Electroolfactogram responses from organotypic cultures of the olfactory epithelium from postnatal mice

Organotypic cultures of the mouse olfactory epithelium connected to the olfactory bulb were obtained with the roller tube technique from postnatal mice aged between 13 and 66 days. To test the functionality of the cultures, we measured electroolfactograms (EOGs) at different days in vitro (DIV), up to 7 DIV, and we compared them with EOGs from identical acute preparations (0 DIV). Average amplitudes of EOG responses to 2 mixtures of various odorants at concentrations of 1 mM or 100 microM decreased in cultures between 2 and 5 DIV compared with 0 DIV. The percentage of responsive cultures was 57%. We also used the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) to trigger the olfactory transduction cascade bypassing odorant receptor activation. Average amplitudes of EOG responses to 500 microM IBMX were not significantly different in cultures up to 6 DIV or 0 DIV, and the average percentage of responsive cultures between 2 and 5 DIV was 72%. The dose-response curve to IBMX measured in cultures up to 7 DIV was similar to that at 0 DIV. Moreover, the percentage of EOG response to IBMX blocked by niflumic acid, a blocker of Ca-activated Cl channels, was not significantly different in cultured or acute preparations.     

Additional minor ecdysteroid components of Leuzea carthamoides

Seventeen additional minor ecdysteroid compounds were isolated and identified from the roots of Leuzea carthamoides (Wild.) DC. Eight of them are new phytoecdysteroids: carthamoleusterone (13) is a new side-chain cyclo-ether with five-membered ring; 14-epi-ponasterone A 22-glucoside (12) is a rare and unusual natural 14 beta-OH epimer; 15-hydroxyponasterone A (11) is also new and rare with its C-15 substituted position, as well as 22-deoxy-28-hydroxymakisterone C (18) possessing secondary hydroxyl in position C-28 and 26-hydroxymakisterone C (20) with hydroxy groups in positions 25 and 26. New are also 1 beta-hydroxymakisterone C (21) and 20,22-acetonides of inokosterone (8) and integristerone A (10). Series of already known ecdysteroids: ecdysone (1), 20-hydroxyecdysone 2- and 3-acetates (3 and 4), turkesterone (6), inokosterone (7), 24-epi-makisterone A (14), and amarasterone A (22) are reported here as new constituents of L. carthamoides. Seven earlier reported Leuzea ecdysteroids: 20-hydroxyecdysone (2), ajugasterone C (5), integristerone A (9), 24(28)-dehydromakisterone A (15), 24(28)-dehydroamarasterone B (16), (24Z)-29-hydroxy-24(28)-dehydromakisterone C (17) and makisterone C (19) are also included because they are now better characterized.     

Biological activities of lignans and stilbenoids associated with plant-insect chemical interactions

Lignans and biogenetically-related secondary metabolites derived from phenylpropanoid precursors play a significant role in the defence of plants against insects. They act largely as regulators of insect feeding, but in a few cases they can influence also specific physiological functions of insects. The antifeedant activities of a series of lignans are summarised and compared with previously published data. The compounds represent either natural substances isolated from plants or their chemically transformed structural analogues. The precise mode of action of such compounds is mostly unknown. One possible mechanism might be interaction with, and disruption of, the endocrine system, which is crucial for proper development of insects and is dependent on the action of moulting hormones (ecdysteroids). This hypothesis has been tested using the specific Drosophila melanogaster B_II cell line bioassay for ecdysteroid agonists and antagonists, in which the potency of the test compound reflects the affinity of binding to the ligand-binding site of the D. melanogaster ecdysteroid receptor. The activity data are evaluated in terms of a structure-activity relationship. To explore this phenomenon, the compounds were prepared and tested alongside ecdysteroid analogues and some insect ecdysis/metamorphosis-disturbing steroidal and non-steroidal natural compounds. Several phenylpropanoids, including lignans and stilbenoids (derived from resveratrol), were evaluated with promising results. The results indicate that such phenylpropanoid-derived compounds can possess ecdysteroid antagonistic activity, which could potentially influence insect development.     

Lectinomics II. A highway to biomedical/clinical diagnostics

The review assesses current status and attempts to forecast trends in the development of lectin biorecognition technology. The progressive trend is characterized scientometrically and reflects the current transient situation, when standard low-throughput lectin-based techniques are being replaced by a novel microarray-based techniques offering high-throughput of detection. The technology is still in its infancy (validation phase), but already shows promise as an efficient tool to decipher the enormous complexity of the glycocode that influences physiological status of the cell. Further enhancement in robustness and flexibility of lectin microarrays is predicted by using recombinant and artificial lectins that will render production of lectin microarrays cost-effective and more affordable. Mass spectrometry is expected to play an important role to characterize the binding profile of new lectins. Differences in glycan recognition by lectins and anti-carbohydrate antibodies are given on a molecular basis, and strong and weak points of both biorecognition molecules in diagnosis are briefly discussed.     

