Nicotinic acetylcholine receptor chimeras of rat α7 and Drosophila SAD reveal species-specific agonist binding regions

Species-specific agonist binding regions of nicotinic acetylcholine receptors (nAChR) were examined. Imidacloprid and physostigmine (Phy) selectively activated insect nAChR composed of Drosophila second alpha-like subunit (SAD) and chick β2, in contrast to rat α7 nAChR. The Phy-activated currents were α-bungarotoxin (α-BGT) sensitive, suggesting activation at the agonist binding loop C. Several SAD-α7 chimeras were constructed, by switching agonist binding regions, and expressed in oocytes. Though none of the chimeras was activated by a range of nicotinic agonists, [¹²⁵I]α-BGT binding revealed homomeric assembly of all chimeric cDNAs. Phy differentially displaced [¹²⁵I]α-BGT from the nAChR chimeras, suggesting that the β subunit is not involved in Phy binding, and that Phy targets the insect agonist binding loop C.     

Nitrogen fixation by Azospirillium brasilense in soil and the rhizosphere under controlled environmental conditions

Summary Acetylene reduction activity by Azospirillum brasilense, either free-living in soils or associated with wheat roots, was determined in a sterilised root environment at controlled levels of O₂ tension and with different concentrations of mineral N. In an unplanted, inoculated soil nitrogenase activity remained low, at approximately 40 nmol C₂H₄ h^-1 per 2kg fresh soil, increasing to 300 nmol C₂H₄ h^-1 when malic acid was added as a C source via a dialyse tubing system. The N₂ fixation by A. brasilense in the rhizosphere of an actively growing plant was much less sensitive to the repressing influence of free O₂ than the free-living bacteria were. An optimum nitrogenase activity was observed at 10 kPa O₂, with a relatively high level of activity remaining even at an O₂ concentration of 20 kPa. Both NO _inf3 ^sup- and NH _inf4 ^sup+ repressed nitrogenase activity, which was less pronounced in the presence than in the absence of plants. The highest survival rates of inoculated A. brasilense and the highest rates of acetylene reduction were found in plants treated with azospirilli immediately after seedling emergence. Plants inoculated at a later stage of growth showed a lower bacterial density in the rhizosphere and, as a consequence, a lower N₂-fixing potential. Subsequent inoculations with A. brasilense during plant development did not increase root colonisation and did not stimulate the associated acetylene reduction. By using the ¹⁵N dilution method, the affect of inoculation with A. brasilense in terms of plant N was calculated as 0.067 mg N₂ fixed per plant, i.e., 3.3% of the N in the root and 1.6% in the plant shoot were of atmospheric origin. This ¹⁵N dilution was comparable to that seen in plants inoculated with non-N₂-fixing Psudomonas fluorescens.     

Familial non-rcd1 generalised retinal degeneration in Irish setters

Four Irish setters were diagnosed with bilateral retinal degeneration and cataracts at an age ranging from six to 11 years. In three of these dogs, progressive night blindness was reported from an age of eight to 11 years. In the fourth dog, aged six, no signs of visual impairment had been noticed. In all four dogs, the rod-cone dysplasia type 1 (rcd1) mutation was excluded as a cause, using an allele-specific PCR. From their three-generation pedigrees, a familial relationship was detected in three out of four dogs, which were also related to four additional Irish setter dogs with a history and clinical signs suggestive of late-onset progressive retinal degeneration. These results suggest the existence of a possibly hereditary, late-onset, progressive retinal atrophy in the Irish setter breed, that is distinct from rcd1.     

Breeding with resistant rams leads to rapid control of classical scrapie in affected sheep flocks

ABSTRACT: Susceptibility to scrapie, a transmissible spongiform encephalopathy in sheep, is modulated by the genetic make-up of the sheep. Scrapie control policies, based on selecting animals of resistant genotype for breeding, have recently been adopted by the Netherlands and other European countries. Here we assess the effectiveness of a breeding programme based on selecting rams of resistant genotype to obtain outbreak control in classical scrapie-affected sheep flocks under field conditions. In six commercially-run flocks following this breeding strategy, we used genotyping to monitor the genotype distribution, and tonsil biopsies and post-mortem analyses to monitor the occurrence of scrapie infection. The farmers were not informed about the monitoring results until the end of the study period of six years. We used a mathematical model of scrapie transmission to analyze the monitoring data and found that where the breeding scheme was consistently applied, outbreak control was obtained after at most four years. Our results also show that classical scrapie control can be obtained before the frequency of non-resistant animals is reduced to zero in the flock. This suggests that control at the national scale can be obtained without a loss of genetic polymorphisms from any of the sheep breeds.     

