Outbreeding depression in the common frog, <Emphasis Type=Italic>Rana temporaria</Emphasis>

Theory suggests that parental relatedness is a continuous variable with a fitness optimum that we heretoforth will refer to as optimal outbreeding. In the present paper, we test this proposition from a conservation (translocation) perspective. Amphibians are facing a global decline and many amphibian populations are today small and threatened by extinction. Because genetic differentiation is often high between amphibian populations, they could be particularly sensitive to outbreeding depression, e.g. due to breakdown of locally adapted gene complexes. We tested if outbreeding would reduce fitness in common frogs, Rana temporaria, crossed from a large and an isolated, small population, separated by 130km, using artificial fertilization. For females from the large population, tadpoles were significantly smaller and more malformed in crosses with males from the small population, than with males from the large population. For the small population, however, no significant paternal genetic effects could be found. The difference in response to outbreeding between populations was accompanied with significant differences in the importance of maternal effects. We conclude that care should be taken when translocating frogs between distantly related populations to avoid outbreeding depression.     

Resident and circulating mast cells in propulsative organs of frog <Emphasis Type=Italic>Rana temporaria</Emphasis>

A histochemical and ultrastructural examination of mast cells (MCs) in “blood” and lymph hearts of the adult frog Rana temporaria showed that they are represented by two populations, i.e., resident and circulating MCs. Resident cardiac MCs have an oval or elongated shape and are located in the atrial and ventricular myocardium, as well as in connective tissue of the epicardium. Circulating MCs were identified in heart lumen lacunas and in narrow clefts produced by ventricular trabecular myocardium, as well as in blood-filled atrial and ventricular central cavities. The small round shape circulating MCs resemble lymphocytes, but their cytoplasm is filled with granules that are ultrastucturally similar to granules of cardiac resident MCs. In the lymph heart, elongated resident MCs are located in the interstitial space between the cross-striated muscle fibers and smooth muscle cells of afferent and efferent heart valves. Round circulating MCs are occasionally visible in the cavity of the lymph heart. More commonly, circulating MCs are found in the lymphatic sinus cavity located adjacent to lymph hearts. In certain parts of the lymphatic sinus walls, MCs form clusters that are in tight contact with the mesothelial cells that line the lymphatic cavity. Our histochemical investigation revealed that both resident and circulating MCs of the propulsative organs are strongly alcian blue positive, but are weakly red after safranin staining and weakly metachromatic with toluidine blue. The presence of populations of circulating MCs in adult frogs suggests that there are differences in MC biology in lower and higher vertebrates.     

Morphofunctional changes in the small intestine epithelium of the frog <Emphasis Type=Italic>Rana temporaria</Emphasis> in the course of hibernation

The structure and function of small intestine epithelium have been studied in overwintering frogs at various stages of hibernation. Experiments on the in vitro absorption of arginine vasotocin (AVT) have shown that the epithelium ability for absorption of this nonapeptide without hydrolysis persists during hibernation. However, there is a decline in AVT absorption observed in January–February and later on (as compared with that in October–December) which is most pronounced in March–April. Changes in the epithelial structure appear by midwinter and are growing towards spring. In April–May, enterocyte height decreased by 33%, microvilli length by 40%, and number of mitochondria by 57% relative to the values at the beginning of hibernation. The absence of signs of breakdown attests to the adaptive nature of the observed changes. The dynamics of the studied parameters reveals morphological and functional plasticity of small intestinal epithelium in Rana temporaria over the hibernation period.     

Peculiarities of Ca<Superscript>2+</Superscript>-regulation of functional activity of myocardium of frog <Emphasis Type=Italic>Rana temporaria</Emphasis>

