Relationships between prostaglandin concentrations, a single nucleotide polymorphism in HSD17B12, and reproductive performance in dairy cows.

Prostaglandins are involved in multiple processes important for fertility, with previous work in mice highlighting a potential role for the HSD17B12 gene in prostaglandin biosynthesis. This study aimed to determine the associations among circulating prostaglandin concentrations, a missense SNP in the HSD17B12 gene predicted to disrupt protein function, and fertility traits in first-lactation Holstein-Friesian dairy cows. We used a study population of approximately 500 animals specifically bred to have either a positive (POS, +5%) or negative (NEG, -5%) genetic merit for fertility (FertBV). Genotypes of a previously identified SNP (rs109711583) in HSD17B12 were determined, with 116 animals genotyped as AA, 215 genotyped as AG, and 153 genotyped as GG. Plasma concentrations of prostaglandin E2 and the PGF2α metabolite PGFM were determined at 3 time points (12 mo of age, 4 d postpartum, and 5 wk postpartum during first lactation) in a selection of animals with AA and GG genotypes from both the POS and NEG FertBV groups (n = 33-40 in each genotype for each FertBV group). Binary reproductive traits (yes or no) examined included submission for artificial breeding in the first 3 or 6 wk of the seasonal breeding period; conception to first service; conception during the first 6 wk of the breeding period; and pregnant at the end of the breeding period. Uterine health at 6 wk after calving was examined by evaluating the percentage of polymorphonuclear leukocytes following uterine cytology and by scoring vaginal discharge based on the presence of purulent material. The 3-wk submission rate was increased in animals that carried the G allele of the missense SNP in HSD17B12, but no differences were present among genotypes for 6-wk submission rate. The trait was additive, with each increase of the G allele increasing the 3-wk submission rate by 6 to 7%. We did not observe any consistent associations between SNP alleles and circulating PGE2 concentrations; however, a complex 3-way interaction among time, fertility group, and SNP allele was present for PGFM concentrations. Plasma concentrations of PGE2 were increased approximately 40% at 5 wk postpartum in animals that were submitted for breeding within 3 or 6 wk of the start of the breeding season, and in those that conceived during the first 6 wk of breeding, compared with those that did not. Plasma concentrations of PGFM were decreased approximately 20% in those animals that conceived to their first service and tended to be decreased in animals that were pregnant at the end of the breeding period, compared with those that were not. In summary, associations were observed between the SNP in HSD17B12 and submission rate by d 21 of the breeding season, as well as between circulating prostaglandin concentrations and fertility traits, but the SNP was not consistently linked to changes in prostaglandin concentrations. Thus, the association between submission rate by d 21 of the breeding season and the SNP in HSD17B12 were unlikely driven by changes in prostaglandins.

A linkage map of sheep chromosome X (OARX) aligned to human chromosome X (HSAX).

We have constructed a genetic linkage map of the sheep X chromosome (OARX) containing 22 new gene loci from across the human X chromosome (HSAX). The female OARX linkage map has a total length of 152.6 cM with average gene spacing of 5.5 cM. Comparison with HSAX confirms one previously reported major breakpoint and inversion, and other minor rearrangements between OARX and HSAX. Comparison of the linkage map with sheep sequence data OAR 1.0 reveals a different arrangement of markers on the q arm, which may more accurately reflect the genuine arrangement of this region.

Avian thyroid: effect of p,p'-DDT on size and activity.

Feeding sublethal amounts of p,p'-DDT to pigeons caused an increase in thyroid weight and a reduction in colloid content of the follicles. This may reflect a hyper- or hypo-functioning gland and may be cotnnected with recent reductions in egg shell weights in wild birds. The effect was accompanied by increased liver weight.

Degradation and disappearance of ortho, para isomer of technical DDT in living and dead avian tissues.

The O,p'-DDT in technical DDT is broken down to p,p'-DDT and then to 1,1-dichloro- 2,2-bis(p-chlorophenyl)ethylene in living avian tissue. In the anaerobic conditions existing after death, O,p'-DDT is metabolized to 1,1-dichloro-2-(O-chlorophenyl)-2-(p-chlorophenyl)ethane. The absence of O,p'-DDT and metabolites in field specimens is ascribed to the rapid rate of breakdown and a masking of the 1,1-dichloro-2-(O-chlorophenyl)-2-(p-chlorophenyl)ethane residue during analysis by the relatively large amounts of 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene.

Comparative mapping of sheep chromosome 2q.

