Specific monitoring of cardiomyogenic and endothelial differentiation by dual promoter-driven reporter systems in bone marrow mesenchymal stem cells

Vectors encoding reporter genes driven by cardiac specific myosin light chain-2v (MLC-2v), endothelial cell-specific Flk1 or Tie2 promoters were constructed. The cardiac differentiation-monitoring vector (pMLC-2v-DsRed), endothelial cell-specific monitoring vectors (pFlk1-EGFP, pTie2-EGFP) as well as the dual promoter driven-reporter genes (pMLC-2v-DsRed-Flk1-EGFP, pMLC-2v-DsRed-Tie2-EGFP) were specifically expressed in the Sca-1⁺ bone marrow mesenchymal stem cells (BMMSCs) with cardiomyogenic or endothelial lineage differentiation. The cardiac or endothelial cell-specific promoter-driven reporter vectors provide important tools for the study of stem cell fate and differentiation in vitro and future stem cell therapy for ischemic cardiomyopathy.     

Dynamics of receptor/G protein coupling in living cells

The interaction of activated G protein-coupled receptors with G proteins is a key event in signal transduction. Here, using a fluorescence resonance energy transfer (FRET)-based assay, we measure directly and in living cells the interaction of YFP-labeled alpha(2A)-adrenergic receptors with CFP-labeled G proteins. Upon agonist stimulation, a small, concentration-dependent increase in FRET was observed. No specific basal FRET was detected in the absence of agonist. Kinetics of the onset of receptor/G protein interaction were <100 ms and depended on expression levels of Galpha. Simultaneously recorded G protein-regulated inwardly rectifying K(+) channel currents revealed a maximal current response already at agonist concentrations producing submaximal FRET amplitudes. By analyzing FRET signals in the presence of a Galpha mutant, which dissociates more slowly from activated receptors, it was demonstrated that only a fraction of wild-type G proteins interacts with the activated receptor at any time. Our data suggest that alpha(2A)-adrenergic receptors and G proteins interact by rapid collision coupling and indicate that there is no significant precoupling between these receptors and G proteins.     

Calcium-dependent regulation of protein kinase D revealed by a genetically encoded kinase activity reporter

Protein kinase D (PKD) regulates many diverse cellular functions in response to diacylglycerol. To monitor PKD signaling in live cells, we generated a genetically encoded fluorescent reporter for PKD activity, DKAR (D kinase activity reporter). DKAR expressed in mammalian cells undergoes reversible fluorescence resonance energy transfer changes upon activation and inhibition of endogenous PKD. Surprisingly, we find that agonist-evoked activation of PKD is driven not only by diacylglycerol production, but by Ca(2+). Furthermore, elevation of intracellular Ca(2+), in the absence of any other stimulus, is sufficient to activate PKD. Concurrent imaging of Ca(2+), diacylglycerol, and PKD activity reveals that thapsigargin-mediated elevation of intracellular Ca(2+) is closely followed by a robust increase in diacylglycerol production, in turn followed by PKD activation. The Ca(2+)-induced production of diacylglycerol and accompanying PKD activation is dependent on phospholipase C activity. These data reveal that Ca(2+) is a major contributor to the initiation of PKD signaling through positive feedback regulation of diacylglycerol production, unveiling a new mechanism in PKD activation.     

Dynamics of apelin receptor/G protein coupling in living cells

During our research on apelin receptor (APJ) signalling in living cells with BRET and FRET, we demonstrated that apelin-13 stimulation can lead to the activation of Gαi₂ or Gαi₃ through undergoing a molecular rearrangement rather than dissociation in HEK293 cells expressing APJ. Furthermore, Gαo and Gαq also showed involvement in APJ activation through a classical dissociation model. However, both FRET signal and BRET ratio between fluorescent Gαi₁ subunit and Gβγ subunits demonstrated little change after apelin-13 stimulation. These results demonstrated that stimulation of APJ with apelin-13 causes activation of Gαi₂, Gαi₃, Gαo, Gαq; among which Gαi₂, Gαi₃ were activated through a novel rearrangement process. These results provide helpful data for understanding APJ mediated G-protein signalling.     

Dynamics of multiple trafficking behaviors of individual synaptic vesicles revealed by quantum-dot based presynaptic probe

Although quantum dots (QDs) have provided invaluable information regarding the diffusive behaviors of postsynaptic receptors, their application in presynaptic terminals has been rather limited. In addition, the diffraction-limited nature of the presynaptic bouton has hampered detailed analyses of the behaviors of synaptic vesicles (SVs) at synapses. Here, we created a quantum-dot based presynaptic probe and characterized the dynamic behaviors of individual SVs. As previously reported, the SVs exhibited multiple exchanges between neighboring boutons. Actin disruption induced a dramatic decrease in the diffusive behaviors of SVs at synapses while microtubule disruption only reduced extrasynaptic mobility. Glycine-induced synaptic potentiation produced significant increases in synaptic and inter-boutonal trafficking of SVs, which were NMDA receptor- and actin-dependent while NMDA-induced synaptic depression decreased the mobility of the SVs at synapses. Together, our results show that sPH-AP-QD revealed previously unobserved trafficking properties of SVs around synapses, and the dynamic modulation of SV mobility could regulate presynaptic efficacy during synaptic activity.     