To elucidate role of intra- and extracellular Ca²⁺ in regulation of rhythm and strength of frog heart contractions, there were studied ECC and isometric contraction of myocardium preparations in response to verapamil, adrenaline, and blockers of α- and β-adrenoreceptors. It has been shown that after an intramuscular injection of verapamil (6 mg/kg), bradycardia develops, the heart rate (HR) decreasing by 50–70%. Further, the cardiac arrest occurred; however, administration to the animals of adrenaline (100 mg/kg) restored the cardiac rhythm for a short while. After an intramuscular injection of adrenaline at doses of 0.1–10 mg/kg, no essential changes were observed in the potential action amplitude and HR; an increase of the administered adrenalin concentration to 100 mg/kg was not accompanied by the cardiac rhythm stimulation, as this takes place in homoiothermal animals and human; on the contrary, an essential HR deceleration was revealed. Phentolamine (5 mg/kg) gradually decelerated HR rhythm by 32–45%. The potential amplitude changed insignificantly. A subsequent intracardiac injection of adrenaline (100 mg/kg) on the background of block of α-adrenoreceptors produced acceleration of the rhythm (by 15–21%) and fall of the electrogram amplitude. These results can indicate that in the frog heart phentolamine interacts predominantly with α 1-adrenoreceptors. An intracardial administration of propranolol (1 mg/kg) to frogs promoted inhibition of β-adrenergic receptors and produced a gradual cardiac rhythm deceleration. In experiments on assessment of verapamil effect on the character of contractions this preparation at a concentration of 150 μM was established to produce a significant dose-dependent decrease of the contraction strength. A rise of verapamil concentration in the sample to 200 μM led to a decrease of the amplitude, on average, by 68–70% and in individual preparations—by 80–85%; however, administration into the sample of adrenaline (10 μM) restored the cardiac contraction strength. Adrenaline (1 nM–100 μM) increased markedly the contraction amplitude. Phentolamine (10 μM) did not inhibit transmission of contractile signal to cardiomyocytes; this was manifested in that the contraction amplitude after addition of adrenaline (10 μM) into the sample was approximately the same as in the sample containing no phentolamine. Propranolol (10 μM) eliminated the stimulatory action of adrenaline (10 μM). The results of these experiments indicate that in the frog ventricular cardiomyocytes the main adrenaline acceptors are β-adrenoreceptors.     

Cryopreservation of <Emphasis Type=Italic>Robinia</Emphasis><Emphasis Type=Italic>pseudoacacia</Emphasis>

Cryopreservation of Robinia pseudoacacia explants by vitrification achieved 78% survival following the stepwise preculture of shoot tips in (0.3 + 0.5 + 0.7 M) sucrose with a 80 min incubation in PVS2; compared to 87% survival after desiccation of explants to 30% water content, following 3 days alginate bead (with glycerol and sucrose treatments) preculture in 0.7 M sucrose.     

Functional role of hypothalamic D1 and D2 receptors in organization of some forms of behavior of the common frog <Emphasis Type=Italic>Rana temporaria</Emphasis>

Dopamine is one of the most ancient, widely spread neurotransmitters. It performs a great number of neuromodulator effects in the vertebrates CNS. For the last few years there considerably increases an interest in study of functional role of this neurotransmitter in regulation of various forms of behavior of poikilothermal vertebrates. The present work deals with study of the role of the dopaminergic system, specifically of the hypothalamic dopaminergic system in providing some behavioral frog reactions. We studied behavior of the animals in the “open field” before and after administration to them of antagonists of D1 (SCH 23390) and D2 (haloperidol) receptors as well as of animals with destroyed anterior and posterior parts of hypothalamus. Administration of SCH 23390 to intact frogs caused a statistically significant decrease in the number of exploratory reactions and goal-oriented jumps, whereas haloperidol only moderately increased the number of the above reactions. Destruction of the posterior part of hypothalamus suppressed essentially all kinds of activity, while destruction of the anterior part inhibited them completely. Antagonist of D12 and D2 receptors of dopamine little changed the initial motor and emotional activity of the operated animals. The obtained data are discussed in terms of evolutionary origin of D1 and D2 receptors in vertebrates and allow concluding that D1 and D2 receptors of hypothalamic dopamine of the common frog are located predominantly in the anterior hypothalamic areas and that their effect on behavior can be mediated and is associated with other brain neurotransmitter systems in such brain structures as lateral hypothalamus, locus coereleus, and striatum that provide different aspects of wakefulness of amphibians.     

