Airborne concentrations and chemical considerations of radioactive ruthenium from an undeclared major nuclear release in 2017.

In October 2017, most European countries reported unique atmospheric detections of aerosol-bound radioruthenium (106Ru). The range of concentrations varied from some tenths of µBq·m-3 to more than 150 mBq·m-3 The widespread detection at such considerable (yet innocuous) levels suggested a considerable release. To compare activity reports of airborne 106Ru with different sampling periods, concentrations were reconstructed based on the most probable plume presence duration at each location. Based on airborne concentration spreading and chemical considerations, it is possible to assume that the release occurred in the Southern Urals region (Russian Federation). The 106Ru age was estimated to be about 2 years. It exhibited highly soluble and less soluble fractions in aqueous media, high radiopurity (lack of concomitant radionuclides), and volatility between 700 and 1,000 °C, thus suggesting a release at an advanced stage in the reprocessing of nuclear fuel. The amount and isotopic characteristics of the radioruthenium release may indicate a context with the production of a large 144Ce source for a neutrino experiment.

Potential Source Apportionment and Meteorological Conditions Involved in Airborne 131I Detections in January/February 2017 in Europe.

Traces of particulate radioactive iodine (131I) were detected in the European atmosphere in January/February 2017. Concentrations of this nuclear fission product were very low, ranging 0.1 to 10 µBq m-3 except at one location in western Russia where they reached up to several mBq m-3. Detections have been reported continuously over an 8-week period by about 30 monitoring stations. We examine possible emission source apportionments and rank them considering their expected contribution in terms of orders of magnitude from typical routine releases: radiopharmaceutical production units > sewage sludge incinerators > nuclear power plants > spontaneous fission of uranium in soil. Inverse modeling simulations indicate that the widespread detections of 131I resulted from the combination of multiple source releases. Among them, those from radiopharmaceutical production units remain the most likely. One of them is located in Western Russia and its estimated source term complies with authorized limits. Other existing sources related to 131I use (medical purposes or sewage sludge incineration) can explain detections on a rather local scale. As an enhancing factor, the prevailing wintertime meteorological situations marked by strong temperature inversions led to poor dispersion conditions that resulted in higher concentrations exceeding usual detection limits in use within the informal Ring of Five (Ro5) monitoring network.

Targeting of peptidergic vesicles in cotransmitting terminals.

In the present study, we examined the targeting of neuropeptide-containing vesicles in terminals of neurons that release both neuropeptides and classical transmitters. Single neurons were electrically stimulated with patterns of activity that were recorded in freely behaving animals. The amount of peptide release was measured biochemically using a radioimmunoassay, and the targeting of peptidergic vesicles was quantified using immunoelectronmicroscopy. Repeated electrical stimulation of single neurons produced a very large increase in peptide release. Peptide release is paralleled by a twofold increase in the number of peptidergic vesicles docked at the portion of the terminal membrane that is away from the target muscle. This is in stark contrast to cholinergic vesicles, which aggregate at, and are released from the conventional release sites in close apposition to the muscle. This differential targeting of cholinergic and peptidergic vesicles may play a significant role in the distinct release requirements and spatial and temporal characteristics of the actions of conventional and peptidergic transmitters.

A pair of reciprocally inhibitory histaminergic sensory neurons are activated within the same phase of ingestive motor programs in Aplysia.

Previous studies have shown that each buccal ganglion in Aplysia contains two B52 neurons, one in each hemiganglion. We now show that there are two B52 neurons in a single buccal hemiganglion and four cells in an animal. We also show that the B52 neurons are histamine-immunoreactive and use reverse phase HPLC to show that the histamine-immunoreactive substance is authentic histamine. Previous studies have shown that the B52 neurons make numerous inhibitory synaptic connections with neurons active during the radula closing/retraction phase of ingestive motor programs. A computational model of the Aplysia feeding central pattern generator has, therefore, suggested that the B52 neurons play a role in terminating closing/retraction. Consistent with this idea we show that both B52 neurons fire at the beginning of radula opening/protraction. We also show that both B52 neurons are sensory neurons. They are depolarized when a flap of connective tissue adjacent to the buccal commissural arch is stretched. During ingestive feeding this is likely to occur at the peak of closing/retraction as opening/protraction begins. In the course of this study we compare the two ipsilateral B52 neurons and show that these cells are virtually indistinguishable; e.g., they use a common neurotransmitter, make the same synaptic connections, and are both sensory as well as premotor neurons. Nevertheless we show that the B52 neurons are reciprocally inhibitory. Our results, therefore, strikingly confirm theoretical predictions made by others that neurons that inhibit each other will not necessarily participate in antagonistic phases of behavior.