Sheep chromosome 2q (OAR2q), which is homologous with human chromosome 2q (HSA2q), and cattle chromosome 2 (BTA2), is known to contain several loci contributing to carcass traits. However, the chromosomal rearrangements differentiating these chromosomes among the three species have not yet been determined and thus precise correspondences between the locations of sheep and human genes are not known. Twenty-six genes from HSA2q (2q21.1-->2q36) have been assigned to OAR2q by genetic linkage mapping to refine this area of the sheep genome. Seventy-six genes were initially selected from HSA2q. Sixty-eight percent of the PCR primer sets designed for these genes amplified successfully in sheep, and 34% amplified polymorphic products. Part of the proximal arm of OAR2q was found to be inverted compared with HSA2q. The breakpoint has been localised near the growth differentiation factor 8 gene (GDF8), spanning 380 kb between the positions of the hypothetical protein (FLJ20160) (HSA2:191008944-191075046) and glutaminase (GLS) (HSA2:191453847-191538510) (Build36.1).

NLRP3 regulates a non-canonical platform for caspase-8 activation during epithelial cell apoptosis.

Nod-like receptor, pyrin containing 3 (NLRP3) is characterized primarily as a canonical caspase-1 activating inflammasome in macrophages. NLRP3 is also expressed in the epithelium of the kidney and gut; however, its function remains largely undefined. Primary mouse tubular epithelial cells (TEC) lacking Nlrp3 displayed reduced apoptosis downstream of the tumor necrosis factor (TNF) receptor and CD95. TECs were identified as type II apoptotic cells that activated caspase-8, tBid and mitochondrial apoptosis via caspase-9, responses that were reduced in Nlrp3-/- cells. The activation of caspase-8 during extrinsic apoptosis induced by TNFα/cycloheximide (TNFα/CHX) was dependent on adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC) and completely independent of caspase-1 or caspase-11. TECs and primary human proximal tubular epithelial cells (HPTC) did not activate a canonical inflammasome, caspase-1, or IL-1ß secretion in response to TNFα/CHX or NLRP3-dependent triggers, such as ATP or nigericin. In cell fractionation studies and by confocal microscopy, NLRP3 colocalized with ASC and caspase-8 in speck-like complexes at the mitochondria during apoptosis. The formation of NLRP3/ASC/caspase-8 specks in response to TNFα/CHX was downstream of TNFR signaling and dependent on potassium efflux. Epithelial ASC specks were present in enteroids undergoing apoptosis and in the injured tubules of wild-type but not Nlrp3-/- or ASC-/- mice following ureteric unilateral obstruction in vivo. These data show that NLRP3 and ASC form a conserved non-canonical platform for caspase-8 activation, independent of the inflammasome that regulates apoptosis within epithelial cells.

Effects of certain muscarinic antagonists on the actions of anticholinesterases on cat skeletal muscle.

1. The effects of some muscarinic antagonists, namely, N-ethyl-2-pyrrolidylmethyl-cyclopentylphenyl glycollate (PMCG), N-methyl-4-piperidyl-phenylcyclohexyl glycollate (PPCG, racemate and R and S enantiomers) and 4'-N-methyl-piperidyl-1-phenyl-cyclopentane carboxylate (G3063) on organophosphate (sarin, soman)- and carbamate (neostigmine)-induced twitch augmentation have been studied in cat soleus muscle. 2. The results of a preliminary study comparing the potency of sarin and soman in inhibiting the acetylcholinesterase activity of muscle in relation to the effect on the maximal twitch response indicated that there is not a simple relationship between degree of enzyme inhibition by these drugs and alteration of muscle function. 3. The muscarinic antagonists studied were capable of preventing or reversing sarin-, soman- or neostigmine-induced twitch augmentation. Doses sufficient to give complete protection from the effects of the anticholinesterase agents had little or no effect on the twitch response of normal muscle. 4. The protective action of these muscarinic antagonists is dose-dependent but independent of known antagonist actions at muscarinic receptors. 5. The effects of some local anaesthetics (lignocaine, prilocaine, cinchocaine, procaine) and other membrane stabilizers (quinine, ketamine, chlorpromazine, triflupromazine) were compared with those of the muscarinic antagonists in an attempt to elucidate the mode of action of these acetylcholine antagonists. The evidence is insufficient to exclude the involvement of a membrane stabilizing action.

The hydrolysis of succinyldithiocholine and related thiocholine esters by human plasma and purified cholinesterase.

