Exposure of burrowing mammals to 222Rn.

Estimates of absorbed dose rates to wildlife from exposure to natural background radionuclides are required to put estimates of dose rates arising from regulated releases of radioactivity and proposed benchmarks into context. Recent review papers have estimated dose rates to wildlife from (40)K, and (238)U and (232)Th series radionuclides. However, only one study previous has considered the potential dose rates to burrowing animals from inhaled (222)Rn and its daughter products. In this paper we describe a study conducted at seven sites in northwest England. Passive track etch detectors were used to measure the (222)Rn concentrations in artificial burrows over a period of approximately one year. Results suggest that absorbed dose rates to burrowing mammals as a consequence of exposure to (222)Rn are likely to be at least an order of magnitude higher than those suggested in previous evaluations of natural background exposure rates which had omitted this radionuclide and exposure pathway. Dose rates in some areas of Great Britain will be considerably in excess of incremental no-effects benchmark dose rates suggested for use as screening levels. Such advised benchmark dose rates need to be better put into context with background dose rates, including exposure to (222)Rn, to ensure credibility; although the context will be determined by the purpose of the benchmark and the assessment level.

Soil microorganisms determine the sorption of radionuclides within organic soil systems.

The potential of soil microorganisms to enhance the retention of (137)Cs and (85)Sr in organic systems was assessed in a series of experiments. A biologically active, 'mineral-free', organic material, produced under laboratory conditions from leaves, was used as the uptake matrix in all experiments to minimise potential interference from competing clay minerals. Biological uptake and release were differentiated from abiotic processes by comparing the sorption of radionuclides in sterilised organic material with sterile material inoculated with soil extracts or single fungal strains. Our results show conclusively that living components of soil systems are of primary importance in the uptake of radionuclides in organic material. The presence of soil microorganisms significantly enhanced the retention of Cs in organic systems and approximately 70% of the Cs spike was strongly (irreversibly) bound (remained non-extractable) in the presence of microorganisms compared to only approximately 10% in abiotic systems. Sorption of (85)Sr was not significantly influenced by the presence of soil microorganisms. A non-linear temperature response was observed for the retention in biotic systems with increased uptake at between 10 and 30 degrees C and lower retention at temperatures above or below the optimum range. The optimum temperatures for biological uptake were between 15 and 20 degrees C for Cs, and 25 and 30 degrees C for Sr. Our results indicate that single strains of soil and saprotrophic fungi make an important contribution to the sorption of Cs and Sr in organic systems, but can only account for part of the strong, irreversible binding observed in biotic systems. Single strains of soil fungi increased the amount of non-extractable (137)Cs (by approximately 30%) and (85)Sr (by approximately 20%) in the organic systems as compared to abiotic systems, but the major fraction of (137)Cs and (85)Sr sorbed in systems inoculated with saprotrophic fungi remained extractable.

Assessing soil biodiversity across Great Britain: national trends in the occurrence of heterotrophic bacteria and invertebrates in soil.

An assessment of the biodiversity of soils was a component of the Countryside Survey 2000 (CS2000). This was the first integrated survey of soil biota and chemical properties at a national scale. A total of 1052 soil samples were collected across Great Britain during CS2000 and analysed for a range of soil microbial and invertebrate characteristics resulting in the production of a series of robust datasets. A principal objective was to use these datasets to investigate relationships between soil biota and environmental factors such as geographical location, vegetation, land use, land cover, soil type and pollutant levels as first stages in characterising the inherent biodiversity of British soils and investigating the potential of soil biodiversity as indicators of soil health at a regional or national scale. Preliminary results for culturable heterotrophic, invertebrate taxa, Acari, Collembola and Oribatid mites are presented here to illustrate the nature of the data collected and the patterns of soil biodiversity in relation to large-scale regional, vegetation and soil characteristics across the British countryside.

Automated method for isolation of adrenal medullary chromaffin cells from neonatal porcine glands.

