Recovery-from-extinction effects in an anuran amphibian: renewal effect, but no reinstatement.

Recovery-from-extinction effects in which a conditioned response returns after extinction have been shown in mammals, birds and fish. Thus, these effects appear to be conserved among vertebrates; however, they have yet to be investigated in amphibians. Using prey catching conditioning in the fire-bellied toad (Bombina orientalis), we tested if renewal and reinstatement occurred after extinction when subjects were respectively re-exposed to the context or reinforcer used during conditioning. For renewal, a different context was used during extinction and thus renewal tests assessed if external contextual cues associated during conditioning stimulated prey catching performance. For reinstatement, the reinforcer withheld during extinction was simply delivered again prior to a test assessing if internal cues associated with recent prey consumption stimulated prey catching performance. Conditioning followed a fixed ratio 5 schedule of reinforcement, where five attempts to capture a cricket stimulus displayed on a computer screen were reinforced by delivery of a single live cricket. Performance was measured as the time to reach five prey catching attempts. A significant improvement in prey catching performance during conditioning followed by deterioration with extinction was seen in the experiments. Upon return to the context used for conditioning after extinction, toads showed a renewal effect whereby they displayed faster performance during testing compared to the end of extinction. Conversely, toads showed no reinstatement effect because pre-feeding of a cricket did not influence performance during the test that followed extinction. Reinstatement could have been lost in amphibian phylogeny due to secondary simplification of the nervous system.

Studies on calcium release and H2O2 level produced by the elicitor induced plant cell by fluorescence probing.

Using fluorescence probing technology, we studied the mechanism and interrelations of calcium release and H(2)O(2) production in situ in living tissues of tobacco and cotton plants which were induced by pathogen elicitor, salicylic acid (SA) and pectinase respectively. Results showed that (1) pathogen elicitors could induced H(2)O(2) response in epidermis cells regardless of environmental calcium, but in mesophyll protoplast, H(2)O(2) response could only be induced at calcium condition. Similarly, SA and pectinase induced H(2)O(2) response could only be observed at calcium condition; (2) pathogen elicitors could induce calcium response in both epidermis cells and protoplasts regardless of environmental calcium, while calcium response couldn't be induced at non-calcium condition by SA and pectinase; (3) H(2)O(2) response and calcium response in protoplast were faster than that in the whole cell. These results indicated that pathogen elicitors can induce the release of cell wall calcium and the cell wall calcium release is independent to pectinase. And it is concluded that free calcium influx is necessary for the oxidative burst and cell wall calcium has an irreplaceable role in defense signal transduction.

Influence of the carbon source concentration on the nitrate removal rate in groundwater.

At present, groundwater nitrate pollution in China is serious. The use of microorganisms for biological denitrification has been widely applied, and it is a universal and efficient in situ groundwater remediation technique, but this approach is influenced by many factors. In this study, glucose was adopted as the carbon source, four different concentrations of 0, 2, 5 and 10 g/L were considered, and natural groundwater with a nitrate concentration of 300.8 mg/L was employed as the experimental solution. The effect of the carbon source concentration on the nitrate removal rate in groundwater was examined through heterotrophic anaerobic denitrification experiments. The results showed that the nitrate removal rate could be improved by the addition of an external carbon source in the process of biological denitrification, and an optimal concentration was observed. At a glucose concentration of 2 g/L, the denitrification effect was the best.

Enhanced optical property of Au coated polystyrene beads for multi-color quantum dots encoding.

A novel quantum dots (QDs) fluorescent encoding method was demonstrated in this paper by using Au coated polystyrene (Au @ PS) beads. In the experiments, Au nanoparticles were deposited onto the polystyrene bead to form a stable Au coating through Layer-by-Layer assembly, and the surface morphology of the Au @ PS beads was studied by Scanning Electronic Microscope (SEM). Furthermore, the QDs encoding abilities, including the loading of QDs, the anti-photo bleaching ability and the multi-color encoding feasibility were studied using Au @ PS beads. The QDs leakage from doped QDs encoded Au @ PS was also studied. This study shows that Au particles improve the QDs encoding performance and the QD-encoded Au @ PS beads are the ideal material for the optical encoding.

Fluorophore-assisted carbohydrate electrophoresis as detection method for carbohydrate-protein interactions.

