Comparative phylogeography of meroplanktonic species, <Emphasis Type="Italic">Aurelia</Emphasis> spp. and <Emphasis Type="Italic">Rhizostoma pulmo</Emphasis> (Cnidaria: Scyphozoa) in European Seas

Mass occurrences of scypozoan medusae have become increasingly common in recent decades in European seas, including species in the genera Aurelia and Rhizostoma. We inferred the phylogeographic patterns of metagenetic scyphozoa Aurelia spp. and Rhizostoma pulmo from mitochondrial COI and nuclear ITS regions. No genetic structure was detected in R. pulmo over the Mediterranean Sea. By contrast, the phylogeographic analyses confirmed the separation of Aurelia spp. to several proposed cryptic species. Our results do not support the null hypothesis that both genera have concordant phylogeographic patterns. The resolvable parsimony network of haplotypes was retrieved for Aurelia aurita, Aurelia sp. 5, and Aurelia sp. 8 without connectivity between them and no genetic structure were found within those groups. Even though evidence of hybridization was found between A. aurita and Aurelia sp. 5, that did not break down the phylogenetic separation among them. The lowest haplotype and nucleotide diversity were found in samples of Aurelia sp. 8 and R. pulmo from the northern Adriatic, which acts as a sink area due to strong genetic drift. These new findings will facilitate linking the phenotype of the organism and its ability to survive in a particular environment—which shapes phylogeographic patterns.     

Molecular-Level Characterization of Lipid Membrane Electroporation using Linearly Rising Current

We present experimental and theoretical results of electroporation of small patches of planar lipid bilayers by means of linearly rising current. The experiments were conducted on ~120-μm-diameter patches of planar phospholipid bilayers. The steadily increasing voltage across the bilayer imposed by linearly increasing current led to electroporation of the membrane for voltages above a few hundred millivolts. This method shows new molecular mechanisms of electroporation. We recorded small voltage drops preceding the breakdown of the bilayer due to irreversible electroporation. These voltage drops were often followed by a voltage re-rise within a fraction of a second. Modeling the observed phenomenon by equivalent electric circuits showed that these events relate to opening and closing of conducting pores through the bilayer. Molecular dynamics simulations performed under similar conditions indicate that each event is likely to correspond to the opening and closing of a single pore of about 5 nm in diameter, the conductance of which ranges in the 100-nS scale. This combined experimental and theoretical investigation provides a better quantitative characterization of the size, conductance and lifetime of pores created during lipid bilayer electroporation. Such a molecular insight should enable better control and tuning of electroporation parameters for a wide range of biomedical and biotechnological applications.     

Transmission of Podisus maculiventris tremulatory signals through plants

Males of the predaceous stink bug Podisus maculiventris (Say) (Heteroptera: Pentatomidae: Asopinae) emit low frequency tremulatory signals. Laser vibrometry was used to record and analyze naturally emitted signals, focusing on variation in signal velocity and frequency during transmission through plants (Phaseolus vulgaris L. and Plumbago auriculata Lam.) as a function of distance from the vibrational source. Signal velocity varied individually between 2 and 15 mm/s recorded on a plant close to the calling male and decreased by 0.3 to 1.5 dB/cm on bean and 0.3 to 0.9 dB/cm on plumbago. The dominant frequency of signals was variable at frequencies below 50 Hz. On bean frequencies centered around 10 Hz or 20 Hz were dominant for signals recorded at the source. Transmission through bean resulted in an increase in the 20 Hz peak relative to other frequencies in the signal. Variation of the dominant frequencies of signals transmitted through plumbago stems were more predictable, showing typical changes in amplitude relative to the distance from the source. The regular variation of the dominant frequency along the stem with linear increase of signal velocity at decreasing distance from the source may provide plant-dwelling insects with information about the distance to the calling individual.     

Effects of pH and Oxygen and Ammonium Concentrations on the Community Structure of Nitrifying Bacteria from Wastewater