An automated method for the isolation of neonatal porcine adrenal chromaffin cells is described. Adrenal chromaffin cells are potentially useful for therapeutic transplantation, but current isolation methodology suffers from labor intensiveness and variability in yield and viability due to imprecision of manual techniques, enzyme purity, and gland age and species. The described approach utilizes an adaptation of an automated procedure previously described for isolation of pancreatic islets. Results from neonatal porcine adrenal glands revealed consistent cell yields with high (approximately 99%) viability. Catecholamine assays showed that the resultant cultures continue to produce and secrete norepinephrine and epinephrine. Immunocytochemical analysis indicated that the majority of cells in the preparation are chromaffin cells and adrenal cortical cells. The procedure meets the following requirements: 1) minimal traumatic action on the adrenal chromaffin cells, 2) continuous digestion in which the adrenal cells that are progressively liberated can be saved from further mechanical action, 3) minimal human intervention in the digestion process, and 4) high yield and viability of the isolated adrenal chromaffin cells.

Cytokine-induced conversion of mesencephalic-derived progenitor cells into dopamine neurons.

We have previously shown that a combination of the cytokines interleukin (IL)-1, IL-11, leukemia inhibitory factor (LIF), and glial cell line-derived neurotrophic factor (GDNF) can convert rat fetal (E14.5) mesencephalic progenitor cells into tyrosine hydroxylase (TH)-immunoreactive (ir) neurons in vitro. The experiments described here characterize the mesencephalic progenitor cells and their cytokine-induced conversion into dopamine (DA) neurons. For all experiments, we used bromodeoxyuridine (BrdU)-ir cultures of (E14.5) mesencephalic progenitor cells that had been expanded at least 21 days. We first demonstrated that IL-1 induced DA neuron conversion in mesencephalic progenitors, but not in striatal progenitors (P < 0.001). Thus, these cells should be classified as lineage-restricted progenitors, and not omnipotent stem cells. To further characterize cell populations in these cultures, we used monoclonal antibodies against Hu (an early marker for neurons), growth-associated protein (GAP)-43 (a marker for neuronal process extension), TH (a marker for DA neurons), and glial fibrillary acidic protein (GFAP, a marker for astrocytes). We assessed (E14.5) mesencephalic progenitor cell cultures (plated at 125,000 cells/cm2) incubated in the cytokine mixture (described above) or in complete media (CM, negative control). Following 7 days incubation, GFAP-positive cells formed a nearly confluent carpet in both types of cultures. However, numbers of Hu-ir and GAP-43-ir cells in the cytokine-incubated cultures far exceeded those in CM-incubated controls (P = 0.0003, P = 0.0001, respectively), while numbers of TH-ir cells were 58-fold greater in the cytokine-incubated cultures versus CM-incubated controls. The TH phenotype persisted for 7 days following withdrawal of the differentiation media. Numerous double-labeled cells that were BrdU-ir and also TH-ir, or Hu-ir and also TH-ir, were observed in the cytokine-incubated cultures. These data suggest that cytokines "drive" the conversion of progenitor cells into DA neurons.

Differentiation of mesencephalic progenitor cells into dopaminergic neurons by cytokines.

