Validated methods for the quantification of biologically active constituents of poplar-type propolis

The validation of rapid, low-cost spectrophotometric procedures for the quantification of the three main groups of bioactive substances (flavones and flavonols, flavanones and dihydroflavonols, and total phenolics) in poplar-type propolis has been performed. A spectrophotometric assay based on the formation of an aluminium chloride complex was applied for the quantification of total flavones and flavonols using galangin as standard. Because of the high amount of flavanones and dihydroflavonols in "poplar type" propolis, the introduction of a distinct procedure for their quantification was considered of special significance and the DAB9 colorimetric method was applied for the purpose. Total phenolic content was measured by the Folin-Ciocalteu procedure using a mixture of pinocembrin and galangin as a reference. The procedures were validated using a model mixture of compounds representing the poplar-type propolis composition as found in previous studies. The accuracy (recovery) varied in the range 84-109%, and the relative standard deviation was 0.5-6.2%. The developed spectrophotometric procedures were applied to six poplar type propolis samples. The results were verified independently by a HPLC procedure. The two sets of results agreed satisfactory, as proven by Student's t-test.     

Structure of S6 kinase 1 determines whether raptor-mTOR or rictor-mTOR phosphorylates its hydrophobic motif site

The mTOR protein kinase is the target of the immunosuppressive and anti-cancer drug rapamycin and is increasingly recognized as a key regulator of cell growth in mammals. S6 kinase 1 (S6K1) is the best characterized effector of mTOR, and its regulation serves as a model for mTOR signaling. Nutrients and growth factors activate S6K1 by inducing the phosphorylation of threonine 389 in the hydrophobic motif of S6K1. As phosphorylation of Thr(389) is rapamycin sensitive and mTOR can phosphorylate the same site in vitro, it has been suggested that mTOR is the physiological Thr(389) kinase. This proposal is not supported, however, by the existence of mutants of S6K1 that are phosphorylated in vivo on Thr(389) in a rapamycin-resistant fashion. Here, we demonstrate that the raptor-mTOR complex phosphorylates the rapamycin-sensitive forms of S6K1, while the distinct rictor-mTOR complex phosphorylates the rapamycin-resistant mutants of S6K1. Phosphorylation of Thr(389) by rictor-mTOR is independent of the TOR signaling motif and depends on removal of the carboxyl terminal domain of S6K1. Because many members of the AGC family of kinases lack an analogous domain, rictor-mTOR may phosphorylate the hydrophobic motifs of other kinases.     

Redox regulation of the nutrient-sensitive raptor-mTOR pathway and complex

The raptor-mTOR protein complex is a key component of a nutrient-sensitive signaling pathway that regulates cell size by controlling the accumulation of cellular mass. How nutrients regulate signaling through the raptor-mTOR complex is not well known. Here we show that a redox-sensitive mechanism regulates the phosphorylation of the raptor-mTOR effector S6K1, the interaction between raptor and mTOR, and the kinase activity of the raptor-mTOR complex. In cells treated with the oxidizing agents diamide or phenylarsine oxide, S6K1 phosphorylation increased and became insensitive to nutrient deprivation. Conversely, the reducing reagent BAL (British anti-Lewisite, also known as 2,3-dimercapto-1-propanol) inhibits S6K1 phosphorylation and stabilizes the interaction of mTOR and raptor to mimic the state of the complex under nutrient-deprived conditions. Our findings suggest that a redox-based signaling mechanism may participate in regulating the nutrient-sensitive raptor-mTOR complex and pathway.     

Estimating intracellular calcium concentrations and buffering without wavelength ratioing

