Chemiluminescence of Pholasin caused by peroxynitrite

The kinetics of the oxidation of Pholasin by peroxynitrite, which leads to emission of light, were studied. The reaction shows a lag phase, which is smaller at higher peroxynitrite-to-Pholasin ratios. The total light emission approximately doubles from pH 5 to 9 and decreases precipitously to half the pH 5 value at pH 10. Dioxygen and carbon dioxide accelerate the reaction course, but they do not change significantly the reaction yield. Chemiluminescence of Pholasin is suppressed by antioxidants, but no significant shift is noticed in the time at which light emission is maximal. The chemiluminescence intensity is strongly dependent on the potassium concentration, although it is not significantly affected by lithium, cesium, or magnesium; potassium decreases luminescence. The mechanism of the peroxynitrite-induced oxidation of Pholasin may start with the reversible formation of a protein-peroxynitrite intermediate, or a first oxidation product, followed in subsequent steps by decomposition and light emission. However, many questions concerning the mechanism of the light emission remain to be elucidated.     

ERK1 activation is required for S-phase onset and cell cycle progression after fertilization in sea urchin embryos

Fertilization of sea urchin eggs results in a large, transient increase in intracellular free Ca2+ concentration that is responsible for re-initiation of the cell division cycle. We show that activation of ERK1, a Ca2+-dependent MAP kinase response, is required for both DNA synthesis and cell cycle progression after fertilization. We combine experiments on populations of cells with analysis at the single cell level, and develop a proxy assay for DNA synthesis in single embryos, using GFP-PCNA. We compare the effects of low molecular weight inhibitors with a recombinant approach targeting the same signalling pathway. We find that inhibition of the ERK pathway at fertilization using either recombinant ERK phosphatase or U0126, a MEK inhibitor, prevents accumulation of GFP-PCNA in the zygote nucleus and that U0126 prevents incorporation of [3H]-thymidine into DNA. Abrogation of the ERK1 signalling pathway also prevents chromatin decondensation of the sperm chromatin after pronuclear fusion, nuclear envelope breakdown and formation of a bipolar spindle.     

Short Time-Response of Psammic Communities of Rotifera to Abiotic Changes in their Habitat

Sandy microhabitats and their fauna are usually characterised as extremely unstable and unpredictable. The aim of the paper is to evaluate the scale and the character of these phenomena. Rotifer composition and density and abiotic variables were investigated every half a day in the hygroarenal of the eutrophic Mikołajskie Lake.In total, 62 species were found with only two species occurring permanently throughout the study period. Densities in general were extremely variable and strong fluctuations were observed even at 12 h intervals. On average, psammophilic rotifers dominated the community (74%) while psammoxenes and psammobionts constituted only 18 and 8%, respectively. The most stable group were the psammophiles. The results support the presumption that fluctuating environments are ideal for organisms that reproduce quickly, may colonize vacant habitats rapidly and have wide niches, i.e. eurybionts.     

Developmental-stage-specific assembly of ParB complexes in Streptomyces coelicolor hyphae

In Streptomyces coelicolor ParB is required for accurate chromosome partitioning during sporulation. Using a functional ParB-enhanced green fluorescent protein fusion, we observed bright tip-associated foci and other weaker, irregular foci in S. coelicolor vegetative hyphae. In contrast, in aerial hyphae regularly spaced bright foci accompanied sporulation-associated chromosome condensation and septation.     

Involvement of glial cells in rhythmic size changes in neurons of the housefly's visual system

