Agrobacterium-mediated transformation of Asparagus officinalis L. long-term embryogenic callus and regeneration of transgenic plants

Summary Twenty-three independent kanamycin resistant lines were obtained after cocultivation of longterm embryogenic cultures of three Asparagus officinalis L. genotypes with an Agrobacterium tumefaciens strain harboring ß-glucuronidase and neomycin phosphotransferase II genes. All the lines showed ß-glucuronidase activity by histological staining. DNA analysis by Southern blots of the kanamycin resistant embryogenic lines and of a plant regenerated from one of them confirmed the integration of the T-DNA.Abbreviations GUS ß-glucuronidase - X-Gluc 5-bromo-4-chloro-3indolyl ß-D-glucuronic acid - NPT II neomycin phosphotransferase II     

Dual mechanism of laminin modulation of ecto-5′-nucleotidase activity

The myoblast cell surface activity of ecto-5′-nucleotidase was stimulated by a laminin substrate, whereas fibronectin and gelatin did not increase the AMPase activity of ecto-5′-nucleotidase. This increase was related to a higher expression of ecto-5′-nucleotidase on the surface of cells seeded on a laminin substrate, but without the mobilization of an intracellular pool of enzyme. Furthermore, laminin and its fragments E′₁ and E₈ modified the AMPase activity of the ecto-5′-nucleotidase purified from chicken striated muscle and reconstituted in liposomes. Over the range of concentrations used, intact laminin and its fragment E₈, consisting of the distal half of the long arm, stimulated the AMPase activity of ecto-5′-nucleotidase. By contrast, the large fragment derived from the short arms, designated E′₁, inhibited the AMPase activity. Furthermore, the monoclonal anti-ecto-5′-nucleotidase antibody, CG37, abolished the stimulatory effect of fragment E₈ on the AMPase activity of ecto-5′-nucleotidase but did not reverse the inhibitory effect of fragment E′₁. In conclusion, laminin stimulates the AMPase activity of ecto-5′-nucleotidase by two mechanisms: inducing the expression of ecto-5′-nucleotidase to the cell surface and direct modulation of the enzymatic activity.     

Construction and characterization of a YAC library with a lowfrequency of chimeric clones from flow-sorted human chromosome 9

Human chromosome 9 DNA, flow-sorted from somaticcell hybrid PK-87-9, has been used to construct two complete digest YAC libraries. The combined representation of chromosome 9 in these libraries, estimated by hybridization of chromosome 9-specific sequences to YAC colony grids, is 95%. The frequency of chimeric clones, analyzed by fluorescence in situ hybridization of chromosome 9 YACs to human metaphase chromosomes, was estimated to be 4%. These libraries provide a resource for physical mapping and for moving from genetic markers to disease loci on chromosome 9.     

Human pluripotent stem cell-derived extracellular vesicles: From now to the future

Extracellular vesicles (EVs) are nanometric particles that enclose cell-derived bioactive molecules in a lipid bilayer and serve as intercellular communication tools. Accordingly, in various biological contexts, EVs are reported to engage in immune modulation, senescence, and cell proliferation and differentiation. Therefore, EVs could be key elements for potential off-the-shelf cell-free therapy. Little has been studied regarding EVs derived from human pluripotent stem cells (hPSC-EVs), even though hPSCs offer good opportunities for induction of tissue regeneration and unlimited proliferative ability. In this review article, we provide an overview of studies using hPSC-EVs, focusing on identifying the conditions in which the cells are cultivated for the isolation of EVs, how they are characterized, and applications already demonstrated. The topics reported in this article highlight the incipient status of the studies in the field and the significance of hPSC-EVs’ prospective applications as PSC-derived cell-free therapy products.     

Bioguided Fractionation and Isolation of an Antiplasmodial Saponin from the Roots of Nauclea xanthoxylon (A.Chev.) Aubrév. (Rubiaceae)

The root extract of Nauclea xanthoxylon (A.Chev.) Aubrév. displayed significant 50 % inhibition concentration (IC₅₀s) of 0.57 and 1.26 μg/mL against chloroquine resistant and sensitive Plasmodium falciparum (Pf) Dd2 and 3D7 strains, respectively. Bio-guided fractionation led to an ethyl acetate fraction with IC₅₀s of 2.68 and 1.85 μg/mL and subsequently, to the new quinovic acid saponin named xanthoxyloside ( 1 ) with IC₅₀s of 0.33 and 1.30 μM, respectively against the tested strains. Further compounds obtained from ethyl acetate and hexane fractions were the known clethric acid ( 2 ), ursolic acid ( 3 ), quafrinoic acid ( 4 ), quinovic acid ( 5 ), quinovic acid 3-O-β-D-fucopyranoside ( 6 ), oleanolic acid ( 7 ), oleanolic acid 3-acetate ( 8 ), friedelin ( 9 ), β-sitosterol ( 10a ), stigmasterol ( 10b ) and stigmasterol 3-O-β-D-glucopyranoside ( 11 ). Their structures were characterised with the aid of comprehensive spectroscopic methods (1 and 2D NMR, Mass). Bio-assays were performed using nucleic acid gel stain (SYBR green I)-based fluorescence assay with chloroquine as reference. Extracts and compounds exhibited good selectivity indices (SIs) of >10. Significant antiplasmodial activities measured for the crude extract, the ethyl acetate fraction and xanthoxyloside ( 1 ) from that fraction can justify the use of the root of N. xanthoxylon in ethnomedicine to treat malaria.     