We describe a method for determining intracellular free calcium concentration ([Ca(2+)]) from single-wavelength fluorescence signals. In contrast to previous single-wavelength calibration methods, the proposed method does not require independent estimates of resting [Ca(2+)] but relies on the measurement of fluorescence close to indicator saturation during an experiment. Consequently, it is well suited to [Ca(2+)] indicators for which saturation can be achieved under physiological conditions. In addition, the method requires that the indicators have large dynamic ranges. Popular indicators such as Calcium Green-1 or Fluo-3 fulfill these conditions. As a test of the method, we measured [Ca(2+)] in CA1 pyramidal neurons in rat hippocampal slices using Oregon Green BAPTA-1 and 2-photon laser scanning microscopy (BAPTA: 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid). Resting [Ca(2+)] was 32-59 nM in the proximal apical dendrite. Monitoring action potential-evoked [Ca(2+)] transients as a function of indicator loading yielded estimates of endogenous buffering capacity (44-80) and peak [Ca(2+)] changes at zero added buffer (178-312 nM). In young animals (postnatal days 14-17) our results were comparable to previous estimates obtained by ratiometric methods (, Biophys. J. 70:1069-1081), and no significant differences were seen in older animals (P24-28). We expect our method to be widely applicable to measurements of [Ca(2+)] and [Ca(2+)]-dependent processes in small neuronal compartments, particularly in the many situations that do not permit wavelength ratio imaging.     

Functional interaction between RAFT1/FRAP/mTOR and protein kinase cdelta in the regulation of cap-dependent initiation of translation

Hormones and growth factors induce protein translation in part by phosphorylation of the eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1). The rapamycin and FK506-binding protein (FKBP)-target 1 (RAFT1, also known as FRAP) is a mammalian homolog of the Saccharomyces cerevisiae target of rapamycin proteins (mTOR) that regulates 4E-BP1. However, the molecular mechanisms involved in growth factor-initiated phosphorylation of 4E-BP1 are not well understood. Here we demonstrate that protein kinase Cdelta (PKCdelta) associates with RAFT1 and that PKCdelta is required for the phosphorylation and inactivation of 4E-BP1. PKCdelta-mediated phosphorylation of 4E-BP1 is wortmannin resistant but rapamycin sensitive. As shown for serum, phosphorylation of 4E-BP1 by PKCdelta inhibits the interaction between 4E-BP1 and eIF4E and stimulates cap-dependent translation. Moreover, a dominant-negative mutant of PKCdelta inhibits serum-induced phosphorylation of 4E-BP1. These findings demonstrate that PKCdelta associates with RAFT1 and thereby regulates phosphorylation of 4E-BP1 and cap-dependent initiation of protein translation.     

Histochemical demonstration of cholesterol in embryonic chick gonads differentiated “in vitro”

Summary Gonads from six and seven day chick embryos were cultured with the common watch-glass technique using natural and synthetic media. The presence of cholesterol in these gonads was determined with the digitonine histochemical technique. A large number of the explanted gonads showed positive reactions with the same localization as previously observed in gonads differentiating in vivo. The possible interpretation of these results is discussed.This work was supported by a grant from the Consejo Nacional de Investigaciones Cientificas y Técnicas of Argentina. Dr. Narbaitz holds a research position and Dr. Sabatini a fellowship of the same Institution.     

Aging influences cellular and molecular responses of apoptosis to skeletal muscle unloading

The influence of aging on skeletal myocyte apoptosis is not well understood. In this study we examined apoptosis and apoptotic regulatory factor responses to muscle atrophy induced via limb unloading following loading-induced hypertrophy. Muscle hypertrophy was induced by attaching a weight to one wing of young and aged Japanese quails for 14 days. Removing the weight for 7 or 14 days after the initial 14 days of loading induced muscle atrophy. The contralateral wing served as the intra-animal control. A time-released bromodeoxyuridine (BrdU) pellet was implanted subcutaneously with wing weighting to identify activated satellite cells/muscle precursor cells throughout the experimental period. Bcl-2 mRNA and protein levels decreased after 7 days of unloading, but they were unchanged after 14 days of unloading in young muscles. Bcl-2 protein level but not mRNA level decreased after 7 days of unloading in muscles of aged birds. Seven days of unloading increased the mRNA level of Bax in muscles from both young and aged birds. Fourteen days of unloading increased mRNA and protein levels of Bcl-2, decreased protein levels of Bax, and decreased nuclear apoptosis-inducing factor (AIF) protein level in muscles of aged birds. BrdU-positive nuclei were found in all unloaded muscles from both age groups, but the number of BrdU-positive nuclei relative to the total nuclei decreased after 14 days of unloading compared with 7 days of unloading. The TdT-mediated dUTP nick end labeling (TUNEL) index was higher after 7 days of unloading in both young and aged muscles and after 14 days of unloading in aged muscles. Immunofluorescent staining revealed that almost all of the TUNEL-positive nuclei were also BrdU immunopositive, suggesting that activated satellite cell nuclei (both fused and nonfused) underwent nuclear apoptosis during unloading. There were significant correlations among levels of Bcl-2, Bax, and AIF and TUNEL index. Our data are consistent with the hypothesis that apoptosis regulates, at least in part, unloading-induced muscle atrophy and loss of activated satellite cell nuclei in previously loaded muscles. Moreover, these data suggest that aging influences the apoptotic responses to prolonged unloading following hypertrophy in skeletal myocytes. satellite cells; Bcl-2 protein family     