Subpopulation-specific metabolic pathway usage in mixed cultures as revealed by reporter protein-based 13C analysis

Most large-scale biological processes, like global element cycling or decomposition of organic matter, are mediated by microbial consortia. Commonly, the different species in such consortia exhibit mutual metabolic dependencies that include the exchange of nutrients. Despite the global importance, surprisingly little is known about the metabolic interplay between species in particular subpopulations. To gain insight into the intracellular fluxes of subpopulations and their interplay within such mixed cultures, we developed here a (13)C flux analysis approach based on affinity purification of the recombinant fusion glutathione S-transferase (GST) and green fluorescent protein (GFP) as a reporter protein. Instead of detecting the (13)C labeling patterns in the typically used amino acids from the total cellular protein, our method detects these (13)C patterns in amino acids from the reporter protein that has been expressed in only one species of the consortium. As a proof of principle, we validated our approach by mixed-culture experiments of an Escherichia coli wild type with two metabolic mutants. The reporter method quantitatively resolved the expected mutant-specific metabolic phenotypes down to subpopulation fractions of about 1%.     

Age-related alterations in prolactin secretion by individual cells as assessed by the reverse hemolytic plaque assay

The objectives of this study were to determine: 1) if lactotropes from old rats, compared to those from young rats, secrete a greater amount of prolactin (PRL) per cell, 2) if the percentage of pituitary cells secreting PRL changes with age; and 3) how estradiol (E2), dopamine (DA), or thyrotropin-releasing hormone (TRH), or the combination of these factors influences both of these parameters in old rats. To meet these objectives we used the reverse hemolytic plaque assay (RHPA), because this method allows us to determine both the percentage of pituitary cells secreting prolactin during the experimental period and the amount of hormone released by each secreting pituitary cell. These parameters were measured in young (2-3 mo old) or old (17-19 mo old) female Sprague-Dawley rats. Animals were ovariectomized (OVX) for 10 days or OVX for 1 wk and then treated with E2 for 3 days. Rats were killed, anterior pituitaries were removed, and cells were enzymatically dispersed and prepared for use in the RHPA. Pituitary cells were treated in vitro with vehicle, DA, or PRL, old OVX and E2-treated rats exhibited a greater percentage of secretory cells than young at both 1 and 2 h of incubation. Administration of E2 increased the percentage of cells secreting PRL in both young and old rats. DA reduced the percentage of cells secreting PRL at the highest dose tested (10(-5) M) regardless of age or E2 status following incubation for 1 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

The multifunctional oxidase activity of ceruloplasmin as revealed by anion binding studies.

The effect of multiple binding of azide, N3-, on the structural and functional properties of ceruloplasmin (CP) has been reinvestigated by means of both spectroscopic and enzymatic techniques. High affinity binding of the anion to human CP resulted in a dramatic increase of the absorbance at 610 nm and in a concomitant decrease of the optical density at 330 nm. The oxidase activity toward Fe(II) was essentially unaffected, while turnover parameters versus nonferrous substrates dramatically changed, with an approximately 100-fold enhancement of the kcat/Km parameter. Chloride at physiological concentration proved to behave very similarly to N3- bound with high affinity, in that it not only induced the spectroscopic changes previously interpreted in terms of an intramolecular electron transfer from reduced type 1 to type 3 copper ions [Musci, G., Bonaccorsi di Patti, M.C. & Calabrese, L. (1995) J. Protein Chem. 14, 611-617], but it also enhanced some 60-fold the kcat/Km value. A different behavior was observed with chicken CP, where a decrease at 330 nm occurred without a concomitant modification at 603 nm. The chicken enzyme was less sensitive also in terms of enzymatic activity, which was nearly unchanged in the presence of either high affinity N3- or Cl-. At higher N3- concentrations, optical changes of both human and chicken CP were mainly focussed on the appearance of ligand-to-metal charge transfer bands below 500 nm, and the anion behaved as an inhibitor of the oxidase activity versus Fe(II) as well as noniron substrates. The well known bleaching of the blue chromophore could be observed, at neutral pH, only at very high N3-/CP ratios. The data presented in this paper are consistent with a mechanism of structural and functional modulation of CP by anions, that would be able to dictate the substrate specificity of the cuproprotein, and suggest the possibility that CP may act in vivo as a multifunctional oxidase.     