Shifts in nitrifying community structure and function in response to different ammonium concentrations (50, 500, 1,000, and 3,000 mg of N liter⁻¹), pH values (pH 6.0, 7.0, and 8.2), and oxygen concentrations (1, 7, and 21%) were studied in experimental reactors inoculated with nitrifying bacteria from a wastewater treatment plant. The abilities of the communities selected for these conditions to regain their original structures after conditions were returned to the original conditions were also determined. Changes in nitrifying community structure were determined by performing an amplified ribosomal DNA (rDNA) restriction analysis of PCR products obtained with ammonia oxidizer-specific rDNA primers, by phylogenetic probing, by small-subunit (SSU) rDNA sequencing, and by performing a cellular fatty acid analysis. Digestion of ammonia-oxidizer SSU rDNA with five restriction enzymes showed that a high ammonium level resulted in a great community structure change that was reversible once the ammonium concentration was returned to its original level. The smaller changes in community structure brought about by the two pH extremes, however, were irreversible. Sequence analysis revealed that the highest ammonium environment stimulated growth of a nitrifier strain that exhibited 92.6% similarity in a partial SSU rRNA sequence to its nearest relative, Nitrosomonas eutropha C-91, although the PCR product did not hybridize with a general phylogenetic probe for ammonia oxidizers belonging to the β subgroup of the class Proteobacteria. A principal-component analysis of fatty acid methyl ester data detected changes from the starter culture in all communities under the new selective conditions, but after the standard conditions were restored, all communities produced the original fatty acid profiles.Autotrophic nitrifying bacteria that oxidize ammonium to nitrite and nitrate are found in soils, sediments, wastewaters, freshwater, and marine water and on building facades. They are essential components of the nitrogen (N) cycle, linking the most reduced and most oxidized forms of inorganic N. Nitrification occurs as a two-step process carried out by two distinct groups of bacteria; ammonia-oxidizing bacteria convert ammonia to nitrite, and then nitrite oxidizers convert nitrite to nitrate (22, 30). Environmental factors control the rate of nitrification. The most significant environmental factors are substrate concentration, pH, temperature, and oxygen availability (12, 23). Nitrifying bacteria exhibit different substrate concentration sensitivities (26). Media containing low substrate concentrations (10 mg of NH₄⁺ liter⁻¹) can give larger most-probable-number counts of ammonia oxidizers than media containing higher NH₄⁺ concentrations (6, 26). Also, ammonia oxidation is inhibited at high substrate concentrations. The growth rates of Nitrosomonas spp. cultures were reduced in the presence of 1,050 to 2,800 mg of NH₄⁺-N liter⁻¹ (16). Substrate inhibition of ammonia oxidation has also been observed in studies of wastewater systems (23). Natural environments, such as soil and water, usually contain 1 to 10 mg of NH₄⁺-N liter⁻¹ (22), yet liquid wastes from animal farms give rise to concentrations up to 1,600 or 5,600 mg of NH₄⁺-N liter⁻¹ (5, 17). Free ammonia (NH₃) rather than the total ammonium concentration inhibits ammonia oxidizers (1). As the ratio between the ionized form and the nonionized form depends on pH, the toxicity of ammonium also depends on the environmental pH.The pH range for growth of pure cultures of ammonia oxidizers is 5.8 to 8.5, and the pH range for growth of nitrite oxidizers is 6.5 to 8.5 (30). Nitrification was inhibited at pH values below 5.8 in our preliminary experiments performed with an enriched culture of nitrifiers obtained from wastewater. Yet in natural environments, such as soil, nitrification has been reported to occur at pH values below 4.0 (7, 29).Limiting amounts of dissolved oxygen (concentrations below 2 mg liter⁻¹) inhibit nitrification and cause nitrite accumulation or nitrous and nitric oxide production (9, 21). Ammonia-oxidizing bacteria are the key functional group in removing ammonium from wastewaters. Knowledge of the effect of oxygen on nitrification and nitrifying populations has economic importance since aeration of activated sludge is one of the most costly items in the operation of a wastewater treatment plant (21).In environments with high inputs of ammonium, such as wastewaters, biooxidation of this substrate increases the oxygen uptake and lowers the pH. Such modifications of the environment not only affect the production of nitrite and nitrate but can also select a different nitrifying community that is perhaps specialized for these new conditions. Nitrification does occur in extreme environments that pure cultures of nitrifiers cannot tolerate (4). In this study we examined extreme environments in which nitrifying bacteria may be viable but have not been cultured thus far.Because of the difficulty of obtaining nitrifier isolates, nucleic acid-based methods have greatly aided studies of the diversity of nitrifiers (11, 20, 27, 28). Recent molecular investigations have provided valuable information concerning the diversity of ammonia oxidizers in natural environments (5, 15, 20, 25). However, no previous study has focused on the structural or compositional responses of nitrifying communities to perturbations in the environment. In the present laboratory study we examined the effects of high ammonium concentrations, different pH values, and different oxygen concentrations on nitrification and on the community structure of nitrifying bacteria from wastewater. To test the abilities of the communities to regain their original structures, growth of nitrifying communities under the new conditions was followed by incubation under the original conditions.     

Persistant immunodominant anti-gag SLYNTVATL responses in HIV-patients with up to 7 years of HAART

We analyzed Gag-specific CD8+ T-cells in HIV-patients on long-term HAART and in untreated chronically-infected patients by using iTAg MHC class I tetramers (HLA-A*0201) specific for SLYNTVATL. Gag SLYNTVATL-specific CD8+ T-cells were detectable in 18 of 26 treated patients (median 5.2 years of HAART) and in 10 of 14 untreated patients. Median percentage of Gag SLYNTVATL-specific CD8+ T-cells in treated patients was 0.10 (range 0.00-0.70%). Median number of Gag SLYNTVATL-specific CD8+ T-cells per 50,000 CD8+ T-cells was 56.0 cells (range 2.0-344.0 cells) and was not significantly different compared with untreated patients (p = 0.978). Numbers of Gag SLYNTVATL-specific CD8+ T-cells were inversely correlated with the duration of undetectable plasma viremia (p = 0.02, Rho = -0.430). Chronically-infected HIV-patients on HAART (for up to 7.7 years) maintained a stable subpopulation of Gag SLYNTVATL-specific CD8+ T-cells. This finding is relevant for the analysis of treatment-induced immune reconstitution and, possibly, for future therapeutic strategies in HIV-disease.     

