Endothelin-induced, long lasting, and Ca2+ influx-independent blockade of intrinsic secretion in pituitary cells by Gz subunits

The G protein-coupled receptors in excitable cells have prominent roles in controlling Ca2+-triggered secretion by modulating voltage-gated Ca2+ influx. In pituitary lactotrophs, spontaneous voltage-gated Ca2+ influx is sufficient to maintain prolactin release high. Here we show that endothelin in picomolar concentrations can interrupt such release for several hours downstream of spontaneous and high K+-stimulated voltage-gated Ca2+ influx. This action occurred through the Gz signaling pathway; the adenylyl cyclase-signaling cascade could mediate sustained inhibition of secretion, whereas rapid inhibition also occurred at elevated cAMP levels regardless of the status of phospholipase C, tyrosine kinases, and protein kinase C. In a nanomolar concentration range, endothelin also inhibited voltage-gated Ca2+ influx through the G i/o signaling pathway. Thus, the coupling of seven-transmembrane domain endothelin receptors to Gz proteins provided a pathway that effectively blocked hormone secretion distal to Ca2+ entry, whereas the cross-coupling to G i/o proteins reinforced such inhibition by simultaneously reducing the pacemaking activity.     

Barley aleurone cells contain two types of vacuoles. Characterization Of lytic organelles by use of fluorescent probes

Light microscopy was used to study the structure and function of vacuoles in living protoplasts of barley (Hordeum vulgare cv Himalaya) aleurone. Light microscopy showed that aleurone protoplasts contain two distinct types of vacuole: the protein storage vacuole and a lysosome-like organelle, which we have called the secondary vacuole. Fluorescence microscopy using pH-sensitive fluorescent probes and a fluorogenic substrate for cysteine proteases showed that both protein storage vacuoles and secondary vacuoles are acidic, lytic organelles. Ratio imaging showed that the pH of secondary vacuoles was lower in aleurone protoplasts incubated in gibberellic acid than in those incubated in abscisic acid. Uptake of fluorescent probes into intact, isolated protein storage vacuoles and secondary vacuoles required ATP and occurred via at least two types of vanadate-sensitive, ATP-dependent tonoplast transporters. One transporter catalyzed the accumulation of glutathione-conjugated probes, and another transported probes not conjugated to glutathione.     

Small mammals as sentinels of antimicrobial-resistant staphylococci

A total of 39 coagulase-negative staphylococci and seven Staphylococcus aureus strains were isolated from small mammal feces, i.e., the striped field mouse (Apodemus agrarius) and the yellow-necked mouse (A. flavicollis) in two sampling areas, deciduous forest and karst plains. MALDI-TOF analysis revealed five species of coagulase-negative staphylococci: S. sciuri, S. hominis, S. warneri, S. haemolyticus, and S. xylosus. All strains were susceptible to tetracycline, linezolid, vancomycin, and teicoplanin. Three MRSA strains with the mecA gene were detected. The beta-lactamase gene blaZ was detected in ampicillin-resistant staphylococci and in the high-level resistant strains (oxacillin over 2 mg/L) mecA gene. The mecC gene was not detected by PCR. Erythromycin-resistant staphylococci harbored the ermC gene and/or the efflux gene msrA. There were no detectable dfr genes in trimethoprim-resistant staphylococci and the rifampicin-resistant strains were without mutation in the rpoB gene. In summary, wild small mammals may serve as sentinels of mecA-positive S. aureus with erythromycin resistance genes ermC and efflux msrA. Small mammals appear to be useful indicators of antibiotic resistance.     