Effect of oxidative processes in mitochondria on contractility of heart muscle of the frog <Emphasis Type=Italic>Rana temporaria</Emphasis>. Actions of Cd<Superscript>2+</Superscript>

The inotropic Cd²⁺ action on frog heart is studied with taking into account its toxic effects upon mitochondria. Cd²⁺ at concentrations of 1, 10, and 20 mM is established to decrease dose dependently (21.3, 50.3, and 72.0%, respectively) the muscle contraction amplitude; this is explained by its competitive action on the potential-controlled Na²⁺-channels of the L-type (Ca_v 1.2). In parallel experiments on isolated rat heart mitochondria (RHM) it was shown that Cd²⁺ at concentrations of 15 and 25 mM produces swelling of non-energized and energized mitochondria in isotonic (with KNO₂ and NH₂NO₃) and hypoosmotic (with 25 mM CH₃COOK) media. Study of oxidative processes in RHM by polarographic method has shown 20 mM Cd²⁺ to disturb activity of respiratory mitochondrial chain. The rate of endogenous respiration of isolated mitochondria in the medium with Cd²⁺ in the presence of malate and succinate was approximately 5 times lower than in control. In experimental preparations, addition into the medium of DNP—uncoupler of oxidation and phosphorylation did not cause an increase of the oxygen consumption rate. Thus, the obtained data indicate that a decrease in the cardiac muscle contractility caused by Cd²⁺ is due not only to its direct blocking action on Ca²⁺-channels, but also is mediated by toxic effect on rat heart mitochondria, which was manifested as an increase in ion permeability of the inner mitochondrial membrane (IMM), acceleration of the energy-dependent K⁺ transport into the matrix of mitochondria, and inhibition of their respiratory chain.     

Life cycle of <Emphasis Type=Italic>Aylax hypecoi</Emphasis> (<Emphasis Type=Italic>Insecta: Hymenoptera: Cynipidae</Emphasis>), a gall inducer on <Emphasis Type=Italic>Hypecoum</Emphasis> ssp. (<Emphasis Type=Italic>Papaveraceae</Emphasis>)

The life cycle and developmental stages of Aylax hypecoi (Trotter, 1913, Hymenoptera: Cynipidae: Aylacini) were studied in detail. Aylax hypecoi is known to induce galls in fruits of two Hypecoum species — H. imberbe and H. geslini (Papaveraceae) and the larva develops in host plant fruits. The morphology and development of egg, larva and pupa were investigated, which has previously not been done. The shape and size of terminal-instar larvae and associated galls are sex-specific. Overwintering stage, adult emergence and flying periods, and egg productivity were studied also.     

Regulation of the <Emphasis Type=Italic>ALBINO3</Emphasis>-mediated transition to flowering in <Emphasis Type=Italic>Arabidopsis</Emphasis> depends on the expression of <Emphasis Type=Italic>CO</Emphasis> and <Emphasis Type=Italic>GA1</Emphasis>

ALBINO3, a homologue of PPF1 in Arabidopsis, encodes a chloroplast protein, and is essential for chloroplast differentiation. In the present study, ALBINO3(−) transgenic plants exhibited a significant decrease in both the number of rosette leaves at bolting and the days before bolting, suggesting the important roles of ALBINO3 in regulating flowering during non-inductive short-day photoperiods. ALBINO3 mRNA was apparently accumulated in shoot apical meristem and floral meristems around the shoot apical meristem in wild-type plants. ALBINO3 might be predominantly involved in inducing the floral repression pathway by activating the expression of TFL1, and by suppressing the expression of LFY, respectively, in the shoot apical meristem. Moreover, the function of ALBINO3 in regulating flowering transition depended on the expression of CO and GA1, because ALBINO3 might function in the downstream integration of the photoperiod-dependent and the photoperiod-independent pathways. These results suggest that ALBINO3 may have an important integrative function in the flowering process in Arabidopsis.     

Attenuation of fibrosis <Emphasis Type=Italic>in vitro</Emphasis> and <Emphasis Type=Italic>in vivo</Emphasis> with <Emphasis Type=Italic>SPARC</Emphasis> siRNA

Introduction  

SPARC is a matricellular protein, which, along with other extracellular matrix components including collagens, is commonly over-expressed in fibrotic diseases. The purpose of this study was to examine whether inhibition of SPARC can regulate collagen expression in vitro and in vivo, and subsequently attenuate fibrotic stimulation by bleomycin in mouse skin and lungs.     

