Assessing toxicity of Lake Diefenbaker (Saskatchewan,Canada) sediments using algal and nematode bioassays

Lake Diefenbaker, on the South Saskatchewan River, Saskatchewan, Canada, receives, on average, 90% of its inflow from snowmelt and rainfall in the Rocky Mountains. The inflowing rivers also receive irrigation return flows and municipal and industrial effluents which may result in the contamination of lake sediments. The sediments were assessed by nematode and algal bioassays.The toxicity of five chemical fractions of the sediment was determined using the nematode Panagrellus redivivus as the test organisms. The results suggest that the sediment chemical fractions frequently inhibit growth and maturation, while lethality was observed at 4 of 12 sites.Samples from 3 of these sites were further evaluated using conventional elutriate Algal Fractionation Bioassays (AFB) with both natural Lake Diefenbaker phytoplankton and a mixed laboratory grown algal culture. The natural phytoplankton showed inhibition at sediment: water ratios of 10: 1; whereas the algal cultures showed both enhancement and inhibition. Evidently, the sediments are frequently toxic to the species tested except for the algal culture. The AFB assesses the mitigative and synergistic effects of contaminants and nutrients and being a conventional elutriate, is more realistic and potentially more acceptable than the chemical fractionation/nematode bioassay technique which essentially considers potential trace organic contaminant effects.     

Identification of four novel splice site mutations in the ornithine transcarbamylase gene

Ornithine transcarbamylase (OTC) deficiency, the most common inborn error of the urea cycle, shows X-linked inheritance with frequent new mutations. Using polymerase chain reaction (PCR) amplification of the individual exons including adjacent intron sequences followed by direct sequencing of the amplimers we identified four new mutations affecting donor splice sites of introns 2, 5, 6, and 8. The mutation at the first position of intron 2 was a G to A exchange associated with acute neonatal hyperammonemia in a male patient at the age of 5 months. A G to C substitution in intron 5 was detected in a boy who developed 2 days after birth hypotonia, and respiratory distress, followed by severe hyperammonemia and terminal coma. The intron 6 mutation, a G to T substitution, was detected in a girl presenting with first episodes of vomiting and agitation at the age of 2 months. The mutation in intron 8, also a G to T transition, caused fatal hyperammonemia and early death at the age of 15 days in a male patient. We present four donor splice site mutations resulting in severe neonatal or very early onset of the disease in three boys and in one female patient. As the GT dinucleotide of the 5 donor splice site is invariant and required for correct splicing the described mutations may lead to improperly spliced mRNAs and aberrant gene products.     

A Quantitative Microtiter Plate Nuclease Assay Based on Ethidium/DNA Fluorescence

A microtiter plate assay was developed to quantitate the nuclease activity of the extracellularSerratia marcescensendonuclease under different buffer conditions. Substrate cleavage was followed as decrease in ethidium/DNA fluorescence using a uv-transilluminator and a video documentation system. Time courses of DNA cleavage were recorded and cleavage rates determined very precisely within a factor of 1.2. The assay has a linear dynamic range covering three orders of magnitude of nuclease activity and can be carried out very quickly within a few minutes. It can also be used with RNA as substrate. With appropriate modifications it should be possible to adapt this assay for other enzymatic reactions which are accompanied by changes in absorbance or fluorescence.     

Dynamic properties of cortical evoked (10 Hz) oscillations: theory and experiment

Experiments probed the dynamic properties of stimulus-evoked (10 Hz) oscillations in somatosensory cortex of anesthetized rats. Experimental paradigms and statistical time series analysis were based on theoretical ideas from a dynamic approach to temporal patterns of neuronal activity. From the results of a double-stimulus paradigm we conclude that the neuronal response contains two components with different dynamics and different coupling to the stimulus. Based on this result a quantitative dynamic model is derived, making use of normal form theory for bifurcating vector fields. The variables used are abstract, but measurable, dynamic components. The model parameters capture the dynamic properties of neuronal response and are related to experimental results. A structural interpretation of the model can be given in terms of the collective dynamics of neuronal groups, their mutual interaction, and their coupling to peripheral stimuli. The model predicts the stimulusdependent lifetime of the oscillations as observed in experiment. We show that this prediction relies on the basic concept of dynamic bistability and does not depend on the modeling details.     

Calcitonin- and somatostatin-positive cells in thyroid gland of pigs at different ages

Immunohistochemical studies were carried out on calcitonin-, somatostatin- and serotonin-reactive cells in newborn pigs and pigs at 3 weeks and 7 months old. The aim of these studies was to examine if the expression of various bioactive substances by parafollicular cells in the pig thyroid varied during development. The volume density of the follicular epithelium was nearly the same in newborn and 3-week-old piglets and significantly lower in 7-month-old animals. The volume density of calciton-in-positive cells, expressed as a percentage of the follicular epithelium density, was similar in young animals, being 12.10% and 13.03% in newborn and 3-week-old piglets, respectively. A small but significant increase to 14.40% was seen in 7-month-old pigs. Somatostatin-positive cells formed a much smaller population at all time points, but these also showed a significant increase with age (0.13%, 0.17% and 0.52% of follicular epithelium density in newborn, 3-week- and 7-month-old pigs, respectively). However the changes in the volume density of somatostatin-positive cells correlated inversely with thyroid activity, the density being highest when the activation index was lowest, suggesting that thyroid activity may be regulated by an increase in the synthesis of this inhibitory peptide. Serotonin-positive cells were extremely rare at all time points and their volume density was not calculated.     

Fluorescence in situ hybridization with cosmid clones for the detection of human cytomegalovirus DNA in peripheral blood leukocytes

 Radioactive in situ hybridization techniques or enzymatic detection procedures of hapten-modified human cytomegalovirus (HCMV) probes have been widely used for studying the infection of peripheral blood leukocytes with HCMV. This report describes significant improvements in terms of signal resolution which can be obtained by applying a highly sensitive fluorescence in situ hybridization (FISH) technique in conjunction with a large subgenomic HCMV DNA probe. Three cosmid clones spanning 119.1 kb of the HCMV genome (230 kb) were used to construct the digoxigenin-11-dUTP-labeled probe which was found to be superior to a total HCMV probe representing the entire genome. Crucial hybridization parameters were analyzed systematically in order to ensure optimal resolution power and sensitivity. The protocol was successfully applied to HCMV-infected fibroblasts and peripheral blood leukocytes of 12 transplant patients and unambiguously facilitated the precise intracellular localization of HCMV genomes in infected cells. Because of its excellent resolution properties, accompanied by the virtual absence by any background staining, we recommend the use of this protocol as a sensitive approach for further virological analyses of the interactions between HCMV and peripheral blood leukocytes at the single-cell level. Accepted: 16 February 1996     

Localization of C-S lyase activity in the root cells ofAlbizzia lophanta

Summary The distribution of C-S lyase activity in root cells ofAlbizzia lophanta Benth. plantlets was investigated histochemically. H₂S formed upon cleavage of exogenously applied L-cysteine was precipitated by Pb⁺⁺ in a capture reaction at the site of its formation. Enzyme activity was found to be localized at the root tip and in a layer of cortex cells adjacent to the endodermis throughout the whole length of the root. Distinct areas within the exodermis, distributed in a regular pattern on the root surface, also exhibited the specific reaction. In vivo roots ofAlbizzia lophanta actively excrete the strongly smelling methylene dithiol, formed by enzymatic cleavage of djenkolic acid, the natural substrate of C-S lyase inAlbizzia. The physiological meaning of this compound, as well as the localization and intracellular distribution of C-S lyase activity are discussed.