The kinetic properties of cholinesterase (ChE) present in plasma were compared with those of purified human ChE using the substrates succinyldithiocholine (SDTCh), acetylthiocholine (AcTCh) and butyrylthiocholine (BuTCh). SDTCh was hydrolysed at two sites; a site with a low Km (Km1 11.4 +/- 3.3 microM) with a Vmax of 0.06 mumol/min/ml and a site with a high Km (Km2 132.4 +/- 14.8 microM) and a Vmax of 0.107 mumol/min/ml. The Km2 site was absent in the sample of purified ChE. The related thiocholine esters, AcTCh and BuTCh were hydrolysed at two sites by both plasma and purified ChE. This indicated that the Km2 site which hydrolysed SDTCh was not ChE. The identity of this component in plasma remains unknown but it was shown not to be albumin. The anticholinesterase agents soman and pyridostigmine were used to demonstrate the direct relationship between inhibition of plasma ChE and hydrolysis of SDTCh at the low concentrations present clinically (20 microM). Whereas high concentrations of SDTCh (200 micron) could be partly hydrolysed by an enzyme present in plasma which is insensitive to ChE inhibitors. In a limited study on the plasma from two "atypical" individuals (Dibucaine number less than 20) all three substrates were hydrolysed at a single site with a higher Km than the Km2 site present in normal plasma. The clinical implications of these results are discussed.

A comparison of the decarbamoylation rates of physostigmine-inhibited plasma and red cell cholinesterases of man with other species.

Plasma and red cells from a variety of animal species were used to demonstrate that there is a relationship between the decarbamoylation rates of physostigmine-inhibited plasma and red cell cholinesterases in vitro and the effectiveness of carbamate pretreatment against nerve agent poisoning reported in the literature. Decarbamoylation rates were faster in the non-human primates than in the guinea-pig, and carbamate pretreatment is more effective in these species than in the guinea-pig. The data for the decarbamoylation rates of physostigmine-inhibited enzymes suggests that the non-human primates are the best animal model for extrapolation of protection studies from animal species to man. Control values for red cell acetylcholinesterase (AChE) activity (mumol/min/mL blood) using acetylthiocholine (1 mM) were higher in the human (4.98) and the rhesus monkey (4.14) than in the marmoset (0.84) and the guinea-pig (0.83). Plasma cholinesterase (ChE) activity (mumol/min/mL plasma) using butyrylthiocholine (10 mM) was highest in the rhesus monkey (9.29), intermediate in human (5.10) and guinea-pig (6.06), and lowest in the marmoset (4.07). There was a species difference in the relative activity of AChE: ChE in blood, human (65:35), rhesus monkey (45:55), marmoset (30:70) and guinea-pig (20:80). The rate of recovery of red cell AChE and plasma ChE activities, following incubation of whole blood with physostigmine (1 x 10(-7) M), was in the order human greater than rhesus monkey greater than marmoset greater than guinea-pig. During the incubation of red cells with physostigmine there was little recovery of AChE activity for 3-4 hr in any species. During the incubation of plasma with physostigmine there was complete recovery of ChE activity by 2-3 hr in the human and rhesus monkey and a significant recovery by 3 hr in the marmoset and guinea-pig. This suggests that a component of plasma, possibly ChE, was responsible for the degradation of physostigmine, presumably by hydrolysis. There was a marked species difference in the decarbamoylation rates of physostigmine-inhibited enzyme. In the red cell the t1/2 values (min) were 14.8 (human), 21.2 (rhesus monkey), 17.9 (marmoset) and 31.9 (guinea-pig). In the plasma the t1/2 values (min) were 11.2 (human), 32.9 (rhesus monkey), 44.1 (marmoset) and 52.4 (guinea-pig).

Brain acetylcholine concentration and acetylcholinesterase activity in selectively-bred strains of rats.

Significant differences in whole brain and in brain area acetylcholine concentration have been observed between the two strains of rats selectively bred for differences in speed of acquisition of conditioned avoidance. Brain acetylcholinesterase activity was similar in the strains. The strain showing poor conditioned avoidance behaviour possessed the highest brain concentration of acetylcholine. The finding supports a theory which describes a central cholinergic inhibitory system which may be active in the control of certain types of learning behaviour.

Effect of acute physostigmine-hyoscine pretreatment on the neurochemical changes produced by soman in the guinea-pig.

The protective effects of two dose regimes of the organophosphate pretreatment combination, physostigmine and hyoscine, were assessed on the central neurochemical changes produced following soman intoxication. The lower dose combination (physostigmine 20 ?g/kg, hyoscine 10 ?g/kg, s.c.) inhibited brain regional acetylcholinesterase (AChE) by between 13.5 and 37.6% in all regions except the striatum, where there was no statistically significant inhibition. This low dose pretreatment failed to protect a measureable proportion of brain AChE from soman and did not prevent the neurotransmitter changes produced by soman. Signs of intoxication were indistinguishable from those seen with soman alone, however more animals survived longer than 24 h. The higher dose combination (physostigmine 196 ?g/kg and hyoscine 113 ?g/kg s.c.) inhibited brain regional AChE by between 56.8 and 67.3%, but had significant effects alone on the levels of ACh and NA. The high dose pretreatment effectively protected 20-30% of the enzyme following soman challenge. This protected enzyme became available as the physostigmine was eliminated from the body after 60-120 min. Animals exhibited mild signs of poisoning, such as hyperactivity and chewing, during the first 30-60 min, after which they recovered. Transmitter changes following soman were completely prevented with high dose pretreatment. Both dose levels of pretreatment decreased lethality but only the high dose, which protected a measurable amount of AChE and prevented neurotransmitter changes, decreased incapacitation.