Fluorophore-assisted carbohydrate electrophoresis (FACE) is a straightforward, sensitive method for determining the presence and relative abundance of individual (oligo)saccharide in a(n) (oligo)saccharide mixture. The single terminal aldehydes of (oligo)saccharides were tagged with the charged fluorophore 8-aminonaphthalene-1,3,6-trisulfonate (ANTS), and separated with high resolution on the basis of size by polyacrylamide gel electrophoresis. ANTS fluorescence labeling is not biased by (oligo)saccharide length. Therefore, band fluorescence intensity is directly related to the relative abundance of individual (oligo)saccharide moieties in heterogeneous sample. In the same time, it also indicates that FACE can be used to investigate the interactions of carbohydrates and proteins.

Preparation of Au coated polystyrene beads and their application in an immunoassay.

A novel immunoassay method based on polystyrene beads coated with Au nanoparticles (Au@PS) is described. Au nanoparticles were prepared by reductive reaction, and then deposited on the surface of polystyrene beads to form Au coatings. Results indicated that the Au coatings had good stability and that human IgG was immobilized at a concentration of 16 microg/g Au@PS. FITC-labeled rabbit-anti-human IgG and FITC-labeled rabbit-anti-goat IgG were employed to react with the human IgG on Au@PS. Fluorescence imaging results showed that the reaction had good immuno-specificity. In addition, further experiments at the single-bead level indicated that the linear range was 0.05-15 microg/ml, and that the FITC signal could be detected even when the target antibody concentration was as low as 0.01 microg/ml. The assay results were compared with an enzyme-linked immunosorbent assay (ELISA), and showed relatively good reliability.

Quantum dot optical encoded polystyrene beads for DNA detection.

A novel multiplex analysis technology based on quantum dot (QD) optical encoded beads was studied. Carboxyl functionalized polystyrene beads, about 100 microm in size, were precisely encoded by the various ratios of two types of QDs whose emission wavelengths are 576 and 628 nm, respectively. Then the different encoded beads were covalently immobilized with different probes in the existing of sulfo-NHS and 1-[3-(Dimethylamino) propyl]-3-ethylcarbodiimide methiodide, and the probe density could reach to 3.1 mmol/g. These probe-linked encoded beads were used to detect the target DNA sequences in complex DNA solution by hybridization. Hybridization was visualized using fluorescein isothiocynate-labeled DNA sequences. The results show that the QDs and target signals can be obviously identified from a single-bead-level spectrum. This technology can detect DNA targets effectively with a detection limit of 0.2 microg/mL in complex solution.

Synthesis of (R)-2,3-epoxypropyl(1-->3)-beta-D-pentaglucoside.

The title pentasaccharide was synthesized via a 2+3 strategy. The disaccharide donor, 3-O-acetyl-2-O-benzoyl-4,6-O-benzylidene-beta-D-glucopyranosyl-(1-->3)-2-O-benzoyl-4,6-O-benzylidene-alpha-D-glucopyranosyl trichloroacetimidate (8), was obtained by selective coupling of allyl 2-O-benzoyl-4,6-O-benzylidene-alpha-D-glucopyranoside with 3-O-acetyl-2-O-benzoyl-4,6-O-benzylidene-alpha-D-glucopyranosyl trichloroacetimidate (4), followed by deallylation, and trichloroacetimidation. Meanwhile, the trisaccharide acceptor, allyl 2-O-benzoyl-4,6-O-benzylidene-beta-D-glucopyranosyl-(1-->3)-2-O-benzoyl-4,6-O-benzylidene-beta-D-glucopyranosyl-(1-->3)-2-O-benzoyl-4,6-O-benzylidene-beta-D-glucopyranoside (12), was prepared by coupling of allyl 2-O-benzoyl-4,6-O-benzylidene-beta-D-glucopyranosyl-(1-->3)-2-O-benzoyl-4,6-O-benzylidene-beta-D-glucopyranoside with 4, followed by deacetylation. Condensation of 8 with 12, followed by epoxidation, and deprotection, gave the target pentaoside.

Synthesis, (1-->3)-beta-D-glucanase-binding ability, and phytoalexin-elicitor activity of a mixture of 3,4-epoxybutyl (1-->3)-beta-D-oligoglucosides.