Efficient separation and purification of allophycocyanin from <Emphasis Type=Italic>Spirulina</Emphasis> (<Emphasis Type=Italic>Arthrospira</Emphasis>) <Emphasis Type=Italic>platensis</Emphasis>

Allophycocyanin (APC) is a minor component of phycobiliproteins in cyanobacteria and red algae. This paper describes a simple and inexpensive extracting method for isolating APC from Spirulina (Arthrospira) platensis with high efficiency. The crude phycobiliprotein extract was pretreated by ammonium sulfate fractionation. Then, by adding hydroxylapatite into crude phycobiliprotein extract dissolved in 20 mM phosphate buffer (pH 7.0), APC was selectively adsorbed by hydroxylapatite but C-phycocyanin (C-PC) was not. The hydroxylapatite was collected and APC was extracted from the crude phycobiliprotein extract. Then, the enriched APC was washed off from the hydroxylapatite using 100 mM phosphate buffer (pH 7.0). In this simple extracting method it was easy to remove C-PC and isolate APC in large amounts. The absorbance ratio A ₆₅₀/A ₂₈₀ of extracted APC reached 2.0. The recovery yield was 70%, representing 4.61 mg · g⁻¹ wet weight. The extracted APC could be further purified by a simple anion-exchange chromatography with a pH gradient from 5.6 to 4.0. The absorbance ratio A ₆₅₀/A ₂₈₀ of the purified APC reached 5.0, and the overall recovery yield was 43%, representing 2.83 mg · g⁻¹ wet weight. Its purity was confirmed by native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate-PAGE.     

<Emphasis Type=Italic>In vitro</Emphasis> organogenesis of <Emphasis Type=Italic>Passiflora alata</Emphasis>

Passiflora alata in vitro organogenesis was studied based on explant type, culture medium composition, and incubation conditions. The results indicated that the morphogenic process occurred more efficiently when hypocotyl segment-derived explants were cultured in media supplemented with cytokinin and AgNO₃ incubated under a 16-h photoperiod. The shoot bud elongation and plant development were obtained by transferring the material to MSM culture medium supplemented with GA₃ and incubated in flasks with vented lids. Histological analyses of the process revealed that the difficulties in obtaining plants could be related to the development of protuberances and leaf primordia structures, which did not contain shoot apical meristem. Roots developed easily by transferring elongated shoots to 1/2 MSM culture medium. Plant acclimatization occurred successfully, and somaclonal variation was not visually detected. The efficiency of this organogenesis protocol will be evaluated for genetic transformation of this species to obtain transgenic plants expressing genes that can influence the resistance to Cowpea aphid borne mosaic virus.     

Interaction of <Emphasis Type=Italic>Bdellovibrio bacteriovorus</Emphasis> with bacteria <Emphasis Type=Italic>Campylobacter jejuni</Emphasis> and <Emphasis Type=Italic>Helicobacter pylori</Emphasis>

Interaction of Bdellovibrio bacteriovorus 100NCJB with bacteria Campylobacter jejuni (strains 1, 2, 3, 4, and 5) and Helicobacter pylori, strain TX30a, was confirmed. The results indicate that lytic activity of bdellovibrios both in liquid media and cells attached to a surface was observed. The potential use of the antimicrobial activity of predatory bacteria for environmental bioprotection and public health is discussed.     

Probiotic <Emphasis Type=Italic>Lactobacillus</Emphasis> strains: <Emphasis Type=Italic>in vitro</Emphasis> and <Emphasis Type=Italic>in vivo</Emphasis> studies

Three Lactobacillus strains (LOCK 0900, LOCK 0908, LOCK 0919) out of twenty-four isolates were selected according to their antagonistic activity against pathogenic bacteria, resistance to low pH and milieu of bile salts. Intragastric administration of a mixture of these strains to Balb/c mice affected cytokine T_H1-T_H2 balance toward nonallergic T_H1 response. Spleen cells, isolated from lactobacilli-treated mice and re-stimulated in vitro with the mixture of heat-inactivated tested strains, produced significantly higher amounts of anti-allergic tumor necrosis factor- and interferon-γ than control animals whereas the level of pro-allergic interleukin-5 was significantly lower. Lactobacillus cells did not translocate through the intestinal barrier into blood, liver and spleen; a few Lactobacillus cells found in mesenteric lymph nodes could create antigenic reservoir activating the immune system. The mixture of Lactobacillus LOCK 0900, LOCK 0908 and LOCK 0919 strains represents a probiotic bacterial preparation with possible use in prophylaxis and/or therapy of allergic diseases.     