Calcium signaling and secretory responses in agonist-stimulated pituitary gonadotrophs

In cultured pituatary gonadotrophs, gonadotropin-releasing hormone (GnRH) caused dose-dependent and biphasic increases in cytoplasmic calcium concentration ([Ca²⁺]_i) and LH release. Both extra- and intracellular calcium pools participate in GnRH-induced elevation of [Ca²⁺]_i and LH secretion. The spike phase of the [Ca²⁺]_i response represents the primary signal derived predominantly from the rapid mobilization of intracellular Ca²⁺. In contrast, the prolonged phase of the Ca²⁺ signal depends exclusively on Ca²⁺ entry from the extracellular pool. The influx of Ca²⁺ occurs partially through dihydropyridine-sensitive calcium channels. Both [Ca²⁺]_i and LH responses to increasing concentrations of GnRH occur over very similar time scales, suggesting that increasing degrees of receptor occupancy are transduced into amplitude-modulated Ca²⁺ responses, which in turn activate exocytosis in a linear manner. However, several lines of evidence indicated the complexity over the relationship between Ca²⁺ signaling and LH exocytosis. In contrast to [Ca²⁺]_i measurements in cell suspension, single cell Ca²⁺ measurements revealed the existence of a more complicated pattern of Ca²⁺ response to GnRH, with a biphasic response to high agonist doses and prominent oscillatory responses to lower GnRH concentrations, with a log-linear correlation between GnRH dose and the frequency of Ca²⁺ spiking. In addition, analysis of the magnitudes of the magnitudes of the [Ca²⁺]_i and LH responses of gonadotrophs to a wide range of GnRH concentrations in the presence and absence of extracellular Ca²⁺, and to K⁺ and phorbol ester stimulation, showed non-linearity between these parameters with amplification of [Ca²⁺]_i-mediated exocytosis. Studies on cell depleted of protein kinase C under conditions that did not change the LH pool suggested the participation of protein kinase C in this amplication, especially during the plateau phase of the secretory response to GnRH.     

Induction of free radicals in seeds by high intensive flashes and the relevant phosphorus metabolism in the seedling

Influence of high intensive flashes on the yield of free radicals in intact seeds and excised embryonic axis, endosperm, and seed coat, and its resulting effect on seedling growth, total biomass production and phosphorus metabolism in wheat (Triticum aeativum), vetch (Vicia sativa L.) and onion (Allium cepa L.) was studied. Free radicals (f.r.) were formed mainly in seed coat and not in the endosperm. Vetch seeds after irradiation had 20.76 X 10¹³ f.r. g^-1 dry intact seed and 17.30 X 10¹³ f.r. g^-1 dry seed coat. Excised seed coats exposed to irradiation also yielded 17.28 × 10¹³ f.r. g^-1 dry matter. High irradiance “white light” flashes induced more f.r. than a monochromatic one of the same photon content. Red (650 nm), farred (750 nm) and even infra-red (1100 nm) radiation did not initiated f.r. formation but resulted in their decay in samples irradiated earlier by “white”, blue and green parts of the spectrum. Blue irradiation of seeds led to the decrease in the length of shoots and roots in comparison to “white”, green and red irradiation but their biomass increased faster than in the seedlings obtained from non-irradiated or irradiated with “white” and green radiation. The quantity of total acid soluble phosphorus followed a sequence with respect to wavelength of radiation: 436 nm > 650 nm> > 540 nm > non-irradiated > 300–800 nm. Quantity of inorganic phosphorus remained unaffected by different spectral character of radiation. The quantity of organic acid soluble nucleic phosphorus and acid insoluble polyphosphates was higher in samples irradiated with red beams (650 ± 6 nm).     

Structural Insights into the Function of P2X4: An ATP-Gated Cation Channel of Neuroendocrine Cells

The P2X4 receptor (P2X4R) is a member of a family of ATP-gated cation channels that are composed of three subunits. Each subunit has two transmembrane (TM) domains linked by a large extracellular loop and intracellularly located N- and C-termini. The receptors are expressed in excitable and non-excitable cells and have been implicated in the modulation of membrane excitability, calcium signaling, neurotransmitter and hormone release, and pain physiology. P2X4Rs activate rapidly and desensitize within the seconds of agonist application, both with the rates dependent on ATP concentrations, and deactivate rapidly and independently of ATP concentration. Disruption of conserved cysteine ectodomain residues affects ATP binding and gating. Several ectodomain residues of P2X4R were identified as critical for ATP binding, including K67, K313, and R295. Ectodomain residues also account for the allosteric regulation of P2X4R; H140 is responsible for copper binding and H286 regulates receptor functions with protons. Ivermectin sensitized receptors, amplified the current amplitude, and slowed receptor deactivation by binding in the TM region. Scanning mutagenesis of TMs revealed the helical topology of both domains, and suggested that receptor function is critically dependent on the conserved Y42 residue. In this brief article, we summarize this study and re-interpret it using a model based on crystallization of the zebrafish P2X4.1 receptor.     

