Image analysis and in vivo imaging as tools for investigation of productivity dynamics in anthocyanin-producing cell cultures of Daucus carota

An anthocyanin-producing suspension culture of Daucus carota (L.) cv. Flakkese was used as model system to study secondary metabolite production in cell culture at the individual cell level. An approach was set up in which growth and production of anthocyanins were investigated using a combination of biochemical analysis, image (colour) analysis and in vivo imaging. This novel approach was used to segment the culture in different subpopulations and dissect the productive process in the cell culture grown under two different conditions, known to differ mainly for oxygen supply and mixing intensity (volume of 50 ml or 20 ml in 250 ml flasks). The 20 ml batch cultures gave a higher content and yield of anthocyanins, which depended on a complex balance between events that positively or negatively affected anthocyanin production. A model is proposed in which the different ability of cells to respond to environmental stimuli and stress depends on the different amount of anthocyanins accumulated within cells.     

The prion protein and its paralogue Doppel affect calcium signaling in Chinese hamster ovary cells

The function of the prion protein (PrP(c)), implicated in transmissible spongiform encephalopathies (TSEs), is largely unknown. We examined the possible influence of PrP(c) on Ca(2+) homeostasis, by analyzing local Ca(2+) fluctuations in cells transfected with PrP(c) and Ca(2+)-sensitive aequorin chimeras targeted to defined subcellular compartments. In agonist-stimulated cells, the presence of PrP(c) sharply increases the Ca(2+) concentration of subplasma membrane Ca(2+) domains, a feature that may explain the impairment of Ca(2+)-dependent neuronal excitability observed in TSEs. PrP(c) also limits Ca(2+) release from the endoplasmic reticulum and Ca(2+) uptake by mitochondria, thus rendering unlikely the triggering of cell death pathways. Instead, cells expressing Doppel, a PrP(c) paralogue, display opposite effects, which, however, are abolished by the coexpression of PrP(c). These findings are consistent with the functional interplay and antagonistic role attributed to the proteins, whereby PrP(c) protects, and Doppel sensitizes, cells toward stress conditions.     

Quantitative appraisal of murine filariasis confirms host strain differences but reveals that BALB/c females are more susceptible than males to Litomosoides sigmodontis

Litomosoides sigmodontis, a rodent filarial nematode, can infect inbred laboratory mice, with full development to patency in the BALB/c strain. Strains such as C57BL/6 are considered resistant, because although filarial development can occur, circulating microfilariae are never detected. This model system has, for the first time, allowed the power of murine immunology to be applied to fundamental questions regarding susceptibility to filarial nematode infection. As this is a relatively new model, many aspects of the biology remain to be discovered or more clearly defined. We undertook a major analysis of 85 experiments, to quantitatively assess differences in filarial survival and reproduction in male versus female and BALB/c versus C57BL/6 mice over the full course of infection. This large dataset provided hard statistical support for previous qualitative reviews, including observations that the resistant phenotype of C57BL/6 mice is detectable as early as 10 days postinfection (dpi). An unexpected finding, however, was that filarial survival was reduced in male BALB/c mice compared to their female counterparts. Worm recovery as well as the prevalence and density of microfilariae were higher in female compared with male BALB/c mice. Therefore, L. sigmodontis bucks the filarial trend of increased susceptibility in males. This could be partially explained by the different anatomical locations of adult L. sigmodontis versus lymphatic filariae. Interestingly, the effects of BALB/c sex upon microfilaremia were independent of worm number. In summary, this study has significantly refined our understanding of the host-L. sigmodontis relationship and, critically, has challenged the dogma that males are more susceptible to filarial infection.     

Identification of the main oxidation products of 8-methoxy-2'-deoxyguanosine by singlet molecular oxygen

It is now well established that oxidation of 2'-deoxyguanosine (dGuo) in DNA by singlet molecular oxygen [O2 (1Delta(g))] produces 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), whereas the main degradation products of free dGuo in aqueous solution have been identified as the two diastereomers of spiroiminodihydantoin nucleoside. Interestingly, O2 (1Delta(g))-mediated oxidation of free 8-oxodGuo gives rise to a pattern of degradation products that is different from that observed when the nucleoside is inserted into DNA. The reasons for these differences and the mechanisms involved in the oxidation reactions are not yet completely understood for either dGuo or 8-oxodGuo, either free or within DNA. In the present work, we report a study of the reaction of O2 (1Delta(g)) toward a modified nucleoside, 8-methoxy-2'-deoxyguanosine (8-MeOdGuo), either free or incorporated into an oligonucleotide. The reason for the choice of 8-MeOdGuo as a chemical model to study in more detail the oxidation pathways of 8-oxodGuo or, more precisely, of the tautomeric 8-hydroxy-2'-deoxyguanosine was dictated by the fact that only the 7,8-enolic tautomer is present in the molecule. The thermolysis of an endoperoxide of a naphthalene derivative as a clean chemical source of 18O-labeled O2 (1Delta(g)) was used to oxidize 8-MeOdGuo. The main O2 (1Delta(g)) oxidation products that were separated and analyzed by HPLC coupled to tandem mass spectrometry were identified as the 2'-deoxyribonucleoside derivatives of 2,2,4-triamino-5-(2H)oxazolone, 2,5-diamino-4H-imidazol-4-one together with the methyl-substituted derivatives of spiroiminodihydantoin, oxidized iminoallantoin and urea. On the other hand, O2 (1Delta(g)) oxidation of 8-MeOdGuo-containing oligonucleotide generated imidazolone as the predominant degradation product. These results provided new mechanistic insights into the reactions of O2 (1Delta(g)) with purine nucleosides.     

