Rapid Global Fitting of Large Fluorescence Lifetime Imaging Microscopy Datasets

Fluorescence lifetime imaging (FLIM) is widely applied to obtain quantitative information from fluorescence signals, particularly using Förster Resonant Energy Transfer (FRET) measurements to map, for example, protein-protein interactions. Extracting FRET efficiencies or population fractions typically entails fitting data to complex fluorescence decay models but such experiments are frequently photon constrained, particularly for live cell or in vivo imaging, and this leads to unacceptable errors when analysing data on a pixel-wise basis. Lifetimes and population fractions may, however, be more robustly extracted using global analysis to simultaneously fit the fluorescence decay data of all pixels in an image or dataset to a multi-exponential model under the assumption that the lifetime components are invariant across the image (dataset). This approach is often considered to be prohibitively slow and/or computationally expensive but we present here a computationally efficient global analysis algorithm for the analysis of time-correlated single photon counting (TCSPC) or time-gated FLIM data based on variable projection. It makes efficient use of both computer processor and memory resources, requiring less than a minute to analyse time series and multiwell plate datasets with hundreds of FLIM images on standard personal computers. This lifetime analysis takes account of repetitive excitation, including fluorescence photons excited by earlier pulses contributing to the fit, and is able to accommodate time-varying backgrounds and instrument response functions. We demonstrate that this global approach allows us to readily fit time-resolved fluorescence data to complex models including a four-exponential model of a FRET system, for which the FRET efficiencies of the two species of a bi-exponential donor are linked, and polarisation-resolved lifetime data, where a fluorescence intensity and bi-exponential anisotropy decay model is applied to the analysis of live cell homo-FRET data. A software package implementing this algorithm, FLIMfit, is available under an open source licence through the Open Microscopy Environment.     

Homologous and heterologous ligands downregulate follicle‐stimulating hormone receptor mRNA in porcine granulosa cells

We investigated homologous and heterologous downregulation of FSH receptor mRNA in porcine granulosa cells from ovaries of immature pigs. Cultures were treated with 0, 40, or 200 ng/ml porcine FSH or medium and terminated at 24 hr intervals for Northern analysis of FSH receptor and cytochrome P450 side chain cleavage (P450scc) mRNA, and for radioimmunoassay of progesterone. Cells luteinized over 96 hr, and control cultures displayed increases in P450scc (8–10 fold) and FSH receptor (2 fold) mRNA and progesterone (100 fold). FSH reduced FSH receptor mRNA by 50–90%, increased P450scc mRNA 8 fold within 48 hr, and elevated progesterone logarithmically over 96 hr. Luteinized cells, (after 96 hr) received FSH or LH (1–200 ng/ml) or prostaglandin E₂ (0.01–1.0 mg/ml) for 6 hr resulting in increased P450scc mRNA (2–8 fold), and progesterone (2–5 fold), and reduced FSH receptor mRNA. FSH (200 ng/ml) or the cAMP analog, dbcAMP (1 mM) for 0–24 hr reduced FSH receptor mRNA to 15% of control from 4–24 hr and elevated P450scc mRNA at 4 and 6 hr, respectively, to maxima at 12–24 hr. Forskolin (1–10 mM) increased P450scc mRNA (2–3 fold) and downregulated FSH receptor mRNA, effects reversed by the inhibitor of cAMP, rpcAMPs. Both epidermal growth factor, and the activator of the protein kinase C pathway, phorbol 12‐myristate, 13‐acetate (PMA) at 10 nM reduced FSH receptor mRNA. We conclude that downregulation of FSH receptor mRNA in luteinized granulosa cells is mediated by both homologous and heterologous ligands which employ cAMP, and that growth factors that activate the PKC pathway reduce FSH receptor and P450scc mRNA abundance. Mol. Reprod. Dev. 53:198–207, 1999. © 1999 Wiley‐Liss, Inc.     