Electrogene therapy with p53 of murine sarcomas alone or combined with electrochemotherapy using cisplatin

The aim of our study was to evaluate feasibility and therapeutic potential of electrogene therapy with p53 alone or combined with electrochemotherapy using cisplatin on two murine sarcomas with different p53 status. Antitumor effectiveness of three consecutive electrogene treatments with p53 was more effective in wild-type LPB tumors than mutated SA-1 tumors, resulting in 21.4% of tumor cures in LPB tumors and 12.5% in SA-1 tumors. Pretreatment of tumors with electrogene therapy with p53 enhanced chemosensitivity of both tumor models treated by electrochemotherapy with cisplatin. After only one application of this treatment combination in the LPB tumor model, specific tumor growth delay was prolonged in the combined treatment group compared to electrogene therapy with p53 or electrochemotherapy with cisplatin alone, whereas in SA-1 tumors this treatment combination resulted in 31.6% of cured animals. Results of our study show that electrogene therapy with p53 alone or combined with electrochemotherapy is feasible and effective treatment of tumors. The combination of electrogene therapy and electrochemotherapy after only one application resulted in complete regression of tumors.     

Molecular Recognition Theory of the complementary (antisense) peptide interactions

Molecular Recognition Theory is based on the finding of Blalock et al. (Biochem. Biophys. Res. Commun. 121 (1984) 203–207; Nature Med. 1 (1995) 876–878; Biochem. J. 234 (1986) 679–683) that peptides specified by the complementary RNAs bind to each other with higher specificity and efficacy. This theory is investigated considering the interaction of the sense peptides coded by means of messenger RNA (read in 5′→3′ direction) and antisense peptides coded in 3′→5′ direction. We analysed the hydropathy of the complementary amino acid pairs and their frequencies in 10 peptide–receptor systems with verified ligand–receptor interaction. An optimization procedure aimed to reduce the number of possible antisense peptides derived from the sense peptide has been proposed. Molecular Recognition Theory was also validated by an “in vivo” experiment. It was shown that 3′→5′ peptide antisense of -MSH abolished its cytoprotective effects on the gastric mucosa in rats. Molecular Recognition Theory could be useful method to simplify experimental procedures, reduce the costs of the peptide synthesis, and improve peptide structure modelling.     

Nicardipine diminished equinatoxin II-induced decrease of coronary flow in isolated rat and pig hearts

Equinatoxin II (EqT II) is a basic, cardiotoxic polypeptide. The vasoconstrictory effect of the toxin on isolated porcine coronary arteries was diminished by nicardipine, an L-type calcium channel antagonist. A comparison was made of the effects of EqT II alone and EqT II in the presence of nicardipine on the coronary flow in porcine and rat hearts isolated according to Langendorff's method. In both models EqT II decreased coronary flow in a dose-dependent manner and there were no statistically significant differences between the two models (p>0.05). However, 1 M nicardipine diminished the effects of EqT II on coronary flow in isolated porcine hearts more than in isolated rat hearts (p<0.05). The results suggest that the activation of L-type calcium channels is one of the mechanisms involved in the lowering of coronary flow induced by EqT II.     

Effects of electrogenetherapy with p53wt combined with cisplatin on curvival of human tumor cell lines with different p53 status

The aim of our study was to evaluate electrogenetherapy with p53wt alone or combined with cisplatin on two colorectal (HT-29 and LoVo) and two prostatic (PC-3 and Du145) carcinoma cell lines with different p53 status. In addition, the feasibility of electrogenetherapy with p53wt was tested also in vivo on PC-3 prostatic cancer xenografts. Electrogenetherapy with p53wt was dependent on the p53 status of the cell lines used. Electrogenetherapy was the most effective on the PC-3 (p53 null) and Du145 (p53mt) cells, and to the much lesser extent in LoVo cells (p53wt). The exception was the HT-29 cell line with overexpressed mutated p53, where electrogenetherapy with p53wt was the least effective. Sensitivity of the cell lines to cisplatin was independent of the p53 status. Furthermore, the presence of exogenous p53 due to electrogenetherapy did not enhance cisplatin cytotoxicity, since the combination of these therapies resulted in additive cytotoxic effect. The effectiveness of electrogenetherapy with p53wt was also demonstrated in vivo by successful treatment of subcutaneous PC-3 tumors in mice. In conclusion, our study shows that electrogenetherapy with p53wt is feasible, and resulted in comparable cytotoxic and antitumor effectiveness to viral-mediated p53wt gene therapy. This therapy was effective and dependent on the p53 status of the tumor cell lines. Combination of electrogenetherapy and cisplatin resulted in additional cell kill by cisplatin, and was not dependent on the p53 status.