Analysis of phenolic compounds in the dissolved and suspended phases of Lake Balaton water by gas chromatography-tandem mass spectrometry.

As a novel approach to characterize the phenolic pollutants of Lake Balaton (Central Europe, western Hungary), 26 endocrine disrupting phenols (chlorophenols, nitrophenols, alkylphenols, triclosan, bisphenol-A) were quantified in dissolved and suspended particulate matter (SPM) phases, alike. Sample collection was performed in the western and eastern basins, at 20 sites in April and October 2014. Solid-phase and ultrasound-assisted extractions to withdraw target phenols from dissolved and suspended phases were employed. Compounds were derivatized with hexamethyldisilazane and trifluoroacetic acid for their quantification as trimethylsilyl derivatives by gas chromatography-tandem mass spectrometry. In Lake Balaton's dissolved phase, 2-chlorophenol (103-164 ng/L), 4-chlorophenol (407-888 ng/L), 2,4-dichlorophenol (20.2-72.0 ng/L), 2,4,6-trichlorophenol (10.4-38.1 ng/L), 2-nitrophenol (31.0-66.5 ng/L), 4-nitrophenol (31.5-94.1 ng/L), and bisphenol-A (20.6-112 ng/L), while in its SPM, 4-chlorophenol (

Potassium channels in the central nervous system of the snail, Helix pomatia: localization and functional characterization.

The distribution and functional presence of three voltage-dependent potassium channels, Kv2.1, Kv3.4, Kv4.3, respectively, were studied in the central nervous system of the snail Helix pomatia by immunohistochemical and electrophysiological methods. Cell clusters displaying immunoreactivity for the different channels were observed in all parts of the CNS, although their localization and number partly varied. Differences were also found in their intracellular, perikaryonal and axonal localization, as well as in their presence in non-neuronal tissues nearby the CNS, such as the perineurium and the aorta wall. At ultrastructural level Kv4.3 channel immunolabeling was observed in axon profiles containing large 80-100nm granular vesicles. Blotting analyses revealed specific signals for the Kv2.1, Kv3.4 and Kv4.3 channels, confirming the presence of the channels in the Helix CNS. Voltage-clamp recordings proved that outward currents obtained from neurons displaying Kv3.4 or Kv4.3 immunoreactivity contained transient components while Kv2.1 immunoreactive cells were characterized by delayed currents. The distribution of the K(+)-channels containing neurons suggests specific roles in intercellular signaling processes in the Helix CNS, most probably related to well-defined, partly local events. The cellular localization of the K(+)-channels studied supports their involvement in both pre- and postsynaptic events at perikaryonal and axonal levels.

Immunodetection and localization of nitric oxide synthase in the olfactory center of the terrestrial snail, Helix pomatia.

The procerebrum of stylommatophoran snails produces nitric oxide (NO)-modulated oscillatory local field potentials which are considered the basis of olfactory information processing. Although the function of NO is well characterized in the PC, the identification and distribution of NO synthase (NOS) has not known completely. In the present study, applying a mammalian anti-NOS antibody, a 170 kDa molecular weight NOS-like protein was demonstrated in the procerebrum homogenate of Helix pomatia. NOS-like immunolabeling of the globuli cells, the internal and terminal neuropils displayed an identical distribution compared to that of NADPH-diaphorase reactive material, confirming the specificity of immunohistochemistry. The detailed characteristics of the immunostaining (different intensity of the neural perikarya, a gradual appearance in the terminal neuropil and in the axon bundles of the tentacular nerve, as well as an intense, homogeneous distribution of NOS-like immunoreactivity in the internal neuropil) suggest that NOS is expressed constitutively, maintaining a high level of the enzyme in neuropil areas. NOS accumulation in the internal neuropil suggests that NO plays an important role in delivering olfactory signals extrinsic to the procerebrum, and integrating them with other sensory modalities, respectively. Our results are the first, demonstrating unequivocally the presence of NOS and resolving its differential distribution in the Helix procerebrum.

Immunological and pharmacological identification of the dopamine D1 receptor in the CNS of the pond snail, Lymnaea stagnalis.

We investigated the presence and distribution of the D1 dopamine receptor in the CNS of Lymnaea stagnalis applying immunobloting and immunocytochemistry. We also investigated the effect of dopamine as well as the specific D1 receptor blocker, SCH23390, on the firing activity of the feeding modulator serotonergic neuron, CGC, which displayed D1 immunoreactivity. Immunoblot experiments showed one specifically labeled band with 62 kDa mw which is close to that of the mammalian D1 receptor. Neurons displaying D1-like immunoreactivity can be observed in each ganglion of the CNS but particularly in the pedal ganglia which are the center for locomotion. Dopamine regularly evokes burst activity in the serotonergic CGC at 1 mM and this effect could be antagonized by SCH23390. These observations suggest that a D1-like receptor molecule is present in the CNS of Lymnaea.

