Current knowledge of the genetic mechanisms underlying the inheritance of photosynthetic activity in forest trees is generally limited, yet it is essential both for various practical forestry purposes and for better understanding of broader evolutionary mechanisms. In this study, we investigated genetic variation underlying selected chlorophyll a fluorescence (ChlF) parameters in structured populations of Scots pine (Pinus sylvestris L.) grown on two sites under non-stress conditions. These parameters were derived from the OJIP part of the ChlF kinetics curve and characterize individual parts of primary photosynthetic processes associated, for example, with the exciton trapping by light-harvesting antennae, energy utilization in photosystem II (PSII) reaction centers (RCs) and its transfer further down the photosynthetic electron-transport chain. An additive relationship matrix was estimated based on pedigree reconstruction, utilizing a set of highly polymorphic single sequence repeat markers. Variance decomposition was conducted using the animal genetic evaluation mixed-linear model. The majority of ChlF parameters in the analyzed pine populations showed significant additive genetic variation. Statistically significant heritability estimates were obtained for most ChlF indices, with the exception of DI0/RC, φD0 and φP0 (Fv/Fm) parameters. Estimated heritabilities varied around the value of 0.15 with the maximal value of 0.23 in the ET0/RC parameter, which indicates electron-transport flux from QA to QB per PSII RC. No significant correlation was found between these indices and selected growth traits. Moreover, no genotype × environment interaction (G × E) was detected, i.e., no differences in genotypes' performance between sites. The absence of significant G × E in our study is interesting, given the relatively low heritability found for the majority of parameters analyzed. Therefore, we infer that polygenic variability of these indices is selectively neutral.
- MeSH
- Pinus sylvestris genetics physiology MeSH
- Chlorophyll physiology MeSH
- Fluorescence MeSH
- Photosynthetic Reaction Center Complex Proteins physiology MeSH
- Photosynthesis genetics MeSH
- Photosystem II Protein Complex physiology MeSH
- Genetic Variation * MeSH
- Genotype * MeSH
- Quantitative Trait, Heritable * MeSH
- Forests MeSH
- Genes, Plant MeSH
- Trees genetics physiology MeSH
- Light MeSH
- Electron Transport MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Úvod: Tularémie je onemocnění, které ve své typické formě nečiní diagnostické problémy, ale její netypické formy jsou někdy diagnostikovány obtížně a pozdě. Materiál a metodika: U 6 pacientů se sérologicky potvrzenou tularémií jsme na průtokovém cytometru stanovili počty B lymfocytů (CD 19+), T lymfocytů (CD3+), T helperů (CD3+/CD4+), T cytotoxických (CD3+/CD8+) a aktivovaných T lymfocytů (CD3+/HLADr+). Výsledky: Ve všech případech jsme nalezli zvýšené počty dvojitě negativních T lymfocytů (CD3+/CD4-/CD8-) a u většiny pacientů mírné změny v dalších parametrech buněčné imunity. Diskuse a závěr: Dvojitě negativní T lymfocyty jsou u zdravých osob málo početnou subpopulací, jejíž zvýšení je charakteristické pro tularémii a další intracelulární infekce. Tento jev se objevuje záhy po infekci a mohl by tak být cenným vodítkem pro stanovení diagnózy. Tomu nasvědčuje fakt, že u dvou pacientů byla tularemic sérologicky potvrzena až po vyšetření buněčné imunity.
Introduction: Tularemia is a disease which poses little diagnostic problems when manifesfing in its typical form. However, atypical forms may sometimes be diagnosed with difficulty and delay. Materials and methods: We determined counts of B lymphocytes (CD 19+), T lymphocytes (CD3+), T helpers (CD3+/CD4+) and T cytotoxic (CD3+/CD8+) and activated T lymphocytes (CD3+/HLADr+) of with flow cytometry in 6 pafients with serologically confirmed tularemia. Results: In all cases we detected elevated counts of double negative T lymphocytes (CD3+/CD4-/CD8-) and minor changes of other cell immunity parameters in most patients. Conclusion: Double negative T lymphocytes comprise a small sub-population in healthy individuals; elevated counts, manifesfing rapidly post infection, are characteristic for tularemia and other intracellular infecfions and may be a valuable aid for diagnosis. This is parficularly underlined by the fact that in two pafients tularemia was serologically confirmed after cell immunity tesfing.