Rat progenitor cells from the germinal region of the fetal mesencephalon were isolated and expanded in media containing the mitogen epidermal growth factor. These cells remained mitotically active (up to 8 months), were immunoreactive for the progenitor cell marker nestin, and were readily infected with the BAG alpha retrovirus. When incubated in complete media containing serum in poly-L-lysine-coated plates, these cells spontaneously converted to neurons and glia but rarely expressed the dopamine (DA) neuron phenotype. Nineteen different cytokines were screened for their ability to induce the DA phenotype and only interleukin (IL)-1 was found to induce the expression of the DA neuron marker tyrosine hydroxylase (TH). The addition of IL-1, IL-11, leukemia inhibitory factor (LIF), and glial cell line-derived neurotrophic factor (GDNF) were found to further increase the number of TH immunoreactive (TH-ir) cells. The addition of mesencephalic membrane fragments and striatal culture-conditioned media along with the cytokine mixture induced the expression of morphologically mature TH-ir cells that were also immunoreactive for dopa-decarboxylase, the DA transporter, and DA itself. The DA neuron cell counts were approximately 20-25% of the overall cell population and 50% of the neurofilament population. Astrocytes and oligodendrocytes were also present. These data suggest that hematopoietic cytokines participate in the development of the DA neuron phenotype. Parallels between the function of hematopoietic cytokines in bone marrow and the central nervous system may exist and be useful in understanding the factors which regulate the differentiation of neurons in the brain.

The effects of storage conditions and trophic supplementation on the survival of fetal mesencephalic cells.

It is estimated that only 5-10% of dopamine (DA) neurons implanted into the striatum of patients undergoing fetal-nigral transplantation as a treatment for Parkinson's Disease survive. Because it is often necessary to store fetal tissue prior to transplantation, we evaluated various storage parameters that could influence DA neuron viability in rostral mesencephalic tegmentum (RMT) cultures using tyrosine hydroxylase immunoreactive (THir) cell counts as an index of DA neuron survival. A high K+ hibernation media (HM) was used in all studies. We found that RMT cell viability and THir cell counts decreased as storage duration increased (up to 120 h). Storage at 37 degrees C in HM killed all cells, while storage at 10 degrees C yielded higher survival rates than 4 degrees C. In comparison to trypsinization, mechanical dissociation of tissue increased cell viability. Neutral pH and a storage density of at least 1 x 10(6) cells/mL were found to be optimal, while striatal coculture of RMT cells with striatal feeder layers increased THir viability up to 16-fold in comparison to monocultures. The nurturing effect of striatal coculture may be explained by the release of autotrophic factors, and we tested this hypothesis by supplementing the HM with human placental cord serum (HPCS, 8%), glial-derived neurotrophic factor (GDNF; 10 microg/mL), and brain-derived neurotrophic factor (BDNF; 10 microg/mL). GDNF and HPCS supplements increased RMT cell viability by 10-15%, while GDNF, BDNF, and HPCS increased viability of THir cells by approximately 40% at all time points studied. As Klenow enzyme labeling technique indicated that 33% of stored RMT cells were undergoing apoptosis, we found that GDNF, BDNF, and HPCS reduced apoptosis by 50%. DNA laddering and DAPI nuclear stain confirmed the presence of apoptosis in hibernated RMT cells, leading us to postulate that the high viability counts seen with trypan blue exclusion are misleading.

Cloning and widespread distribution of the rat rod-type cyclic nucleotide-gated cation channel.

We used Northern blot analysis, ribonuclease protection assay (RPA), reverse transcriptase-polymerase chain reaction, and in situ hybridization to investigate the hypothesis that the CNG1 isoform of the cyclic nucleotide-gated nonselective cation channel may be widely distributed in tissues of the rat. A cDNA encoding the CNG1 isoform was isolated from rat eye and human retina, and partial sequences were isolated from rat pineal gland and human kidney. Northern blot analysis revealed a 3.1-kilobase (kb) CNG1 transcript in rat eye, pineal gland, pituitary, adrenal gland, and spleen, and a larger transcript of 3.5 kb was found in testis. RPA confirmed the identity of CNG1 mRNA in rat eye, lung, spleen, and brain. Polymerase chain reaction-based detection of the mRNA for CNG1 indicates that the channel is expressed in lower abundance in many other tissues, including thymus, skeletal muscle, heart, and parathyroid gland. The cellular distribution of CNG1 was further studied by in situ hybridization, which demonstrated expression of mRNA in lung, thymus, pineal gland, hippocampus, cerebellum, and cerebral cortex but not in heart or kidney.

cGMP stimulates sodium and chloride currents in rat tracheal airway epithelia.

