide D-4F promotes human endothelial progenitor cell proliferation,migration, adhesion though eNOS/NO pathway

Circulating endothelial progenitor cells (EPCs) have a critical role in endothelial maintenance and repair. Apolipoprotein A-I mimetic peptide D-4F has been shown to posses anti-atherogenic properties via sequestration of oxidized phospholipids, induction of remodeling of high density lipoprotein and promotion of cholesterol efflux from macrophage-derived foam cells. In this study, we test the effects of D-4F on EPC biology. EPCs were isolated from the peripheral venous blood of healthy male volunteers and characterized by 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine-labeled acetylated LDL uptake and ulex europaeus agglutinin binding and flow cytometry. Cell proliferation, migration, adhesion, nitric oxide production and endothelial nitric oxide synthase (eNOS) expression in the absence and presence of D-4F or simvastatin (as a positive control), were assayed. We demonstrated that D-4F significantly enhanced EPC proliferation, migration and adhesion in a dose-dependent manner compared with vehicle. However, all of the favorable effects of D-4F on EPCs were dramatically attenuated by preincubation with NOS inhibitor L-NAME. Further, D-4F also increased nitric oxide production in culture supernatant and the levels of eNOS expression and phosphorylation. The stimulatory effects of D-4F (10 μg/ml) on EPC biology were comparable to 0.5 μM simvastatin. These results suggest that eNOS/NO pathway mediates the functional modulation of EPC biology in response to D-4F treatment and support the notion that the beneficial role of D-4F on EPCs may be one of the important components of its anti-atherogenic potential.     

Flocculation in ale brewing strains of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>: re-evaluation of the role of cell surface charge and hydrophobicity

Flocculation is an eco-friendly process of cell separation, which has been traditionally exploited by the brewing industry. Cell surface charge (CSC), cell surface hydrophobicity (CSH) and the presence of active flocculins, during the growth of two (NCYC 1195 and NCYC 1214) ale brewing flocculent strains, belonging to the NewFlo phenotype, were examined. Ale strains, in exponential phase of growth, were not flocculent and did not present active flocculent lectins on the cell surface; in contrast, the same strains, in stationary phase of growth, were highly flocculent (>98%) and presented a hydrophobicity of approximately three to seven times higher than in exponential phase. No relationship between growth phase, flocculation and CSC was observed. For comparative purposes, a constitutively flocculent strain (S646-1B) and its isogenic non-flocculent strain (S646-8D) were also used. The treatment of ale brewing and S646-1B strains with pronase E originated a loss of flocculation and a strong reduction of CSH; S646-1B pronase E-treated cells displayed a similar CSH as the non-treated S646-8D cells. The treatment of the S646-8D strain with protease did not reduce CSH. In conclusion, the increase of CSH observed at the onset of flocculation of ale strains is a consequence of the presence of flocculins on the yeast cell surface and not the cause of yeast flocculation. CSH and CSC play a minor role in the auto-aggregation of the ale strains since the degree of flocculation is defined, primarily, by the presence of active flocculins on the yeast cell wall.     

Chitosan enhanced gene delivery of cationic liposome via non-covalent conjugation

Two new types of stable ternary complexes were formed by mixing chitosan with DOTAP/pDNA lipoplex and DOTAP with chitosan/pDNA polyplex via non-covalent conjugation for the efficient delivery of plasmid DNA. They were characterized by atomic force microscopy, gel retarding, and dynamic light scattering. The DOTAP/CTS/pDNA complexes were in compacted spheroids and irregular lump of larger aggregates in structure, while the short rod- and toroid-like and donut shapes were found in CTS/DOTAP/pDNA complexes. The transfection efficiency of the lipopolyplexes showed higher GFP gene expression than DOTAP/pDNA and CTS/pDNA controls in Hep-2 and Hela cells, and luciferase gene expression 2–3-fold than DOTAP/pDNA control and 70–120-fold than CTS/pDNA control in Hep-2 cells. The intracellular trafficking was examined by confocal laser scanning microscopy. Rapid pDNA delivery to the nucleus enchanced by chitosan was achieved after 4 h transfection.     