Expression and intracellular localization of catechol O-methyltransferase in transfected mammalian cells.

The intracellular localization of soluble and membrane-bound isoforms of rat and human catechol O-methyltransferase (COMT) was studied by expressing the recombinant COMT proteins either separately or together in mammalian cell lines (HeLa and COS-7 cells) and in rat primary neurons. The distribution of soluble and membrane-bound COMT enzyme was visualized by immunocytochemistry. For comparison, the localization of native COMT was studied in rat C6 glioma cells by immunoelectron microscopy. Staining of cells expressing membrane-bound COMT with a COMT-specific antiserum revealed an immunofluorescence signal in intracellular reticular structures and in the nuclear membrane. Double-staining of the cells with antisera against proteins specific for the rough endoplasmic reticulum indicated that they colocalized with membrane-bound COMT, suggesting that it resided in the endoplasmic reticulum. Notably, no COMT-specific fluorescence of plasma membranes was detected. The signal in the endoplasmic reticulum was also evident in the cells expressing both recombinant COMT forms. Intracellular native COMT reaction was detected by immunoelectron microscopy in rat C6 glioma cells and an intense cytoplasmic signal was seen in the primary neurons infected with the recombinant Semliki Forest virus. The cells expressing recombinant soluble COMT revealed intense nuclear staining together with diffuse cytoplasmic immunoreactivity, suggesting that a part of soluble COMT is transported to nuclei. Western blotting from rat liver and brain revealed soluble COMT in the nuclei. Enzyme activity measurements from liver cytoplasmic and nuclear fractions suggested that about 5% of the soluble COMT resided in nuclei. The intracellular localization of both COMT forms implies that COMT acts in the cytoplasm and possibly also in the nuclear compartment, and that the physiological substrates of COMT enzymes may have to be internalized before their methylation by COMT.

Peptide GEGLSS-like immunoreactivity in the rat central nervous system.

A rabbit antiserum was raised against the N-terminal fragment peptide, GEGLSS (Gly-Glu-Gly-Leu-Ser-Ser) of bovine neuropeptide AF (NPAF, A18Famide). NPAF is an octadecapeptide isolated from the bovine brain together with neuropeptide FF (NPFF). GEGLSS-like immunoreactivity was localized with immunofluorescence technique in colchicine-treated rats in neuronal cell bodies of the supraoptic (SON) and paraventricular (PVN) hypothalamic nuclei. A few neurons were also observed in the retrochiasmatic part of the SON. GEGLSS-like immunoreactivity was also localized to nerve terminals of the posterior pituitary. No GEGLSS-ir neuronal cell bodies were observed in the medial hypothalamus, in an area that contains NPFF-ir neurons. GEGLSS immunoreactivity was also seen in the fibers and terminals of nucleus of the solitary tract. We injected a retrograde tracer, fluorogold, to the posterior pituitary gland and visualized GEGLSS-ir neuronal cell bodies double-labeled with the tracer in SON, PVN, and SOR. The pituitary stalk transsection totally abolished the GEGLSS-ir structures from the posterior pituitary. Our results suggest that GEGLSS immunoreactivity in the rat brain has a more limited distribution than NPFF immunoreactivity. GEGLSS immunoreactivity was partially colocalized with arginine-vasopressin and oxytocin in neuronal cell bodies in the SON and PVN. Considering the fact that the known rat NPFF-NPAF precursor does not contain GEGLSS structure, the detected GEGLSS immunoreactivity may be derived from a previously unknown precursor.

Catechol-O-methyltransferase in rat sensory ganglia and spinal cord.