Východisko. Cytognenetické abnormality chromozómu č. 13 patří k důležitým prognostickým faktorům mnohočetného myelomu a jsou asociovány se špatnou prognózou. Metody a výsledky. U 40 pacientů s mnohočetným myelomem jsme určovali prognostický význam delece oblasti q14 na chromozómu č. 13 na separovaných a neseparovaných plazmatických buňkách kostní dřeně v závislosti na standardních prognostických faktorech. Zjišťovali jsme, zda metoda interfázní fluorescenční in situ hybridizace detekující deleci RB1 genu na buňkách získaných imunomagnetickou separací je více citlivá než na buňkách neseparovaných. U neseparovaných vzorků byla delece nalezena v 25,0 % případů, u obohacené suspenze v 62,5 % případů. Byla zjištěna negativní korelace mezi hodnotou fluorescenční in situ hybridizace a albuminu jak u separovaných (p=0,003), tak u neseparovaných (p=0,010) buněk. Nebyl zjištěn rozdíl v celkovém přežití pacientů sledované delece na separovaných a neseparovaných buňkách (p=0,830; p=0,260), stejně tak v případě léčebné odpovědi pacientů a u pacientů po transplantaci na obohacených buňkách (p=0,880; p=0,520) i na kostní dřeni (p=0,370; p=0,190). Závěry. Použitím interfázní fluorescenční in situ hybridizace u imunomagneticky separovaných buněk lze zvýšit záchyt delece oblasti 13q14 v plazmatických buňkách mnohočetného myelomu. Byla nalezana korelace mezi hodnotou fluorescenční in situ hybridizace a hodnotou albuminu u obou typů buněk.
Background. Cytogenetic abnormalities of chromosome 13 are emerging as important prognostic factors in multiple myeloma and have been associated with poor prognosis. Methods and Results. The occurrence of 13q14 deletion and other standard laboratory parameters were determined in 40 patients with multiple myeloma. We found that interphase fluorescence in situ hybridization using a locus specific probe for RB1 gene on immunomagnetically selected myeloma cells was more sensitive than non selected cells. The 13q14 deletion was found in 10 of 40 (25,0%) of bone marrow samples without cell selection and in 25 of 40 (62.5%) of samples with CD138+ enriched myeloma cells. Negative correlation was found between albumin and the 13q14 deletion in separated (p=0,003) as well as in cells without selection (p=0,010). No significant correlation was found in overall survival of separated and unseparated cells (p=0,830; p=0,260) and a similar result was obtained for treatment response after transplantation of separated cells ( p=0,520) or non-separated cells (0,190). Conclusions. Our results confirm that immunomagnetic selection of CD138+ cells increases the probability of detection of the 13q14 deletion in bone marrow samples. The correlation was found between albumin and the 13q14 deletion in both of type of cells.
- MeSH
- Bone Marrow Cells immunology MeSH
- Research Support as Topic MeSH
- In Situ Hybridization, Fluorescence methods utilization MeSH
- Immunomagnetic Separation methods utilization MeSH
- Humans MeSH
- Chromosomes, Human, Pair 13 immunology MeSH
- Multiple Myeloma diagnosis immunology MeSH
- Check Tag
- Humans MeSH
Measurement of Pulse-Amplitude-Modulated (PAM) chlorophyll a fluorescence is widely used method for obtaining information on the functional state of photosystem II (PSII). Recently, it has been shown that some of long-established fluorescence parameters must be interpreted with caution, when the light-induced chloroplast movements occur. In our work we have analyzed the effect of chloroplast movements on these parameters. We have derived new parameters that are independent of the change in PSII absorption occurring during measurement. To verify whether there is a need for new parameters or the difference between the parameters commonly used and the newly derived ones is insignificant, we conducted an experiment with Arabidopsis thaliana wild type plants and its phot1 phot2 mutant defective in chloroplast movement. Plants were exposed to light of different qualities (450, 470, 550 or 660 nm) and quantities (100, 400 or 1200 μmol m-2 s-1) for up to 40 min. Since the blue light-induced chloroplast avoidance reaction is a photoprotective mechanism, we expected that phot1 phot2 mutant will compensate the lack of this mechanism by increasing non-photochemical quenching. However, using the light at both 450 and 470 nm, the calculation of commonly used parameter, ΦNPQ (quantum yield of regulated light-induced thermal energy dissipation in PSII) based on Hendrickson et al. [L. Hendrickson, R.T. Furbank, W.S. Chow, Photosynth. Res. 82 (2004) 73-81] showed the opposite. On the other hand, the results obtained using our newly proposed formulae to determine quantum yield of PSII thermal energy dissipation were in line with our assumption. Thus, the experimental data showed that some formulae of fluorescence parameters are dependent on the change in PSII absorption and need to be interpreted carefully. On the contrary, the formulae introduced by us can remove the effect of changes in PSII absorption that occur during measurement, without additional measurements, and give the real estimate of light-induced non-photochemical quenching.