We describe a approach for the synthesis of a mixture of 3,4-epoxybutyl (1-->3)-beta-D-oligoglucosides. The particular (1-->3)-beta-D-glucan isolated from the cell walls of Saccharomyces cerevisiae was recovered from the aqueous medium as water-insoluble particles by the spray drying (GS) method, and it was characterized by FTIR spectroscopy. The acid-solubilized (1-->3)-beta-D-oligoglucosides were prepared by partial acid hydrolysis of glucan particles, which were qualitatively analyzed by fluorophore-assisted carbohydrate electrophoresis (FACE). The peracetylated 3-butenyl (1-->3)-beta-D-oligoglucosides were synthesized by treating peracetylated (1-->3)-beta-D-oligoglucosides with the 3-butenyl alcohols and a Lewis acid (SnCl4) catalyst. Epoxidation of the peracetylated 3-butenyl oligoglucosides took place with m-chloroperoxybenzoic acid (m-CPBA). NaOMe in dry methanol was used for the deacetylation of the blocked derivatives, to give the 3,4-epoxybutyl (1-->3)-beta-D-oligoglucoside mixture in an overall yield of 21%. The sample was analyzed by positive-ion electrospray ionization mass spectrometry (ESIMS). In a 3,4-epoxybutyl (1-->3)-beta-D-oligoglucoside-binding (1-->3)-beta-D-glucanase assay, we found that the (1-->3)-beta-D-glucanase was obviously inactivated by the 3,4-epoxybutyl (1-->3)-beta-D-oligoglucosides. At the same time, we found the 3,4-epoxybutyl (1-->3)-beta-D-oligoglucoside mixture was more active as compared to the underivatized oligoglucoside mixture in eliciting phytoalexin accumulation in tobacco cotyledon tissue. Furthermore, it could be kept for a longer time than a (1-->3)-beta-D-oligoglucoside mixture, which indicated it is much more stable than (1-->3)-beta-D-oligoglucosides.

Preparation of silica encapsulated quantum dot encoded beads for multiplex assay and its properties.

Novel -COOH modified polystyrene beads were prepared by sulfonation grafting, and the surface area and pore volume are greatly improved in comparison with the swelling-treated beads. The optimization coating time is 4 h, and the corresponding -COOH content is approximately 2.1 mmol/g. The scanning electron microscope results show that the silica particles deposited on the beads and formed a silica shell that decreases the leakage of quantum dots (QDs) preferably and improves the bar code stability greatly. The anti-photobleaching of silica-coated beads was studied systemically, and the results show that the half-decay time (t1/2) of the coated beads increases to 537 s--seven times longer than that of the uncoated ones. Further DNA probe hybridization experiments indicated that the coding signal and target signal can be detected simultaneously and that the assays based on these probe-conjugated silica/QD/polystyrene beads have good specificity and sensitivity that can detect a concentration as low as 0.01 microg/ml target DNA in denatured calf thymus DNA solution, indicating that it is feasible to use this kind of bead for multiplex analysis.

The application of quantum dots as fluorescent label to glycoarray.

A sensitive, specific, and rapid method for the detection of carbohydrate-protein interactions was demonstrated using quantum dots (QDs) as a fluorescence label coupled with protein. 1,3-Dipolar cycloaddition between azide and alkyne was exploited to attach alpha-d-glucopyranoside to a C(14) hydrocarbon chain that noncovalently binds to the microtiter well surface, and the product formation was detected by both electrospray ionization-mass spectrometry (ESI-MS) and QD- (or fluorescein isothiocyanate (FITC))-conjugated lectin binding. It indicated that the peak intensity of the fluorescence emission was proportional to the initial concanavalin A (Con A) concentration in the range of 2 x 10(-3) micromol/L to 2 x 10(-2)mmol/L with a detection limit at least 100 times lower than that of the FITC-based method.

Synthesis, immunological activities, and scavenging ability toward superoxide anion of (1-->3)-beta-D-pentaglucoside and its epoxyalkyl derivatives.

The epoxyalkyl (1-->3)-beta-D-pentaglucosides 2 and 3 were synthesized in order by acetylation, glycosidation, oxidation, and deacetylation of 1. The immunological activities (superoxide anion production activity, phagocytic activity, and lymphocyte proliferation) and scavenging ability toward superoxide anion of (1-->3)-beta-D-pentaglucoside (1) and its epoxyalkyl derivatives (2 and 3) were investigated. Superoxide anion released from human blood monocytes was measured by the reduction of ferricytochrome c. Phagocytosis by peritoneal macrophages was detected through a teal ingesting that measured the chicken red blood cells (CRBC). Lymphocyte proliferation was determined by the MTT method. The scavenging ability of 1, 2, and 3 toward superoxide anions was evaluated by means of chemiluminescence (CL). The results showed that 2 and 3 had a little higher immunological activity and scavenging ability toward superoxide anion than 1, which indicated that the reducing end of the oligoglucosides was quite important for maximum biological activity.

