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81.
Mixing in 96-well microplates was studied using soluble pH indicators and a fluorescence pH sensor. Small amounts of alkali were added with the aid of a multichannel pipet, a piston pump, and a piezoelectric actuator. Mixing patterns were observed visually using a video camera. Addition of drops each of about 1 nL with the piezoelectric actuator resulted in umbrella and double-disklike shapes. Convective mixing was mainly observed in the upper part of the well, whereas the lower part was only mixed quickly when using the multichannel pipet and the piston pump with an addition volume of 5 microL or larger. Estimated mixing times were between a few seconds and several minutes. Mixing by liquid dispensing was much more effective than by shaking. A mixing model consisting of 21 elements could describe mixing dynamics observed by the dissolved fluorescence dye and by the optical immobilized pH sensor. This model can be applied for designing pH control in microplates or for design of kinetic experiments with liquid addition. 相似文献
82.
83.
Barker CJ Leibiger IB Leibiger B Berggren PO 《American journal of physiology. Endocrinology and metabolism》2002,283(6):E1113-E1122
Pancreatic beta-cell function is essential for the regulation of glucose homeostasis in humans, and its impairment leads to the development of type 2 diabetes. Inputs from glucose and cell surface receptors act together to initiate the beta-cell stimulus-response coupling that ultimately leads to the release of insulin. Phosphorylated inositol compounds have recently emerged as key players at all levels of the stimulus-secretion coupling process. In this current review, we seek to highlight recent advances in beta-cell phosphoinositide research by dividing our examination into two sections. The first involves the events that lead to insulin secretion. This includes both new roles for inositol polyphosphates, particularly inositol hexakisphosphate, and both conventional and 3-phosphorylated inositol lipids. In the second section, we deal with the more novel concept of the autocrine role of insulin. Here, released insulin initiates signal transduction cascades, principally through the activity of phosphatidylinositol 3-kinase. This new round of signal transduction has been established to activate key beta-cell genes, particularly the insulin gene itself. More controversially, this insulin feedback has also been suggested to either terminate or enhance insulin secretion events. 相似文献
84.
Generation of activated and antigen-specific T cells with cytotoxic activity after co-culture with dendritic cells 总被引:5,自引:0,他引:5
Märten A Greten T Ziske C Renoth S Schöttker B Buttgereit P Schakowski F von Rücker A Sauerbruch T Schmidt-Wolf IH 《Cancer immunology, immunotherapy : CII》2002,51(1):25-32
Co-culturing of immunological effector cells with antigen-pulsed DC leads to an increase of cytotoxic activity against antigen-expressing tumour cells. Using this approach, we could detect up to 2.8% antigen-specific CTLs after co-culture with antigen-pulsed DC. However, the required high effector cell numbers remain a major obstacle in immunotherapy. In this study, we show an approach for generating activated and antigen-specific effector cells that enables us to decrease effector to target cell ratios. We used an interferon-gamma secretion assay to enrich activated effector cells after co-culture with antigen-pulsed dendritic cells (DC). Purified immunological effector cells lysed 58.3% of antigen-expressing tumour cells at an effector to target ratio of 1:1. Furthermore, using MHC-IgG complexes, we enriched effector cells expressing antigen-specific T-cell receptor after co-culture with DC. Performing ELISpot, flow cytometry and TCR analysis, we could show a significant increase of activated and specific TCR-expressing effector cells after co-culture with DC. 相似文献
85.
Andreas Niemeitz Ralf Kreutzfeldt Manfred Schartl Jakob Parzefall Ingo Schlupp 《Acta ethologica》2002,5(1):45-49
The Tamesí molly, Poecilia latipunctata, has a very limited biogeographical range in northeast Mexico. This area is nested within the ranges of the Atlantic molly,
Poecilia mexicana, and the unisexual Amazon molly, Poecilia formosa. Based on morphology, especially fin shape, the Tamesí molly has been considered to be a "short-fin" molly. We describe the
courtship sequence of P. latipunctata. The courtship clearly places the species into the clade of "long-fin" mollies, a finding that corroborates earlier studies
based on nuclear DNA and mitochondrial DNA. All three species live together in certain habitats. This renders P. latipunctata a potential host species for the sperm-dependent, unisexual Amazon molly. Using behavioural tests, we demonstrate that P. latipunctata males actually copulate with Amazon mollies, despite a pronounced preference for conspecific females. In laboratory experiments
P. latipunctata males are capable of triggering embryogenesis in P. formosa females. Field observations support the hypothesis that P. latipunctata is a third host species for P. formosa, indicating that the Amazon molly effectively exploits all available host species for its gynogenetic mode of reproduction.
Electronic Publication 相似文献
86.