Flow cytometric detection of mitotic cells using the bromodeoxyuridine/DNA technique in combination with 90 degrees and forward scatter measurements

Mitotic cells could be well discriminated from the cells in the G1-, S- and G2-phases of the cell cycle using pulse labeling of S-phase cells with bromodeoxy-uridine (BrdUrd) and staining of the cells for incorporated BrdUrd and total DNA content. Unlabeled G2- and M-phase cells could be measured as two separate peaks according to propidium iodide fluorescence. M-phase cells showed lower propidium iodide fluorescence emission compared to G2-phase cells. The fluorescence difference of M- and G2-phase cells was caused by the different thermal denaturation of their DNA. Best separation of M- and G2-phase cells was obtained after 30-50 min heat treatment at 95 degrees C. Mitotic index could be measured if no unlabeled S-phase cells were present in the cell culture. With additional measurements of 90 degree scatter and/or forward scatter signals, mitotic cells could be clearly discriminated from both unlabeled G2- and S-phase cells. The correct discrimination (about 99%) of mitotic cells from interphase cells was verified by visual analysis of the nuclear morphology after selective sorting. Unlabeled and labeled mitotic cells could be observed as pulse-labeled cells progressed through the cell cycle. We conclude that this modified BrdUrd/DNA technique using prolonged thermal denaturation and the simultaneous measurement of scatter signals may offer additional information especially in the presence of BrdUrd-unlabeled S-phase cells.     

Landscape-scale patterns of biological invasions in shoreline plant communities

Little is known about the patterns and dynamics of exotic species invasions at landscape to regional spatial scales. We quantified the presence (identity, abundance, and richness) and characteristics of native and exotic species in estuarine strandline plant communities at 24 sites in Narragansett Bay, Rhode Island, USA. Our results do not support several fundamental predictions of invasion biology. Established exotics (79 of 147 recorded plant species) were nearly indistinguishable from the native plant species (i.e. in terms of growth form, taxonomic grouping, and patterns of spatial distribution and abundance) and essentially represent a random sub-set of the current regional species pool. The cover and richness of exotic species varied substantially among quadrats and sites but were not strongly related to any site-level physical characteristics thought to affect invasibility (i.e. the physical disturbance regime, legal status, neighboring habitat type, and substrate characteristics). Native and exotic cover or richness were not negatively related within most sites. Across sites, native and exotic richness were positively correlated and exotic cover was unrelated to native richness. The colonization and spread of exotics does not appear to have been substantially reduced at sites with high native diversity. Furthermore, despite the fact that the Rhode Island strandline system is one of the most highly-invaded natural plant communities described to date, exotic species, both individually and as a group, currently appear to pose little threat to native plant diversity. Our findings are concordant with most recent, large-scale investigations that do not support the theoretical foundation of invasion biology and generally contradict small-scale experimental work.     

Quantitative histochemical assay for superoxide dismutase in rat brain.

Superoxide anions are highly reactive radicals overproduced in many pathological situations such as inflammation and ischemia. One of the major factors in the protection from superoxide anions is the enzyme superoxide dismutase (SOD), which catalyzes the dismutation of superoxide to hydrogen peroxide. This study presents a quantitative histochemical method to estimate SOD activity in rat brain tissue sections. This method is based on the cerium capture method and 3,3'-diaminobenzidine amplification of transition cerium compounds. Substrate for SOD was provided by reduction of oxygen during the autoxidation of riboflavin in the presence of UV light. This histochemical method reveals the overall activity of the three different forms of SOD described in mammalian tissues: cytosolic copper-zinc SOD, mitochondrial manganese SOD, and the high molecular weight extracellular SOD. Eventually, this method can be used to quantify SOD activity in tissue sections by image analysis.     

Effects of peptide YY and its analogues on chloride ion secretion in fed and fasted rat jejunum

The aim of our study was to determine whether a meal modifies the antisecretory response induced by PYY and the structural requirements to elicit antisecretory effects of analogue PYY(22–36) for potential antidiarrhea therapy. The variations in short-circuit current (Isc) due to the modification of ionic transport across the rat intestine were assessed in vitro, using Ussing chambers. In fasted rats, PYY induced a dose- and time-dependent reduction in Isc, with a sensitivity threshold at 5 × 10⁻¹¹ M (ΔIsc −2 ± 0.5 μA/cm²). The reduction was maximal at 10⁻⁷ M (Isc −23 ± 2 μA/cm²), and the concentration producing half-maximal inhibition was 10⁻⁹ M. At 10⁻⁷ M, reduction of Isc by PYY reached 90% of response to 5 × 10⁻⁵ M bumetanide. The PYY effect was partly reversed by 10⁻⁵ M forskolin (Isc +13.43 ± 2.91 μA/h·cm², p < 0.05) or 10⁻³ M dibutyryl adenosine 3′,5′ cyclic monophosphate (Isc +12 ± 1.69 μA/cm², p < 0.05). Naloxone and tetrodotoxin did not alter the effect of PYY. In addition, PYY and its analogue P915 reduced net chloride ion secretion to 2.85 and 2.29 μEq/cm² (p < 0.05), respectively. The antisecretory effect of PYY was accompanied by dose- and time-dependent desensitization when jejunum was prestimulated by a lower dose of peptide. The antisecretory potencies exhibited by PYY analogues required both a C-terminal fragment (22–36) and an aromatic amino acid residue (Trp or Phe) at position 27. At 10⁻⁷ M the biological activity of PYY was lower in fed than fasted rats (p < 0.001). Our results confirm the antisecretory effect of PYY, but show that the fed period is accompanied by desensitization, similar to the transient desensitization observed in the fasted period with cumulative doses. This suggests that PYY may act as a physiological mediator that reduces intestinal secretion.