Impaired cutaneous wound healing after sensory denervation in developing rats: effects on cell proliferation and apoptosis.

The role of sensory nociceptor nerves in cutaneous wound healing was investigated following full-thickness 4-mm diameter dorsal cutaneous excision wounding of rats on postnatal day 12. In rats with intact innervation, wounds at 3 days contained large numbers of TUNEL- and BRDU-labeled nuclei, consistent with inflammatory cell death and granulation cell proliferation. Wound area and volume decreased through 11 days in concert with a transient appearance of alpha-smooth muscle actin-immunoreactive myofibroblasts, declining rates of cell division, and increased occurrence of apoptotic cells. Sensory denervation by capsaicin injections on postnatal days 2 and 9 reduced calcitonin gene-related peptide-immunoreactive wound innervation persistently by up to 43%. This was associated with increased wound surface area and volume, and delays in scab loss and re-epithelialization. Relative to control wounds, granulation tissue showed increased myofibroblast content at 5-7 days. Capsaicin-treated rats had more BRDU-labeled cells, including myofibroblasts, through day 7. Numbers of TUNEL apoptotic cells per unit area of tissue section were reduced by denervation in both early and late stages of healing. We conclude that partial loss of sensory innervation impairs cutaneous wound healing in developing rats, as manifested by delayed re-epithelialization and failure of the wound area to decrease normally through at least 21 days. This is associated with an abnormally enlarged wound tissue volume resulting from increased granulation cell proliferation without proportionate increases in apoptosis. These findings suggest that nociceptor innervation plays a critical role in wound healing by regulating wound cellularity.

Synthesis, (1-->3)-beta-D-glucanase-binding ability and phytoalexin-elicitor activity of (R)-2,3-epoxypropyl (1-->3)-beta-D-pentaglucoside.

The (1-->3)-beta-D-pentaglucoside was synthesized as its (R)-2,3-epoxypropyl glycoside via 2+3 strategy. The disaccharide donor 8 was obtained by 3-selective coupling of 2 with 4, followed by deallylation, and trichloroacetimidation. Meanwhile, the trisaccharide acceptor 12 was prepared by coupling of 10 with 4, followed by deacetylation. Condensation of 8 with 12, followed by epoxidation, and deprotection, gave the target pentaoside. The results of these bioassays demonstrated that the (1-->3)-beta-D-glucanase was obviously inactivated by 15 with k(app)=3.79 x 10(-4) min(-1). At the same time, we found that the 15 was more active as compared to the laminaripentaose in eliciting phytoalexin accumulation in tobacco cotyledon tissue, and it could be kept longer time than laminaripentaose, which indicated it is much more stable than laminaripentaose.

Synthesis, protein-binding ability and phytoalexin-elicitor activity of epoxyalkyl (1-->3)-beta-D-oligoglucosides.

We describe a approach for the synthesis of (1-->3)-beta-D-oligosaccharide derivatives 10-18. 1-9 were synthesized by treating peracetylated (1-->3)-beta-D-oligosaccharides with the corresponding alkenyl alcohols and Lewis acid (SnCl(4)) catalyst. Epoxidation of the corresponding alkenyl oligoglucosides took place by m-CPBA. NaOMe in dry methanol was used for the deacetylation of the blocked derivatives, to give 10-18 in an overall yields of 25-32%. In subsequent glucan-binding protein of soybean assays, we found that 16 was most active, with an IC(50) value of 9 mM. However, the activities of 17, 18, 13, 14, 15, 10, 11, and 12 were gradually decreased. At the same time, we found 16 was most active as compared to the other (1-->3)-beta-D- oligoglucoside derivatives in eliciting phytoalexin accumulation in soybean cotyledon tissue, and 16 was kept longer time than (1-->3)-beta-D-glucohexaose, which indicated 16 is much more stable than (1-->3)-beta-D-glucohexaose.