Assessment of the effect of cyclic chalcone analogues on mitochondrial membrane and DNA

The cytotoxic and protective effects of selected synthetic chalcone analogues have been shown in previous studies. We studied their cytotoxic effect on the modification of mitochondrial membrane potential and on DNA. The first spectral information about the methoxy group as well as the dimethylamino substituent in E-2-arylmethylene-1-benzosuberones molecule was obtained by absorption and emission spectra. The cytotoxic effect of both cyclic chalcone analogues on DNA were detected by alkaline single-cell gel electrophoresis. Better fluorescent chalcone analogue E-2-(4′-dimethylamino-benzylidene)-1-benzosuberone was studied further in fresh isolated mitochondria. The decrease of rat liver mitochondria membrane potential (Δψ) was observed by fluorescence emission spectra. For the collapsing of mitochondrial potentials and as the negative control of mitochondrial function the CCCP uncoupler was used. The absorption maximum of the methoxy group was found at a shorter wavelength (λ = 335 nm) than that of the dimethylamino group (λ = 406 nm). The excitation spectra were very similar to the absorption spectra for both molecules but the emission spectra showed a better fluorescence for dimethylamino derivative. After the addition of E-2-(4′-dimethylamino-benzylidene)-1-benzosuberone to the intact mitochondria the decrease of mitochondrial membrane potential Δψ was observed by emisssion fluorescence spectra. Both cyclic chalcone analogues induced DNA damage, which was detected by alkaline comet assay. Mainly the apoptotic cells were detected, but necrotic cells were also present. Similarities in the percentages of DNA migration from the head were observed in both treatment groups. Both benzosuberones, with dimethylamino- and methoxy- substituent, were very active biologically, as shown by DNA results of the comet assay. Due to its better fluorescence properties, only the fluorophore with dimethylamino substituent was selected for further study of the function of rat liver mitochondria. Decline of mitochondrial function as well as mitochondrial DNA damage were evident between experimental and control groups.     

Chemically mimicked hypoxia modulates gene expression and protein levels of the sodium calcium exchanger in HEK 293 cell line via HIF-1α

Up to now a little is known about the effect of hypoxia on the sodium calcium exchanger type 1 (NCX1) expression and function. Therefore, we studied how dimethyloxallyl glycine (DMOG), an activator and stabilizer of the hypoxia-inducible factor (HIF)-1α, could affect expression of the NCX1 in HEK 293 cell line. We also tried to determine whether this activation can result in the induction of apoptosis in HEK 293 cells. We have found that DMOG treatment for 3 hours significantly increased gene expression and also protein levels of the NCX1. This increase was accompanied by a decrease in intracellular pH. Wash-out of DMOG did not result in reduction of the NCX1 mRNA and protein to original - control levels, although pH returned to physiological values. Using luciferase reporter assay we observed increase in the NCX1 promoter activity after DMOG treatment and using wild-type mouse embryonic fibroblast (MEF)-HIF-1(+/+) and HIF-1-deficient MEF-HIF-1(-/-) cells we have clearly shown that in the promoter region, HIF-1α is involved in DMOG induced upregulation of the NCX1. Moreover, we also showed that an increase in the NCX1 mRNA due to the apoptosis induction is not regulated by HIF-1α.     

Is chromatin remodeling required to build sister-chromatid cohesion?

Chromosome segregation during mitosis and meiosis depends on the linkage of sister DNA molecules after replication. These links, known as sister-chromatid cohesion, are provided by a multi-subunit complex called cohesin. Recent papers suggest that chromatin-remodeling complexes also have a role in the generation of sister-chromatid cohesion. It remains unclear whether they do so by facilitating the recruitment of cohesin to specific chromosomal sequences or by modifying an event at replication forks giving rise to cohesion between sister DNAs.     

Influenza A Virus Assembly Intermediates Fuse in the Cytoplasm

Reassortment of influenza viral RNA (vRNA) segments in co-infected cells can lead to the emergence of viruses with pandemic potential. Replication of influenza vRNA occurs in the nucleus of infected cells, while progeny virions bud from the plasma membrane. However, the intracellular mechanics of vRNA assembly into progeny virions is not well understood. Here we used recent advances in microscopy to explore vRNA assembly and transport during a productive infection. We visualized four distinct vRNA segments within a single cell using fluorescent in situ hybridization (FISH) and observed that foci containing more than one vRNA segment were found at the external nuclear periphery, suggesting that vRNA segments are not exported to the cytoplasm individually. Although many cytoplasmic foci contain multiple vRNA segments, not all vRNA species are present in every focus, indicating that assembly of all eight vRNA segments does not occur prior to export from the nucleus. To extend the observations made in fixed cells, we used a virus that encodes GFP fused to the viral polymerase acidic (PA) protein (WSN PA-GFP) to explore the dynamics of vRNA assembly in live cells during a productive infection. Since WSN PA-GFP colocalizes with viral nucleoprotein and influenza vRNA segments, we used it as a surrogate for visualizing vRNA transport in 3D and at high speed by inverted selective-plane illumination microscopy. We observed cytoplasmic PA-GFP foci colocalizing and traveling together en route to the plasma membrane. Our data strongly support a model in which vRNA segments are exported from the nucleus as complexes that assemble en route to the plasma membrane through dynamic colocalization events in the cytoplasm.     

Floral scent and its correlation with AFLP data in Sorbus

Comparisons between floral scent-based and DNA-molecular-based taxonomies are rare, yet such comparisons indicate that scent can provide useful taxonomic information. Here, we correlate the phytochemical differentiation in floral scent to the DNA-molecular-based differentiation in the genus Sorbus. Inflorescence scent patterns of the apomictic and endemic Sorbus latifolia microspecies Sorbus franconica, Sorbus adeana, and Sorbus cordigastensis originated by hybridization as well as their parental taxa Sorbus aria agg. and Sorbus torminalis were investigated with the dynamic headspace method. The scent data (presence/absence of compounds) were used to construct an UPGMA tree, to calculate a similarity matrix, and to correlate them with the published amplified fragment length polymorphism (AFLP) data of the same individuals, populations, and taxa. Flow cytometry was used to estimate the DNA-ploidy level of the taxa. Scent analyses showed a total of 68 substances, among them aromatic compounds, terpenoids, aliphatics, and nitrogen-containing compounds. The scent patterns were taxon-specific, and the number of scent components differed among taxa. The correlations with the published AFLP data on population and individual level are highly significant, indicating that the scent and AFLP data are highly congruent in the plants studied. Scent therefore provides useful taxonomic characters in Sorbus.