The localization of catechol-O-methyltransferase immunoreactivity in rat dorsal root ganglia and in the spinal cord and its co-existence with substance P, calcitonin gene-related peptide and fluoride-resistant acid phosphatase in dorsal root ganglion cells was examined with immunohistochemical and histochemical double-staining methods. Analysis of dorsal of dorsal root ganglia at both cervical and lumbar levels revealed catechol-O-methyltransferase immunoreactivity in numerous dorsal root ganglion cells. Double-staining studies showed that catechol-O-methyltransferase and substance P immunoreactivities were located in different cells with a few exceptions, whereas both catechol-O-methyltransferase and calcitonin gene-related peptide immunoreactivities were detected in about 10% of all labeled cells positive for one of the two markers at both levels studied. The great majority of fluoride-resistant alkaline phosphatase-positive cells were also immunoreactive for catechol-O-methyltransferase. Again, no difference was found between cervical and lumbar levels. Catechol-O-methyltransferase immunoreactivity was also found in the neuropil of the dorsal horn of the spinal cord. The staining was most intense in the superficial laminae (I-III) and overlapped partly with substance P and calcitonin gene-related peptide immunoreactivity. Western blotting analysis revealed that soluble catechol-O-methyltransferase was the clearly dominating form of the enzyme in dorsal root ganglia. The distribution pattern of catechol-O-methyltransferase in dorsal horn and sensory neurons suggests that the enzyme may modulate sensory neurotransmission.

Neuronal and non-neuronal catechol-O-methyltransferase in primary cultures of rat brain cells.

Previous biochemical and histochemical studies have suggested that catechol-O-methyltransferase (COMT) is a predominantly glial enzyme in the brain. The aim of this work was to study its localization and molecular forms in primary cultures, where cell types can be easily distinguished with specific markers, COMT immunoreactivity was studied in primary astrocytic cultures from newborn rat cerebral cortex, and in neuronal cultures from rat brain from 18-day-old rat embryos using antisera against rat recombinant COMT made in guinea pig. Double-staining studies with specific cell markers to distinguish astrocytes, neurons and oligodendrocytes were performed. COMT immunoreactivity colocalized with a specific oligodendrocyte marker galactocerebroside in cells displaying oligodendrocyte morphology, flat cells displaying type-1 astrocyte morphology and glial fibrillary acidic protein, in branched cells displaying type-2 astrocyte morphology and in cell bodies of neurons, the processes of which displayed neurofilament immunoreactivity. Western blots detected both soluble 24 kDa and membrane-bound 28-kDa COMT proteins in neuronal and astrocyte cultures. The results suggest that COMT is synthesized by cultured astrocytes, oligodendrocytes and neurons.

Systemic oral isotretinoin therapy and flow rate, pH, and matrix metalloproteinase-9 activity of stimulated saliva.

Systemic retinoids are known to cause dryness of the mouth and changes in oral and lip mucosa. The purpose of this study was to evaluate changes in salivary variables during treatment with oral isotretinoin in patients receiving the drug for 3 months for cutaneous acne. Patients were examined 1 month after initiation of medication and approximately 3.7 months after its discontinuation. Salivary flow and pH could be measured in 8 and the relative amount of matrix metalloproteinase-9 (MMP-9) of stimulated saliva in 17 patients. The mean flow rate of stimulated saliva was lower during medication than at control examination (P = 0.0277), but no change in the mean pH value was observed during medication. The mean activity of MMP-9 during medication was higher than at control examination (P = 0.0442). The enzyme activity increased in 13 of 17 and decreased in 4 of 17 cases.

Catechol-O-methyltransferase (COMT) in rat brain: immunoelectron microscopic study with an antiserum against rat recombinant COMT protein.

Localization of catechol-O-methyltransferase (COMT) in rat cerebral cortex, neostriatum and cerebellar cortex was studied with preembedding immunoelectron microscopy using a specific antiserum raised against rat recombinant COMT protein. In all areas, immunoreactivity was found both in astrocytes and in neuronal processes. Reaction product was seen in the cytoplasm and in association with tubular structures of dendritic processes. Immunoreactivity was also located postsynaptically in dendritic spines and associated with the postsynaptic membrane. Strong immunoreaction was also seen in the cytoplasm of ependymal cells lining the ventricles, and in tanycytes in median eminence. The results suggest that postsynaptic dendritic spines and astrocytic processes may be the sites of catecholamine inactivation by COMT in rat brain.

O-methylation of L-dopa in melanin metabolism and the presence of catechol-O-methyltransferase in melanocytes.