Effects of sap velocity on the daytime increase of stem CO2 efflux from stems of Schima superba trees

Stem CO₂ efflux (E _s) has been estimated from a temperature-related equation, but sap flux often affects measurements of E _s, which leads to misunderstanding real stem respiration. In order to observe the relationship between E _s and stem temperature and to analyze the effect of sap velocity on E _s, stem temperature, E _s and sap flux were measured from a subtropical Schima superba plantation in South China on three trees for consecutive 3 days in July and October 2009. Stem temperature, E _s and sap velocity were significantly higher in July than in October. Stem temperature could explain 17–41 and 54–75% variations of E _s in July and October, respectively. A negative relationship between E _s and stem temperature was found during 1800–2300 hours in July. The daytime E _s was 9.2, 4.3 and 2.4% higher than the predicted for three trees in July, and this occurred only on Tree 1 in October. Sap velocity was positively correlated with E _s for three trees in July, and the increase of E _s with the increase of sap velocity was only observed on Tree 1 in October. These results demonstrated that the occurrence of sap flux could account for the increase of daytime E _s, and the effect of sap velocity on E _s varied with the seasons from the S. superba stem.     

Amplification activation loop between caspase-8 and -9 dominates artemisinin-induced apoptosis of ASTC-a-1 cells

Although caspases have been demonstrated to be involved in artemisinin (ARTE)-induced apoptosis, their exact functions are not well understood. The aim of this report is to explore the roles of caspase-8, -9 and -3 during ARTE-induced apoptosis in human lung adenocarcinoma (ASTC-a-1) cells. ARTE treatment induces a rapid generation of reactive oxygen species (ROS), and ROS-dependent apoptosis as well as the activation of caspase-8, -9 and -3 via time- and dose-dependent fashion. Of upmost importance, inhibition of caspase-8 or -9, but not caspase-3, almost completely blocks the ARTE-induced not only activation of the caspase-8, -9 and -3 but also apoptosis. In addition, the apoptotic process triggered by ARTE does not involve the Bid cleavage, tBid translocation, significant loss of mitochondrial membrane potential and cytochrome c release from mitochondria. Moreover, silencing Bax/Bak does not prevent the ATRE-induced cell death as well as the activation of caspase-8, -9 and -3. Collectively, our data firstly demonstrate that ARTE triggers a ROS-mediated positive feedback amplification activation loop between caspase-8 and -9 independent of mitochondria, which dominantly mediated the ARTE-induced apoptosis via a caspase-3-independent apoptotic pathway in ASTC-a-1 cells. Our findings imply a potential to develop new derivatives from artemisinin to effectively initiate the amplification activation loop of caspases.     

Cold tolerance of the coconut hispine beetle, <Emphasis Type="Italic">Brontispa longissima</Emphasis> (Coleoptera: Chrysomelidae) in Japan

The coconut hispine beetle, Brontispa longissima (Gestro), supposedly originated from Papua New Guinea and Indonesia but has recently invaded Southeast and East Asian countries where it has been causing serious damage to Cocos nucifera L. This insect also occurs on the Southwest Islands off Kyushu Island in Japan. To evaluate the potential northward range expansion of this insect in Japan, we investigated its cold tolerance at 0, 5, and 10 °C (egg, larva, pupa, and adult), 13 °C (adult), and 15 °C (egg and hatched larva). At 15 °C, few eggs hatched, and the larvae that hatched died within a few days of hatching. At 13 °C, Ltime₉₅ was estimated to be 23 days for adults, with the most cold-tolerant developmental stage at 10 °C. At all developmental stages, Ltime₉₅ of B. longissima was estimated to be 19 days at 10 °C, 8 days at 5 °C, and 5 days at 0 °C, suggesting the cold tolerance of this beetle is very low. Considering average daily temperatures, it is unlikely that B. longissima can establish itself north of Amami-Oshima Island, located in the far south off the main island of Japan.