Effect of photostimulation on the wakefulness-sleep cycle in the common frog Rana temporaria

Effect of daily 30-min photostimulation in the 10 s light: 10 s pause regime (the total of 5 days) on the time structure of the wakefulness-protosleep cycle (WPC) was studied in the common frog Rana temporaria. Changes of EEG wave components were analyzed in three immobility forms of the types of catalepsy (P-1), catatonia (P-2), and cataplexy (P-3) that form protosleep. The first three photostimulations promoted a gradual increase of the P-1 state to 84.16 ± 11.6% (the initial value (IV) 22.9 ± 9.1%) and a decrease of representation of wakefulness to 4.86 ± 2.1% (IV 13.8 ± 7.8 %), of P-2 to 11.1 ± 5.3 (IV 53.3 ± 13.3 %), and of P-3 to 2.21 ± 1.0% (IV 11.1 ± 5.6%). After 4–5 photostimulations and especially after their complete cessation the percentage of P1 in the WPC was restored to initial values, whereas the percentage of the frog WPC P-3 considered to be a precursor of the homoiothermal sleep rose to 20 ± 8.3% after 5 photostimulations and to 38.5 ± 6.7% the next day. Changes in the frog EEG spectra appeared only after one photostimulation and were characterized by a brief increase of power of α-like waves and by inhibition of slow δ-waves. In P-2 the power of the slow δ-waves gradually rose. In P-3 the EEG parameters did not change. In all experimental animals a decrease of the relative thymus and adrenal masses was revealed, which indicates the stress nature of the photostimulation regime used in the work. The obtained data allow thinking that a certain neurohormonal response to stress has already been formed at the amphibian level and that an important role in this response realization is played by a coordinated interaction of the hypothalamic sleep-regulating system providing protosleep manifestations and of the hypothalamic neurosecretory system triggering hormonal cascade of the stress-reaction.     

Effect of photostimulation on the wakefulness-sleep cycle in the common frog Rana temporaria

Effect of daily 30-min photostimulation in the 10 s light: 10 s pause (the total of 5 days) on the time structure of the wakefulness--protosleep cycle (WPC) was studied in the common frog Rana temporaria. Changes were analyzed of EEG wave components in three immobility forms of the type of catalepsy (P-1), catatonia (P-2), and cataplexy (P-3) that form protosleep. The first three photostimulations promoted a gradual increase of the P-1 state to 84.16 +/- 11.6% [the initial value (IV) 22.9 +/- 9.1%] and a decrease of representation of wakefulness to 4.86 +/- 2/1% (IV 13.8 +/- 7.8%), of P-2 to 11.1 +/- 5.3 (IV 53.3 +/- 13.3%), and of P-3 to 2.21 +/- 1.0% (IV 11.1 +/- 5.6%). After 4-5 photostimulations and especially after their complete cessation the percentage of P-1 in the WPC was restored to initial values, whereas the percentage of the frog WPC P-3 considered to be a precursor of the homoiothermal sleep rose to 20 +/- 8.3% after 5 photostimulations and to 38.5 +/- 6.7% the next day. Changes in the frog EEG spectra appeared only after one photostimulation and were characterized by a brief increase of power of alpha-like waves and by inhibition of slow 6-waves. In P-2 the power of the slow delta-waves gradually rose. In P-3 the EEG parameters did not change. In all experimental animals a decrease of the relative thymus and adrenal masses was revealed, which indicates the photostimulation regime used in the work induces stress. The obtained data allow thinking that a certain neurohormonal response to stress has already been formed at the amphibian level and that an important role in this response realization is played by a coordinated interaction of the hypothalamic sleep-regulating system providing protosleep manifestations and of the hypothalamic neurosecretory system triggering the stress-reaction hormonal cascade.     

<Emphasis Type=Italic>Pseudomonas aeruginosa</Emphasis> inhibits <Emphasis Type=Italic>in-vitro Candida</Emphasis> biofilm development

Background  

Elucidation of the communal behavior of microbes in mixed species biofilms may have a major impact on understanding infectious diseases and for the therapeutics. Although, the structure and the properties of monospecies biofilms and their role in disease have been extensively studied during the last decade, the interactions within mixed biofilms consisting of bacteria and fungi such as Candida spp. have not been illustrated in depth. Hence, the aim of this study was to evaluate the interspecies interactions of Pseudomonas aeruginosa and six different species of Candida comprising C. albicans, C. glabrata, C. krusei, C. tropicalis, C. parapsilosis, and C. dubliniensis in dual species biofilm development.