Thoracic aortic rupture in horses

The aorta can rupture at the aortic root or aortic arch. In most breeds, the aortic root is the likely site and rupture leads to aortocardiac fistula with communication between the aorta and the right atrium, right ventricle and/or the interventricular septum. There is a high prevalence of aortic rupture in young Friesian horses and rupture occurs at the aortic arch with pseudoaneurysm and potentially aortopulmonary fistulation. Echocardiographic and post‐mortem techniques must be adapted to identify aortic arch rupture that is not generally identified with standard approaches. Given the narrow genetic base of the Friesian breed and the significant differences found in extracellular matrix composition and metabolism between Friesians and Warmbloods, genetic factors are likely to contribute to the condition in the Friesian breed.     

Heavy Metal Concentration Survey in Soils and Plants of the Les Malines Mining District (Southern France): Implications for Soil Restoration

Mining activities generate spoils and effluents with extremely high metal concentrations of heavy metals that might have adverse effects on ecosystems and human health. Therefore, information on soil and plant metal concentrations is needed to assess the severity of the pollution and develop a strategy for soil reclamation such as phytoremediation. Here, we studied soils and vegetation in three heavily contaminated sites with potential toxic metals and metalloids (Zn, Pb, Cd, As, TI) in the mining district of Les Malines in the Languedoc region (southern France). Extremely high concentrations were found at different places such as the Les Aviniéres tailing basins (up to 160,000 mg kg^?C1 Zn, 90,000 mg kg^?C1 Pb, 9,700 mg kg^?C1 of As and 245 mg kg^?C1 of Tl) near a former furnace. Metal contamination extended several kilometres away from the mine sites probably because of the transport of toxic mining residues by wind and water. Spontaneous vegetation growing on the three mine sites was highly diversified and included 116 plant species. The vegetation cover consisted of species also found in non-contaminated soils, some of which have been shown to be metal-tolerant ecotypes (Festuca arvernensis, Koeleria vallesiana and Armeria arenaria) and several Zn, Cd and Tl hyperaccumulators such as Anthyllis vulneraria, Thlaspi caerulescens, Iberis intermedia and Silene latifolia. This latter species was highlighted as a new thallium hyperaccumulator, accumulating nearly 1,500 mg kg^?C1. These species represent a patrimonial interest for their potential use for the phytoremediation of toxic metal-polluted areas.     

The effect of hemolysis on plasma antithrombin activity as determined by a chromogenic method

BACKGROUND: The concentration of free hemoglobin (Hgb) in plasma can markedly affect the outcome of laboratory tests by interfering with the spectrophotometric absorbance of biochemical tests read at wavelengths within the absorbance range of Hgb (400-440 nm). Little is known about the effects of hemoglobinemia on antithrombin (AT) activity in human plasma samples, and we are unaware of data for canine plasma samples. OBJECTIVES: The objective of this study was to determine the degree of interference by Hgb on plasma AT activity and to determine if the interference is proportional to the concentration of plasma Hgb. METHODS: Two pools of test plasma, designated AT100 and AT70, were prepared. Hemolysate was prepared by washing and freeze-thawing packed red cells in a small volume of saline, followed by collection of the filtrate. Solutions of decreasing Hgb concentration were prepared and added to the test plasma pools to create a series of samples with final calculated and measured Hgb concentrations of 0, 0.5, 1.5, 2.5, 3.5, 4.5, and 5.5 g/L. AT activity, expressed as a percentage of normal human plasma, was determined using a functional chromogenic assay. RESULTS: Increasing concentrations of Hgb resulted in a linear decrease in AT activity. Using linear regression analysis on the 70% and 100% plasma pools, the slopes for samples containing <1.5 g Hgb/L were not significantly different from zero. Slopes for samples containing Hgb concentrations >or=1.5 g/L were significantly (P > .0001) different from zero for both plasma pools, indicating interference with the assay. CONCLUSIONS: The results of this study suggest it may be possible, using a conversion equation, to accurately determine AT activity in hemolyzed samples, facilitating evaluation of coagulation status in patients with intravascular hemolysis.     