To test the hypothesis that guanosine 3',5'-cyclic monophosphate (cGMP) regulates ion transport in airway epithelial cells, we measured short-circuit current (I(sc)) and (22)Na+ fluxes in primary cultured rat tracheal epithelial cells. In Cl- -containing Ringer solution, I(sc) was increased by approximately 17 microA/cm2 after application of 1 mM 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP), whereas, in Cl- -free solutions, the Na+ -mediated component was approximately 5 microA/cm2, suggesting a cGMP stimulation of Cl-secretory current and a smaller Na+ absorptive current. Inward and net mucosal-to-serosal (22)Na+ flux was doubled in the presence of 2 mM 8-BrcGMP. To determine whether nucleotide-gated channels play a role in this transepithelial Na+ absorption, blockers of nucleotide-gated cation channels were used to inhibit I(sc). The cGMP-stimulated Na+-mediated I(sc) was blocked by as little as 500 nM dichlorobenzamil or 50 microM L-cis-diltiazem, which are known blockers for cyclic nucleotide-gated cation channels. These agents also blocked the basal (non-cGMP-stimulated) current when measured in the presence of 10 microM amiloride, which blocks current through 5-pS amiloride-sensitive Na+ channels. To document whether the distribution of nucleotide-gated nonselective cation channels was consistent with a role in airway epithelial transport, in situ hybridization was performed. In situ hybridization of mRNA encoding for nucleotide-gated cation channels was found in epithelial cell layers of rat trachea, bronchi, bronchioles, and alveolar cells but not in smooth muscle layers or tracheal cartilage. Reverse transcriptase-polymerase chain reaction, restriction enzyme analysis, and sequencing of the cDNA transcribed from mRNA of whole lung and tracheal epithelial cells indicate that a channel highly homologous to the retinal nucleotide-gated nonselective cation channel (CNG1) is present. Thus these data, along with evidence supporting the existence of signal transduction pathways elevating intracellular levels of cGMP, indicate that cGMP regulates transepithelial ion transport in lung epithelial tissues.

Development of muscarinic receptor subtypes in the forebrain of the mouse.

Cholinergic mechanisms are involved in the regulation of developmental events in the nervous system. Muscarinic cholinergic receptors are thought to be the predominant mediator of cholinergic neurotransmission in the forebrain; however, their developmental role is less well understood. The present study takes advantage of subtype-specific antibodies to muscarinic receptor proteins to investigate the cellular localization of the subtypes in developing mouse forebrain. Receptor protein expression was assessed between postnatal day (PND) 5 and adulthood by immunocytochemical methods with antibodies to m1, m2, and m4 receptors, the most abundant subtypes in rodent brain. We have found dramatic developmental changes in the distribution of all three receptors. In the adult mouse, m1 and m2 receptor immunoreactivity displayed complementary staining patterns in most forebrain areas with m4 sharing similarities in pattern with both m1 and m2. Furthermore, each receptor was expressed transiently in gray matter areas or fiber bundles at various developmental stages. The m4 receptor was also expressed in developing blood vessels. Such transient immunoreactivity was usually associated with times and areas of dynamic morphogenesis, thus suggesting distinct roles for the receptor subtypes in ontogenetic events.

Overzicht Van Het Hematologisch Kwaliteitskontrole 1973.

It appears from this Q.C. survey that automatic cel counting generally gives good results for erythrocytes, but somewhat less good results for white blood cells. Thrombocytic numeration however, gives unreliable and uncomparable results in a Q.C. survey, regardless of the method used, because of the extreme sensitivity of the platelets to a number of external factors, such as temperature, transportation, etc. As far as morphological examination is concerned it appears that many observers pay too little attention to morphological abnormalities of the erythrocytes. The results with blood group serology point to some difficulties with Rh-factor determination.