Determination of the viability of Aeromonas hydrophila in different types of water by flow cytometry, and comparison with classical methods

The presence of Aeromonas spp. in water can represent a risk for human health. Therefore, it is important to know the physiological status of these bacteria and their survival in the environment. We studied the behavior of a strain of Aeromonas hydrophila in river water, spring water, brackish water, mineral water, and chlorinated drinking water, which had different physical and chemical characteristics. The bacterial content was evaluated by spectrophotometric and plate count techniques. Flow cytometric determination of viability was carried out using a dual-staining technique that enabled us to distinguish viable bacteria from damaged and membrane-compromised bacteria. The traditional methods showed that the bacterial content was variable and dependent on the type of water. The results obtained from the plate count analysis correlated with the absorbance data. In contrast, the flow cytometric analysis results did not correlate with the results obtained by traditional methods; in fact, this technique showed that there were viable cells even when the optical density was low or no longer detectable and there was no plate count value. According to our results, flow cytometry is a suitable method for assessing the viability of bacteria in water samples. Furthermore, it permits fast detection of bacteria that are in a viable but nonculturable state, which are not detectable by conventional methods.     

Vitamin C homeostasis in skeletal muscle cells

In skeletal muscle, vitamin C not only enhances carnitine biosynthesis but also protects cells against ROS generation induced by physical exercise. The ability to take up both ascorbic and dehydroascorbic acid from the extracellular environment, together with the ability to recycle the intracellular vitamin, maintains high cellular stores of ascorbate. In this study, we examined vitamin C transport and recycling, by using the mouse C2C12 and rat L6C5 muscle cell lines, which exhibit different sensitivity to oxidative stress and GSH metabolism. We found that: (1) both cell lines express SVCT2, whereas SVCT1 is expressed at very low levels only in proliferating L6C5 cells; furthermore L6C5 myoblasts are more efficient in ascorbic acid transport than C2C12 myoblasts; (2) C2C12 cells are more efficient in dehydroascorbic acid transport and ascorbyl free radical/dehydroascorbic acid reduction; (3) differentiation is paralleled by decreased ascorbic acid and dehydroascorbic acid transport and reduction and increased ascorbyl free radical reduction; (4) differentiated cells are more responsive to oxidative stress induced by glutathione depletion; indeed, myotubes showed increased SVCT2 expression and thioredoxin reductase-mediated dehydroascorbic acid reduction. From our data, SVCT2 and NADPH-thioredoxin-dependent DHA reduction appears to belong to an inducible system activated in response to oxidative stress.     

JNK/p53 mediated cell death response in K562 exposed to etoposide-ionizing radiation combined treatment

The study of the ability of chemotherapeutic agents and/or ionizing radiation (IR) to induce cell death in tumor cells is essential for setting up new and more efficient therapies against human cancer. Since drug and ionizing radiation resistance is an impediment to successful chemotherapy against cancer, we wanted to check if etoposide/ionizing radiation combined treatment could have a synergic effect to improve cell death in K562, a well-known human erythroleukemia ionizing radiation resistant cell line. In this study, we examined the role played by JNK/SAPK, p53, and mitochondrial pathways in cell death response of K562 cells to etoposide and IR treatment. Our results let us suppose that the induction of cell death, already evident in 15 Gy exposed cells, mainly in 15 Gy plus etoposide, may be mediated by JNK/SAPK pathway. Moreover, p53 is a potential substrate for JNK and may act as a JNK target for etoposide and ionizing radiation. Thus further investigation on these and other molecular mechanisms underlying the cell death response following etoposide and ionizing radiation exposure could be useful to overcome resistance mechanisms in tumor cells.