Potassium channels in the Helix central nervous system: preliminary immunohistochemical studies. Short communication.

Distribution of the potassium channel of Kv4.3 type was investigated in the central nervous system (CNS) of Helix pomatia by immunohistochemistry. Immunopositive neurons were found widely distributed in the CNS, present mostly in smaller groups in the different central ganglia but not in the visceral ganglion. Labeled fibers were characteristic for not only the neuropils of all ganglia but also the connective tissue sheath around the CNS and the aorta wall were richly innervated. Western blot analysis revealed a clear identity with the mammalian Kv4.3 subunit, suggesting an evolutionary conserved structure of this channel type. Our preliminary results provide a steady basis for further experiments aiming partly at the identification of other potassium channel types and partly the ultrastructural localization of Kv4.3.

Anti-TNF-alpha antibody (infliximab) therapy supports the recovery of eNOS and VEGFR2 protein expression in endothelial cells.

The aim of this study is to investigate the effect of sera obtained from patients of Crohn's disease treated by anti-TNF-alpha antibody (Infliximab) on the expression of endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor receptor-2 (VEGFR2) protein in human umbilical vein endothelial cells (HUVEC) cultured in vitro. HUVEC was cultured in the presence of sera derived from patients before and after treatment, or from healthy individuals. Effects of sera on the expression of eNOS and VEGFR2 were monitored by determination of mRNA and protein levels using real time quantitative PCR and Western blot analysis, respectively. The serum of Crohn's patients contained elevated levels of TNF-alpha (34±1.80 pg/mL), which resulted in a decrease in the protein level of eNOS in HUVEC with a simultaneous induction of VEGFR2. Infliximab treatment normalized the expression level of these proteins by decreasing TNF-alpha level, particularly in those cases when clinical healing was also recorded, and it also conferred restitution of the level of angiogenic cytokines. Results suggest that altered angiogenesis possibly contributes to the initiation and perpetuation of inflammatory processes in inflammatory bowel disease (IBD). Endothelial dysfunction, a selective feature of Crohn's disease is beneficially affected by intravascular TNF-alpha neutralization.

Localization of serotonin transporter mRNA in the CNS of Lymnaea stagnalis.

The serotonin transporter, SERT, is reported as a key molecule that regulates serotonergic neurotransmission. In the present study, we analyzed the localization of Lymnaea SERT (LymSERT) mRNA-containing neurons by in situ hybridization using frozen sections of the central nervous system (CNS) of Lymnaea. To precisely demonstrate the distribution of LymSERT mRNA-containing neurons, colocalization with serotonin immunoreactivity was also examined. The results showed that LymSERT mRNA was constitutively expressed and localized in the serotonin-containing neurons in the CNS.

NADPH-diaphorase activity in the nervous system of the embryonic and juvenile pond snail, Lymnaea stagnalis.

Nicotinamide-adenine-dinucleotide-phosphate-diaphorase (NADPH-d) histochemistry has been applied in the present study to determine the distribution of putative nitric oxide (nitric oxide synthase)-producing cells during embryonic and early postembryonic development in the pond snail, Lymnaea stagnalis L., with special reference to the nervous system. The first NADPH-d-positive structures appear as early as 18% of development (E18, trochophore stage) and correspond to the pair of protonephridia. These structures later show disintegration, although after metamorphosis (E26=75%) staining of their individually spreading cells can be observed until hatching. Peripheral sensory neurons in the foot, mantle edge and lips, and their afferents projecting to the central nervous system reveal NADPH-d activity in the postmetamorphosis period (E25-E27=E60%-E80%) of embryogenesis. After hatching (P1-P3), a number of stained sensory cells appear in the pharynx and esophagus. Some NADPH-d positive neuronal perikarya occur in the pedal and pleural ganglia, and a few weakly stained cells in the cerebral and buccal ganglia of juvenile snails. At the same time, a continuous bundle of reactive fibers is formed in the neuropil both through and through around the circumesophageal ganglion ring. The localization of NADPH-d activity in the developing nervous system of Lymnaea suggests that nitric oxide participates mainly in sensory processes. However, its role in specific intraganglionic integrative events cannot be excluded following embryonic metamorphosis.