- MeSH
- Arabidopsis metabolism MeSH
- Chlorophyll A chemistry MeSH
- Chloroplasts physiology MeSH
- Photosystem II Protein Complex chemistry genetics metabolism MeSH
- Quantum Theory MeSH
- Plant Leaves chemistry MeSH
- Mutagenesis MeSH
- Arabidopsis Proteins chemistry genetics metabolism MeSH
- Light MeSH
- Models, Theoretical MeSH
- Thermodynamics MeSH
- Publication type
- Journal Article MeSH
Glutamate carboxypeptidase II (GCPII) is an important target for therapeutic and diagnostic interventions aimed at prostate cancer and neurologic disorders. Here we describe the development and optimization of a high-throughput screening (HTS) assay based on fluorescence polarization (FP) that facilitates the identification of novel scaffolds inhibiting GCPII. First, we designed and synthesized a fluorescence probe based on a urea-based inhibitory scaffold covalently linked to a Bodipy TMR fluorophore (TMRGlu). Next, we established and optimized conditions suitable for HTS and evaluated the assay robustness by testing the influence of a variety of physicochemical parameters (e.g., pH, temperature, time) and additives. Using known GCPII inhibitors, the FP assay was shown to be comparable to benchmark assays established in the field. Finally, we evaluated the FP assay by HTS of a 20 000-compound library. The novel assay presented here is robust, highly reproducible (Z' = 0.82), inexpensive, and suitable for automation, thus providing an excellent platform for HTS of small-molecule libraries targeting GCPII.
- MeSH
- Antigens, Surface genetics metabolism MeSH
- Fluorescent Dyes chemical synthesis MeSH
- Fluorescence Polarization methods MeSH
- Glutamate Carboxypeptidase II antagonists & inhibitors genetics metabolism MeSH
- Small Molecule Libraries pharmacology MeSH
- Humans MeSH
- Ligands MeSH
- High-Throughput Screening Assays methods MeSH
- Protein Binding MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Research Support, N.I.H., Extramural MeSH
The plasma membrane of various mammalian cell types is heterogeneous in structure and may contain microdomains, which can impose constraints on the lateral diffusion of its constituents. Fluorescence correlation spectroscopy (FCS) can be used to investigate the dynamic properties of the plasma membrane of living cells. Very recently, Wawrezinieck et al. (Wawrezinieck, L., H. Rigneault, D. Marguet, and P. F. Lenne. 2005. Biophys. J. 89:4029-4042) described a method to probe the nature of the lateral microheterogeneities of the membrane by varying the beam size in the FCS instrument. The dependence of the width of the autocorrelation function at half-maximum, i.e., the diffusion time, on the transverse area of the confocal volume gives information on the nature of the imposed confinement. We describe an alternative approach that yields essentially the same information, and can readily be applied on commercial FCS instruments by measuring the diffusion time and the particle number at various relative positions of the cell membrane with respect to the waist of the laser beam, i.e., by performing a Z-scan.
A rapid procedure based on a direct extraction and HPLC determination of dihydroergocristine in a pharmaceutical preparation with fluorescence detection has been developed and validated. The optimized chromatographic conditions included a Purospher RP18e column, 5 microm particle size, 250 x 4.0 mm, and 25 mM potassium dihydrogen phosphate buffer (pH 2.8)-acetonitrile (60 + 40, v/v) mobile phase at a flow rate of 1 ml/min. The separation was carried out at 50 degrees C, and the injection volume was 5 microL. Fluorescence detection was performed at an excitation and emission wavelength of 224 and 344 nm, respectively. The mobile phase parameters such as organic solvent composition, temperature, and pH were studied. The proposed method has the advantages of a very simple sample pretreatment and fast HPLC determination.
- MeSH
- Anthraquinones MeSH
- Dihydroergocristine analysis standards MeSH
- Chemistry, Pharmaceutical MeSH
- Spectrometry, Fluorescence MeSH
- Hydrogen-Ion Concentration MeSH
- Reference Standards MeSH
- Temperature MeSH
- Chromatography, High Pressure Liquid methods standards statistics & numerical data MeSH
- Publication type
- Journal Article MeSH
- Validation Study MeSH