Effects of additional starch or fat in late-gestating high nonstarch polysaccharide diets on litter performance and glucose tolerance in sows

The effects of feeding additional starch or fat from d 85 of gestation until parturition on litter performance and on glucose tolerance in sows that were fed a diet with a high level of fermentable nonstarch polysaccharides (NSP) were studied. The day after breeding, 141 multiparous sows were assigned to the experiment. At d 85 of gestation, sows were assigned to the treatments. Sows were fed 3.4 kg/d (as-fed basis) of a high-NSP diet or the same quantity of the high-NSP diet and an additional 360 g of starch (from wheat starch) daily, or the same quantity of the high-NSP diet and an additional 164 g of fat (from soybean oil) daily. During lactation, all sows were given free access to the same lactation diet. Approximately 1 wk before the expected time of parturition, an oral glucose tolerance test was performed in 38 randomly chosen sows by feeding pelleted glucose (3 g/kg BW0.75). Blood samples for glucose analyses were taken at -10, 10, 20, 30, 40, 50, 60, 70, 80, 90, 105, and 120 min after glucose was fed. The supply of additional dietary starch or fat did not increase piglet birth weight or total litter weight at birth. Sows that were fed the high-NSP diet had more (P = 0.097) live-born piglets and fewer (P = 0.084) stillborn piglets than did sows that were fed additional fat, whereas sows that were fed additional starch were intermediate for these variables. Piglet mortality after birth was not affected by dietary treatment. Body weight and backfat gains in the last month of gestation were higher for sows fed additional starch or fat than for sows fed the high-NSP diet (P < 0.001 and P = 0.017, respectively). Feed intake in lactation was greatest by sows fed the high-NSP diet, least by sows fed additional starch at the end of gestation, and intermediate by sows fed additional fat (P = 0.099). The differences in lactation feed intake did not result in differences in BW and backfat losses during lactation. Sows that were fed additional fat had the greatest glucose area under the curve (P = 0.044), indicating that these sows were less tolerant to glucose. In conclusion, feeding additional energy (starch or fat) in late-gestating sows that are fed a high-NSP diet did not increase litter weight at birth or piglet survival, but did increase maternal gain. Feeding sows additional energy from fat might induce glucose intolerance, whereas feeding sows additional energy from starch did not induce glucose intolerance.     

Binding and bioactivity of insulin in primary cultures of carp (Cyprinus carpio) hepatocytes

Isolated carp hepatocytes cultured in serum-free, chemically defined medium were used to investigate within the same cell preparation characteristics of the binding of insulin as well as effects of insulin on cellular metabolism. The binding of human [¹²⁵I]-insulin to carp hepatocytes was studied in kinetic, saturation and displacement experiments. A dependency of insulin binding on the collagenase used for cell isolation was demonstrated. Insulin binding decreased during the first 12h of culture but remained constant during the following 12h. The kinetic experiments revealed that [¹²⁵I]-insulin binding reached a steady state within 20–30 min of incubation. The mathematical analysis of the saturation experiments demonstrated the existence of two populations of binding sites, one with high affinity (K_d1 = 5.5 pM) and low capacity (B_max1 = 0.14 fmol/mg protein or 77 binding sites/cell) and one with low affinity (K_d2 = 2.4 nM) and high capacity (B_max2 = 17.6 fmol/mg protein or 9623 binding sites/cell). In competition experiments, 312 pM [¹²⁵I]-insulin was displaced by cold insulin, IGF-I and IGF-II with IC₅₀ values of 2.2, 7.9 and 20.3 nM, respectively. Glucagon was without effect. Binding of insulin to carp hepatocytes resulted in a significant reduction of glucose release and a significant increase of protein synthesis as of de novo fatty acid synthesis. dedicated to Prof. Dr. W. Hanke on the occasion of his 65^th birthday.