We recently developed the Rosetta algorithm for ab initio protein structure prediction, which generates protein structures from fragment libraries using simulated annealing. The scoring function in this algorithm favors the assembly of strands into sheets. However, it does not discriminate between different sheet motifs. After generating many structures using Rosetta, we found that the folding algorithm predominantly generates very local structures. We surveyed the distribution of beta-sheet motifs with two edge strands (open sheets) in a large set of non-homologous proteins. We investigated how much of that distribution can be accounted for by rules previously published in the literature, and developed a filter and a scoring method that enables us to improve protein structure prediction for beta-sheet proteins. Proteins 2002;48:85-97. 相似文献
87.
Structure-function analysis of the Rho-ADP-ribosylating exoenzyme C3stau2 from Staphylococcus aureus
Exoenzyme C3stau2 from Staphylococcus aureus is a new member of the family of C3-like ADP-ribosyltransferases that ADP-ribosylates RhoA, -B, and -C. Additionally, it modifies RhoE and Rnd3. Here we report on studies of the structure-function relationship of recombinant C3stau2 by site-directed mutagenesis. Exchange of Glu(180) with leucine caused a complete loss of both ADP-ribosyltransferase and NAD glycohydrolase activity. By contrast, exchange of the glutamine residue two positions upstream (Gln(178)) with lysine blocked ADP-ribosyltransferase activity without major changes in NAD glycohydrolase activity. NAD and substrate binding of this mutant protein was comparable to that of the recombinant wild type. Exchange of amino acid Tyr(175), which is part of the recently described "ADP-ribosylating toxin turn-turn" (ARTT) motif [Han, S., Arvai, A. S., Clancy, S. B., and Tainer, J. A. (2001) J. Mol.Biol. 305, 95-107], with alanine, lysine, or threonine caused a loss of or a decrease in ADP-ribosyltransferase activity but an increase in NAD glycohydrolase activity. Recombinant C3stau2 Tyr175Ala and Tyr175Lys were not precipitated by matrix-bound Rho, supporting a role of Tyr(175) in protein substrate recognition. Exchange of Arg(48) and/or Arg(85) resulted in a 100-fold reduced transferase activity, while the recombinant C3stau2 double mutant R48K/R85K was totally inactive. The data indicate that amino acid residues Arg(48), Arg(85), Tyr(175), Gln(178), and Glu(180) are essential for ADP-ribosyltransferase activity of recombinant C3stau2 and support the role of the ARTT motif in substrate recognition of RhoA by C3-like ADP-ribosyltransferases. 相似文献
88.
The H+-ATP synthase from chloroplasts, CF0F1, was isolated, reconstituted into liposomes and ATP synthesis activity was measured after energization of the proteoliposomes with an acid-base transition. The ATP yield was measured as a function of the reaction time after energization, the data were fitted by an exponential function and the initial rate was calculated from the fit parameters. CF0F1 was reconstituted by detergent dialysis in asolectin liposomes and phosphatidylcholine/phosphatidic acid (PtdCho/PtdAc from egg yolk) liposomes. In asolectin liposomes, high initial rates of ATP synthesis (up to 400 s(-1)) were observed with a rapid decline of the rate; in PtdCho/PtdAc liposomes the initial rate is smaller (up to 200 s(-1)), but the decline of the activity is slower. CF0F1 was reconstituted into PtdCho/PtdAc liposomes either by detergent dialysis or into reverse phase liposomes. The dependence of the rate of ATP synthesis on the phosphate concentration was measured with both types of proteoliposomes. The data can be described by Michaelis-Menten kinetics with a K(M) value of 350 microM for reverse phase liposomes and a K(M) value of 970 microM for dialysis liposomes. Both K(M) values depend neither on the magnitude of DeltapH nor on the electric potential difference, whereas V(max) decreases strongly with decreasing energization. At low phosphate concentration, there are small deviations from Michaelis-Menten kinetics. The measured rates are higher than those calculated from the fitted Michaelis-Menten parameters. This effect is interpreted as evidence that more than one phosphate binding site is involved in ATP synthesis. 相似文献
89.
Effects of combined expression of antifungal barley seed proteins in transgenic wheat on powdery mildew infection 总被引:7,自引:0,他引:7
Bieri Stéphane Potrykus Ingo Fütterer Johannes 《Molecular breeding : new strategies in plant improvement》2003,11(1):37-48
Transgenicwheat plants (variety Frisal) constitutively expressing a number of potentialantifungal proteins alone or in combinations were generated and tested forincreased resistance to Blumeria graminis f.sp. tritici(powdery mildew) in a detached leaf infection assay. The most significativerateof protection was obtained with an apoplastic ribosome-inactivation proteinfrombarley seed. Apoplastic Barnase was less efficient and individual plant linesharbouring a barley seed chitinase and -1,3-glucanase showed linespecificphenotypes from increased resistance to increased susceptibility. Combinationbycrossing of three barley seed proteins did not lead to significant improvementof protection. 相似文献
90.