In the housefly's first optic neuropile, or lamina, the axons of two classes of monopolar cell interneurons, L1 and L2, exhibit a daily rhythm of size changes: swelling during the day, and shrinking by night. At least for the L2 cells this rhythm is circadian. Moreover, epithelial glial cells that enwrap each lamina cartridge, its monopolar cell axons, and their surrounding crown of input photoreceptor terminals also change size, but in the opposite direction to the changes in L1 and L2-swelling by night and shrinking by day. The rhythmic changes in glia indicate the possible involvement of these cells in the lamina's circadian system. To examine their role in regulating the rhythmic changes of L1 and L2's axon sizes we have injected three chemicals into the haemolymph of the fly's head: fluorocitrate (FL) and iodoacetate (IAA), which affect the metabolism of glial cells, and octanol (OC), which closes gap junction channels. All chemicals exerted an effect on L1 and L2, which depended on the time of injection, the drug concentration, and the postinjection times at which we examined the fly's brains. Moreover, day/night changes in the axon sizes of L1 and L2 were increased in FL- and IAA-treated flies, indicating that glial cells may normally inhibit these changes by regulating the sizes of L1 and L2's axons during the day and night. In turn, lack of a day/night rhythm in L1 and L2 after OC injections shows that the rhythm's persistence depends on communication between the lamina cells through gap junction channels.     

Effect of Rho-associated kinase inhibition on actin cytoskeleton structure and calcium response in glioma C6 cells

The role of actin cytoskeleton functional state in glioma C6 cell morphology and calcium signaling was investigated through modification of myosin II activity by blocking Rho-associated kinase with the specific inhibitor Y-27632. Treatment of glioma C6 cells with ROCK inhibitor resulted in actin cytoskeleton reorganization and also in the changed shape and distribution of mitochondria. Changes in the distribution of ER, the main calcium store in glioma C6 cells, were not visible. The inhibition of myosin II activity influences the first phase of calcium signaling evoked by agonist, and both phases of thapsigargin-evoked calcium response. We suggest that the observed increase in Ca2+ release from intracellular stores induced by IP3 formation as well as inhibition of SERCA ATPase is at least in part related to severely affected mitochondria. Enhancement of capacitative calcium entry evoked by thapsigargin is probably associated with the reorganization of the acto-myosin II system. ATP-induced calcium response presents no changes in the second phase. We observed that ATP stimulation of Y-27632 pretreated cells leads to immediate morphological rearrangement of glioma C6 cells. It is a consequence of actin cytoskeleton reorganization: formation of stress fibers and relocation of phosphorylated myosin II to actin filaments. It seems that the agonist-evoked strong calcium signal may be sufficient for myosin II activation and the stress fiber organization. This is the first work showing the dependence between the functional state of the acto-myosin II system and calcium signaling stressing the reversible character of this relationship.     

A simplified method for purification of recombinant soluble DnaA proteins

An improved, simplified method for the purification of recombinant, tagged DnaA proteins is described. The presented protocol allowed us to purify soluble DnaA proteins from two different bacterial species: Helicobacter pylori and Streptomyces coelicolor, but it can most likely also be used for the isolation of DnaA proteins from other bacteria, as it was adapted for Mycobacterium tuberculosis DnaA. The isolation procedure consists of protein precipitation with ammonium sulphate followed by affinity chromatography. The composition of the buffers used at each purification step is crucial for the successful isolation of the recombinant DnaA proteins. The universality of the method in terms of its application to differently tagged proteins (His-tagged or GST-tagged) as well as different properties of purified proteins (e.g., highly aggregating truncated forms) makes the protocol highly useful for all studies requiring purified and active DnaA proteins.     

Retrograde and local destination transfer of uterine prostaglandin E2 in early pregnant sow and its physiological consequences

The local destination transfer of prostaglandin E2 (PGE2) from the uterine lymph to arterial blood supplying the ovary and its retrograde transfer to arterial blood supplying the uterine horn and the effect of additional delivery of PGE2 into the ovary on the secretion of steroid hormones was studied in early pregnant gilts. The injection of PGE2 under the perimetrium caused an increase (P<0.001) in PGE2 concentration in both uterine venous effluent and ovarian and uterine arterial blood. The infusion of PGE2 into the ovarian artery increased the concentration of progesterone in ovarian venous blood on day 13 of pregnancy during (P<0.05) and after (P<0.001) infusion, and on day 14 of pregnancy after infusion (P<0.01). In conclusion, local destination transfer of PGE2 from uterine lymph and venous blood to the ovary may affect luteal function, and retrograde transfer of PGE2 to the arterial blood supplying the uterus may contribute to the prevention of regressive changes of the endometrium in early pregnant gilts.