O-Methylation of L-dopa was investigated as a possible regulatory mechanism in melanin metabolism. The methylation product of L-dopa, 3-O-methoxytyrosine was detected in extracts of cultured human melanocytes. The enzyme catechol-O-methyltransferase is responsible for this O-methylation and that of the dihydroxyindolic intermediates of melanogenesis. The enzyme is present in melanocytes in its soluble and membrane-bound isoforms. Immuno-electron microscopy suggests the presence of the membrane-bound enzyme in the endoplasmic reticulum. This localization may indicate a role of catechol-O-methyltransferase in protecting the melanocyte against reactive dihydroxyphenolic intermediates of melanogenesis leaking from the melanogenic compartments. On the other hand, the O-methylation of L-dopa may serve as a regulatory point in melanogenesis during early stage of tyrosinase processing in the endoplasmic reticulum.

Distribution of catechol-O-methyltransferase enzyme in rat tissues.

In the present study we show the distribution of catechol-O-methyltransferase (COMT) in various rat tissues with a highly specific antiserum prepared against recombinant rat COMT. Immunoprecipitation and immunocytochemical controls confirmed the COMT-specificity of the antibodies. The antiserum detected both the 24 KD soluble and the 28 KD membrane-bound forms of the enzyme. By immunohistochemical staining the COMT enzyme was found in most rat tissues. Staining was most intense in the liver and in the kidney, in agreement with previous studies and our immunoblotting results. In the gastrointestinal tract, epithelial cells of the stomach, duodenum, and ileum were immunoreactive for COMT. In pancreas, COMT immunoreactivity was found in insulin-producing beta-cells and somatostatin-producing D-cells but not in glucagon-producing alpha-cells of the islets of Langerhans. In pituitary, COMT immunoreactivity was found in cleft cells, in pituicytes of the posterior lobe, and in the anterior lobe, partly in the same cells containing luteinizing hormone (LH). In other endocrine organs, COMT immunoreactivity was found in epithelial cells of the thyroid gland and in zona glomerulosa of the adrenal cortex. In the brain, brightest immunofluorescence was seen in ependymal cells of the cerebral ventricles and choroid plexus. Weak to moderate immunofluorescence was found in the neuropil of several brain areas, including striatum and cortex. Scattered small neurons in spinal sensory ganglia were also COMT immunoreactive. Previous immunocytochemical studies, enzyme activity determinations, and distribution of the COMT mRNA are in general agreement with the results presented here. The wide distribution of COMT in different tissues suggests an important role for this protein in inactivation of catechol compounds.

High frequency one-step gene replacement in Trichoderma reesei. I. Endoglucanase I overproduction.

The chromosomal cellobiohydrolase 1 locus (cbh1) of the biotechnologically important filamentous fungus Trichoderma reesei was replaced in a single-step procedure by an expression cassette containing an endoglucanase I cDNA (egl1) under control of the cbh1 promoter. CBHI protein was missing from 37-63% of the transformants, showing that targeting of the linear expression cassette to the cbh1 locus was efficient. Studies of expression of the intact cbh1-egl1 cassette at the cbh1 locus revealed that egl1 cDNA is expressed from the cbh1 promoter as efficiently as cbh1 itself. Furthermore, a strain carrying two copies of the cbh1-egl1 expression cassette produced twice as much EG I as the amount of CBHI, the major cellulase protein, produced by the host strain. The level of egl1-specific mRNA in the single-copy transformant was about 10-fold higher than that found in the non transformed host strain, indicating that the cbh1 promoter is about 10 times stronger than the egl1 promoter. The 10-fold increase in the secreted EG I protein, measured with an enzyme-linked immunosorbent assay (ELISA), correlated well with the increase in egl1-specific mRNA.

High frequency one-step gene replacement in Trichoderma reesei. II. Effects of deletions of individual cellulase genes.

Four cellulase genes of Trichoderma reesei, cbh1, cbh2, egl1 and egl2, have been replaced by the amdS marker gene. When linear DNA fragments and flanking regions of the corresponding cellulase locus of more than 1 kb were used, the replacement frequencies were high, ranging from 32 to 52%. Deletion of the major cellobiohydrolase 1 gene led to a 2-fold increase in the production of cellobiohydrolase II; however, replacement of the cbh2 gene did not affect the final cellulase levels and deletion of egl1 or egl2 slightly increased production of both cellobiohydrolases. Based on our results, endoglucanase II accounts for most of the endoglucanase activity produced by the hypercellulolytic host strain. Furthermore, loss of the egl2 gene causes a significant drop in the filter paper-hydrolysing activity, indicating that endoglucanase II has an important role in the total hydrolysis of cellulose.