Estrogen induced prolactin mRNA accumulation in adult male rat pituitary as revealed by in situ hybridization

Adult male rats were injected with different doses (1, 10, and 100 micrograms) of 17 beta-estradiol daily for 5 days, and the changes in prolactin (PRL) mRNA levels were examined by in situ hybridization and cytoplasmic dot blot hybridization using cloned cDNA for rat prolactin mRNA. An increase in cytoplasmic PRL mRNA content was evident in all the animals treated with estrogen as revealed with cytoplasmic dot blot analysis. There were however, no significant differences in PRL mRNA content among the three estradiol treated groups. Cytoplasmic PRL mRNA was also demonstrated by in situ hybridization on the frozen pituitary sections using a 3H-labeled PRL cDNA probe. The number of grains per cell was increased after estrogen treatment. 3H-thymidine uptake into pituitary cells was also examined in vivo using combined techniques of immunocytochemistry and autoradiography. Although the percentage of immunoreactive PRL cells which took up thymidine in their nuclei increased to more than double after estrogen treatment, the increase in the total number of immunoreactive PRL cells was small. These results suggest that the major effect of estrogen on PRL cells is an increase in the accumulation of PRL mRNA in the individual PRL cells. The number of grains per cell was found to vary from cell to cell, both in control and estrogen treated animals. This variability is discussed in relation to the functional heterogeneity within the PRL cell population.     

Dynamics of proteasome distribution in living cells.

Proteasomes are proteolytic complexes involved in non-lysosomal degradation which are localized in both the cytoplasm and the nucleus. The dynamics of proteasomes in living cells is unclear, as is their targeting to proteins destined for degradation. To investigate the intracellular distribution and mobility of proteasomes in vivo, we generated a fusion protein of the proteasome subunit LMP2 and the green fluorescent protein (GFP). The LMP2-GFP chimera was quantitatively incorporated into catalytically active proteasomes. The GFP-tagged proteasomes were located within both the cytoplasm and the nucleus. Within these two compartments, proteasomes diffused rapidly, and bleaching experiments demonstrated that proteasomes were transported slowly and unidirectionally from the cytoplasm into the nucleus. During mitosis, when the nuclear envelope has disintegrated, proteasomes diffused rapidly throughout the dividing cell without encountering a selective barrier. Immediately after cell division, the restored nuclear envelope formed a new barrier for the diffusing proteasomes. Thus, proteasomes can be transported unidirectionally over the nuclear membrane, but can also enter the nucleus upon reassembly during cell division. Since proteasomes diffuse rapidly in the cytoplasm and nucleus, they may perform quality control by continuous collision with intracellular proteins, and degrading those proteins that are properly tagged or misfolded.     

The daily cycle of activity in the mole (Tabu europaea) and its seasonal changes, as revealed by radioactive monitoring of the nest

The daily cycle of activity in the mole (Talpa europaea) and its seasonal changes, as revealed by radioactive monitoring of the nest
Published information on the activity rhythm of the mole ( Talpa europaea ), although limited to observations on a very few moles during February-April, had indicated a basic short-term cycle of approximately 8 hours. The technique used, manual tracking of radioactively tagged individuals with portable monitors, was modified for the present study to record automatically the presence or absence of the tagged mole at its nest site. Data were obtained for all months of the year, with a total of 797 days of records from 14 male and 13 female animals. The most characteristic feature of the records, irrespective of the time of year and thus daylength, was the sharpness of the transition from inactivity to activity at dawn and the reverse shortly prior to dusk. During midwinter, when daylength was short the animals tended to be active throughout the day, with few or no rest periods at the nest, and there was only one other active period, during the night. With increasing daylength there was usually a short rest period about midday so that the animals had the classical 3-cycle daily rhythm. In summer, with the longest days and shortest nights the timing of the daytime rest period was variable and there was often no active period during the night so that the short-term cycles were less distinct.     

Green fluorescent protein as a quantitative reporter of relative promoter activity in E. coli

Green fluorescent protein (GFP) has become a valuable tool for the detection of gene expression in prokaryotes and eukaryotes. To evaluate its potential for quantitation of relative promoter activity in E. coli, we have compared GFP with the commonly used reporter gene lacZ, encoding beta-galactosidase. We cloned a series of previously characterized synthetic E. coli promoters into GFP and beta-galactosidase reporter vectors. Qualitative and quantitative assessments of these constructs show that (a) both reporters display similar sensitivities in cells grown on solid or liquid media and (b) GFP is especially well suited for quantitation of promoter activity in cells grown on agar. Thus, GFP provides a simple, rapid and sensitive tool for measuring relative promoter activity in intact E. coli cells.