Metabolic Pools of ATP in Cultured Bovine Adrenal Medullary Chromaffin Cells

Cultured bovine adrenal chromaffin cells contain a pool of ATP sequestered within the chromaffin vesicles and an extravesicular pool of ATP. In a previous study it was shown that the turnover of ATP in the extravesicular pool was biphasic. One phase occurred with a t1/2 of 3.5-4.5 h whereas the second phase occurred with a t1/2 of several days. The studies described here were undertaken to characterize further the vesicular and extravesicular pools of ATP by examining the effects of metabolic inhibitors, adenosine, and digitonin on ATP utilization and subcellular localization immediately after and 48 h after labeling with [3H]adenosine and 32Pi. Immediately after labeling a combination of cyanide, 2-deoxy-D-glucose, the beta-glucono-1,5-lactone resulted in a 90-95% depletion of the labeled ATP but only a 25% depletion of the endogenous ATP within 30 min. Forty-eight hours after labeling, addition of the inhibitors resulted in a 70% depletion of the [3H]ATP but only a 25% depletion of the [32P]ATP and endogenous ATP. Addition of 10 microM adenosine to the media resulted in a similar loss of [3H]ATP in cells examined immediately after or 48 h after labeling. Adenosine increased the amounts of [32P]ATP when added immediately after labeling but had no effect on the [32P]ATP content when added 48 h after labeling.(ABSTRACT TRUNCATED AT 250 WORDS)     

High concordance between marker profiles of 22 human leukemia-lymphoma cell lines tested with the same monoclonal antibodies before and during the second international workshop on human differentiation antigens

Summary Our laboratory participated in the Second International Workshop and Conference on Human Leucocyte Differentiation Antigens. In this international study the reactivity profiles of monoclonal antibodies were analyzed on normal and malignant hematopoietic cells. The Workshop was divided into three categories: the T-cell, B-cell and myelomonocytic cell studies. We blindly tested 159 coded monoclonal antibodies of the panel for the T-cell study on 22 permanently established leukemia cell lines. The monoclonal antibodies were provided by the Workshop Committee and their reactivity with the target cells was visualized by standardized indirect immunofluorescence. After decoding it was recognized that 11 monoclonal antibodies had been examined on these cell lines prior to the Workshop. The reactivity of these 11 monoclonal antibodies was analyzed and compared with the earlier results. From a total of 217 paired tests done blindly in the Workshop study and prior to the Workshop, 191 tests (88%) did not show significantly different data. The possible reasons for discrepancies include nonspecific Fc-receptor-binding on some cell lines and a relatively nonspecific reactivity of some monoclonal antibodies.This analysis demonstrates the stability of the antigen expression on human leukemia-lymphoma cell lines grown at consistently optimal conditions, for the tests, using the same monoclonal antibodies as in the Workshop, had been performed 0.5–5 years prior to the Workshop study. On the other hand, nonspecific Fc-binding, wide specificity of monoclonal antibodies and a shift in antigen expression of the cells (due to poor growth conditions, involuntary induction of differentiation and other factors) must be taken into consideration upon immunological analysis.This study was supported in part by Veterans Administration RAG GrantH. G. D. is recipient of a Research Training Fellowship awarded by the Deutsche Forschungsgemeinschaft (Dr 157/1-1)     

α-Methyl-l-tryptophan as a tracer to study brain serotonergic system

Special issue devoted to Dr. Leon S. Wolfe.     

Role of proteolytic enzymes in the development of amino acid imbalance in wheat endosperm proteins

Three proteases with caseinolytic activity have been isolated from the developing wheat endosperm. Two have been purified. The activity of protease A, the one that appears early in endosperm development, is inhibited by - SH inhibitors. Protease C, the one that appears late in endosperm development, is not affected. Protease A cleaves polyaspartic acid and polyglutamic acid but not polylysine. Protease C, on the other hand, cleaves polylysine but not polyaspartic acid and polyglutamic acid. Protease C degrades lysine-rich proteins isolated from wheat endosperm more efficiently than protease A.     

The sugar moiety is a major determinant of the absorption of dietary flavonoid glycosides in man

Flavonoids are antioxidants present in plant foods. They occur mainly as glycosides, i.e. linked with various sugars. It is uncertain to what extent dietary flavonoid glycosides are absorbed from the gut. We investigated how the nature of the sugar group affected absorption of one major flavonoid, quercetin. Quercetin linked with glucose, i.e. quercetin glucoside and quercetin linked with rutinose, i.e. quercetin rutinoside, both occur widely in foods. When we fed these compounds to nine volunteers, the peak concentration of quercetin (Cmax) in plasma was 20 times higher and was reached (Tmax) more than ten times faster after intake of the glucoside (Cmax = 3.5+/-0.6 microM (mean +/- SE); Tmax < 0.5 h) than after the rutinoside (Cmax = 0.18+/-0.04 microM; Tmax = 6.0+/-1.2 h). The bioavailability of the rutinoside was only 20% of that of the glucoside. We suggest that quercetin glucoside is actively absorbed from the small intestine, whereas quercetin rutinoside is absorbed from the colon after deglycosylation. Absorption of other food components might also be enhanced by attachment of a glucose group.