Endogenous histamine in cultured bovine adrenal chromaffin cells.

Histamine releases catecholamines and opioids in primary cultured bovine adrenal medullary (BAM) chromaffin cells. We have studied whether histamine is synthesized and localized in BAM cells, and whether it can be released upon activation with secretagogues. In BAM cells histamine is immunohistochemically co-localized with tyrosine hydroxylase in 45 +/- 8% of all cells. Only histamine immunoreactivity was observed in 8 +/- 2% of all BAM cells. No mast-cell-like cells were observed in our system. Histamine can be released from BAM cells by high potassium (56 mM K+) in a calcium-dependent manner. Compound 48/80 did not release histamine from BAM cells but nicotine caused a dose-dependent liberation of the amine. Cultured BAM cells have histidine decarboxylase activity which is inhibited by alpha-fluoromethylhistidine. These results indicate that endogenous histamine is synthesized, stored and released in BAM chromaffin cells in vitro.

Neurotransmitters in the nervous system of Macoma balthica (Bivalvia).

The distribution of histamine-, octopamine-, gamma-aminobutyric acid- (GABA) and taurine-like immunoreactivity in the bivalve mollusc Macoma balthica was studied immunocytochemically with antisera produced in rabbits. Histamine levels in the ganglia and whole animals were also measured by high-performance liquid chromatography using a postcolumn derivatization method. Immunoreactivity for these substances, except for taurine, is found in the central nervous system of this species. The most extensive neuronal system is revealed with the antiserum against histamine. All the main ganglia contain histamine-immunoreactive cell bodies, and a dense network of nerve fibers is seen in the ganglia and nerve roots. Histamine-immunoreactive nerve fibers project to the mantle edge, lips and oesophagus. The basal part of the inhalant siphon is rich in histamine-immunoreactive fibers. Unlike histamine, octopamine- and GABA-like immunoreactivities are restricted to the central nervous system. Taurine-like immunoreactivity is not found in the nervous system of this species. In the nervous system, histamine-immunoreactive cell bodies and fibers are more numerous than those that are octopamine- and GABA-immunoreactive. The distribution of these substances in the ganglia is different. GABA-immunoreactive cells are typically smaller than most of the histamine- and octapamine-immunoreactive cells. Most GABA- and octopamine-immunoreactive cells and fibers are located in the pedal ganglion. Histamine is distributed more evenly in the ganglia and nerve roots. The biochemical measurements of histamine correlate well with the immunohistochemical findings and confirm the predominant location of the amine in the nervous tissue. These results suggest that histamine is more widespread than some other putative transmitters, and support the concept that histamine may have an important role in many physiological processes in molluscs.

Histamine in the nervous system of Macoma balthica (Bivalvia).

The distribution of histamine(HA)-immunoreactivity of the molluscan species Macoma balthica was mapped with an antiserum against a histamine-protein conjugate. The main ganglia of the central nervous system of M. balthica, the cerebropleural ganglia, the pedal ganglion and the visceral ganglion all contained strongly HA-positive fluorescent cell bodies. The positive cell bodies were situated in clusters in the outer region of the ganglia. Immunoreactive fibres were located in the inner neuropil region of the ganglia. Also the commissure and the connectives of the ganglia as well as many nerve roots contained HA-positive fibres. These results agree well with the recent finding of the wide distribution of histamine in the nervous system of two marine gastropod molluscs, Aplysia and Pleurobranchaea supporting the thought that histamine has an important role in many physiological function of molluscs.

Fall in intracellular pH mediated by GABAA receptors in cultured rat astrocytes.

The influence of muscimol (a specific gamma-aminobutyric acid-A (GABAA) receptor agonist) on intracellular pH (pHi) was studied in cultured rat astrocytes by means of fluorescence spectrophotometry with BCECF as the H+ indicator. In an HCO3(-)-free medium, muscimol had little effect on pHi. In a solution containing 22 mM HCO3-, muscimol produced a reversible, concentration-dependent fall in pHi with a maximum of about 0.1-0.15 units. The muscimol-induced fall in pHi was antagonized by an increase in the external K+ concentration, which suggest that the acidosis is an immediate consequence of a net efflux of HCO3- through GABAA receptor channels rather than an indirect effect caused by a change in membrane potential. The present results raise the possibility that astrocytes may participate in the regulation of extracellular pH at GABAergic synapses and contribute to activity-induced pH changes in nervous tissue.