Participation of the Lys313-Ile333 sequence of the purinergic P2X4 receptor in agonist binding and transduction of signals to the channel gate

To study the roles of the Lys(313)-Ile(333) ectodomain sequence of the rat P2X(4) receptor in ATP binding and transduction of signals to the channel gate, the conserved Lys(313), Tyr(315), Gly(316), Ike(317), Arg(318), Asp(320), Val(323), Lys(329), Phe(330), and Ile(333) residues were mutated. Current recordings were done on lifted cells and ATP was applied using an ultrafast solution-switching system. The rates of wild type channel opening and closing in the presence of ATP, but not the rate of washout-induced closing, were dependent on agonist concentration. All mutants other than I317A were expressed in the plasma membrane at comparable levels. The majority of mutants showed significant changes in the peak amplitude of responses and the EC(50) values for ATP. When stimulated with the supramaximal (1.4 mm) ATP concentration, mutants also differed in the kinetics of their activation, deactivation, and/or desensitization. The results suggest a critical role of the Lys(313) residue in receptor function other than coordination of the phosphate group of ATP and possible contribution of the Tyr(315) residue to the agonist binding module. The pattern of changes of receptor function by mutation of other residues was consistent with the operation of the Gly(316)-Ile(333) sequence as a signal transduction module between the ligand binding domain and the channel gate in the second transmembrane domain.     

Habitat variation in species composition of flea assemblages on small mammals in central Europe

We studied patterns of variation in species composition of flea assemblages on small mammals across different habitats of Slovakia and compared flea species composition within and across host species among habitats. We asked (1) how variable the composition of flea assemblages is among different populations of the same host occurring in different habitats and (2) whether the composition of flea assemblages in a habitat is affected either by species composition of hosts or by environmental affinities of this habitat. Between-habitat similarity in flea species composition increased with an increase in the similarity in host species composition. Species richness of flea assemblages of a host species correlated positively with mean number of cohabitating host species but not with the number of habitats occupied by a host species. Results of the ordination of flea collections from each individual host demonstrated that the first five principal components explained most of the variance in species composition of flea assemblages. The segregation between rodent and insectivore flea assemblages was easily discerned from the ordination diagram when flea assemblages were plotted according to their hosts. When flea assemblages were plotted according to their habitat affinities, the distinction of habitats based on variation in flea composition was not as clear. The results of ANOVA of each principal component showed the significant effect of both host species and habitat type. The variation in each principal component was explained better by the factor of host species compared with the factor of habitat type. Multidimensional scaling of flea assemblages within host species across habitats demonstrated that among-habitat variation in flea composition was manifested differently in different hosts.     

Relationship between host abundance and parasite distribution: inferring regulating mechanisms from census data

1. We studied the effect of host abundance on parasite abundance and prevalence using data on 57 associations of fleas (Siphonaptera) and their mammalian hosts from Slovakia. 2. We assumed that flea-induced host mortality could be inferred from the relationship between flea aggregation and flea abundance, whereas host-induced flea mortality could be inferred from the relationship between flea abundance or aggregation and host abundance. 3. Relationships between flea abundance or prevalence and host abundance were either negative (in 23 flea-host associations) or absent (in 34 flea-host associations). Negative relationships between flea abundance and host abundance were always accompanied by negative relationships between flea prevalence and host abundance. 4. The link between flea abundance/prevalence and host abundance was evaluated as the coefficient of determination of the respective regressions. Across flea-host associations, this link decreased with an increase in the degree of flea aggregation (measured as a parameter b of Taylor's power law). 5. Mean crowding of fleas decreased with an increase of host abundance in eight flea-host associations, being asymptotic in four of them. On the other hand, mean crowding of fleas increased with an increase in flea abundance in 49 flea-host associations, being asymptotic in 15 of them. 6. Results of this study suggest that different flea-host associations are governed by different regulating mechanisms, but different regulation mechanisms may act simultaneously within the same flea-host associations.