Photoinactivation of Escherichia coli using porphyrin derivatives with different number of cationic charges

The photodynamic effect of meso-substituted cationic porphyrins, 5-[4-(trimethylammonium)phenyl]-10,15,20-tris(2,4,6-trimethoxyphenyl)porphyrin iodide 1, 5,10-di(4-methylphenyl)-15,20-di(4-trimethylammoniumphenyl)porphyrin iodide 2 and 5-(4-trifluorophenyl)-10,15,20-tris(4-trimethylammoniumphenyl)porphyrin iodide 3, have been investigated in both homogeneous medium bearing photooxidizable substrates and in vitro on a typical gram-negative bacterium Escherichia coli. Absorption and fluorescence spectroscopic studies were compared in N,N-dimethylformamide. Fluorescence quantum yields (varphiF) of 0.10, 0.06 and 0.08 were calculated for porphyrins 1, 2 and 3, respectively. The singlet molecular oxygen, O2(1Deltag), production was evaluated using 9,10-dimethylanthracene yielding values of 0.66, 0.36 and 0.42 for porphyrins 1, 2 and 3, respectively. Guanosine 5'-monophosphate was used as biological substrate model. Similar decomposition of guanosine 5'-monophosphate was obtained using these cationic porphyrins as sensitizer. In biological medium, photosensitized inactivation of E. coli was analyzed using cells without and with one washing step. E. coli cultures were treated with sensitizer at 37 degrees C for 30 min in dark. In both procedures, a higher photoinactivation of cells (>99.999%) was found for cells treated with 10 microM of tricationic porphyrin 3 and irradiated for 5 min with visible light. Porphyrins 1 and 2 only show an important photodamage when the cells are irradiated without washing step. These results indicated that the tetracationic porphyrin 3 could be a promising sensitizer with potential applications in the photoinactivation of bacterial cells by photodynamic therapy.     

Helicobacter pylori, T cells and cytokines: the "dangerous liaisons"

Helicobacter pylori infection is the major cause of gastroduodenal pathologies, but only a minority of infected patients develop chronic and life threatening diseases, as peptic ulcer, gastric cancer, B-cell lymphoma, or autoimmune gastritis. The type of host immune response against H. pylori is crucial for the outcome of the infection. A predominant H. pylori-specific Th1 response, characterized by high IFN-gamma, TNF-alpha, and IL-12 production associates with peptic ulcer, whereas combined secretion of both Th1 and Th2 cytokines are present in uncomplicated gastritis. Gastric T cells from MALT lymphoma exhibit abnormal help for autologous B-cell proliferation and reduced perforin- and Fas-Fas ligand-mediated killing of B cells. In H. pylori-infected patients with autoimmune gastritis cytolytic T cells infiltrating the gastric mucosa cross-recognize different epitopes of H. pylori proteins and H+K+ ATPase autoantigen. These data suggest that peptic ulcer can be regarded as a Th1-driven immunopathological response to some H. pylori antigens, whereas deregulated and exhaustive H. pylori-induced T cell-dependent B-cell activation can support the onset of low-grade B-cell lymphoma. Alternatively, H. pylori infection may lead in some individuals to gastric autoimmunity via molecular mimicry.     

Negative effects of elevated testosterone on female fecundity in zebra finches

Although factors influencing androgen deposition in the avian egg and its effects on nestling fitness are recently receiving considerable attention, little is known about the potential costs of high testosterone levels in the females. Our study aimed at determining the effect of injections of testosterone (T) in female zebra finches (Taeniopygia guttata), on clutch size, egg mass, yolk mass, and yolk androgen content. Females were given a single bolus injection of T in a range of doses after laying the first egg. Results show that administration of T negatively affected clutch size; the strength of this effect increased with increasing doses of T. Females injected with the highest testosterone dose showed suppressed oviposition of the third and the fourth eggs. Interestingly, testosterone administration made females produce eggs with relatively large yolks, suggesting that T may mediate the trade-off between number and size of eggs. Testosterone injection resulted in elevated levels of androgen in the eggs, in contrast to control clutches, which showed a decreasing pattern of androgen concentration along the laying sequence. We conclude that high androgen investment in eggs may be limited by physiological requirements of the ovulatory process.