The reversal by oximes and their de oximinomethyl analogues of neuromuscular block produced by soman.

A series of oximes and related compounds were assessed for their ability to restore soman-induced neuromuscular block in the isolated diaphragm preparation of the rat, guinea-pig and marmoset. In the rat the bispyridinium oximes HS6, HI6 and HS14 were superior to P2S and all other compounds tested. Conversely, in the guinea-pig, most of the compounds tested produced a good reversal of neuromuscular block. In a limited number of experiments in the marmoset, only a partial reversal of neuromuscular block, was obtained with the oximes HI6 and HS6. The restoration of neuromuscular block was due to one or more of the following factors: (i) enzyme reactivation (ii) direct action (iii) adaptation. The results of this study suggest that both the acetylcholine receptor and the rate of 'ageing' of soman inhibited AChE are different in these three species.

The spatial population dynamics of insects exploiting a patchy food resource : II. Movements between patches.

The population dynamics of ten species of phytophagous insects and seven parasitoids inhabiting the flowerheads of two herbaceous plants, Centaurea nigra and Arctium minus, were studied, and three main aspects of their ecology were examined, namely, rates of population extinction, density dependence in population changes from one generation to the next, and movements between populations. The study was based on monitoring the insect populations on more than 50 patches of each plant, scattered over 5 km2 of arable farmland and the results were used to test the relative importance of immigration and population regulation to the persistence of these populations. This paper describes the study of movements between patches of food plant. Experimentally planted new patches of plants were rapidly colonised by all species and this appeared to be unaffected by distance from a source population, up to the maximum distance of 800 m considered in the experiment. Large patches tended to be colonised more readily than small ones. Movements between plant patches were studied with the use of chemical markers (Rb, Sr, Dy and Cs) which were applied as chloride salts to individual patches, and which were translocated to the flowerheads and so to insects feeding on the seed, and to their parasitoids. Initial experiments in the laboratory showed that these elements could be readily detected by ICP (Inductively Coupled Plasma) mass spectrometry in the bodies of all species reared on potted plants sprayed with solutions containing them. Background levels of strontium were patchily high on the study area, but the other elements were naturally either absent or in very low concentrations. Four patches of each plant were marked with a different element in 1991. In 1992, samples of four species of tephritid fly and two parasitoids were collected from all patches, and analysed for the four elements. These analyses showed that individual of all species moved considerable distances, with movements of up to 2 km being commonly recorded. Estimates of rates of immigration to patches showed that movement plays an important role in the population dynamics of these insects. There was some evidence that immigration was density-dependent: it was highest when the resident populations (numbers per flowerhead) were low.

The reversal by pyridostigmine of neuromuscular block produced by soman.

The effect of pyridostigmine on neuromuscular block produced by soman was studied in the isolated phrenic nerve-diaphragm preparation. In the rat, soman produced an irreversible reduction in tetanic tension and functional acetylcholinesterase (AChE) activity. Pretreatment with pyridostigmine before exposure of the diaphragm to soman, followed by removal of the anticholinesterase from the organ bath, produced a return of tetanic tension and an increase of 5% in functional AChE activity. Similar results were obtained in the guinea-pig. The changes in synaptic AChE activity were verified pharmacologically by showing a decrease in the blocking activity of acetylcholine in preparations pretreated with pyridostigmine in comparison to those given soman alone following removal of the anti-cholinesterase. The blocking dose of carbachol did not change in these two groups indicating that desensitization was not a component of the protective action. A comparison was also made of the results obtained by measuring inhibition of AChE in situ with those obtained from muscle homogenates. The implications of these results are discussed.

The protection of primates against soman poisoning by pretreatment with pyridostigmine.

The effectiveness of pyridostigmine pretreatment against soman poisoning has been determined in rhesus monkeys and marmosets receiving atropine therapy. Pretreatment with the maximum sign-free dose (200 microgram kg-1, i.v.) raised the subcutaneous LD50 of soman by a factor of 28 in rhesus monkeys and 15 in marmosets. The protection afforded by a quarter of the sign-free dose of pyridostigmine was not significantly less. These levels of protection are higher than any reported in non-primate species.

Investigation into the effects of ingestion of zinc shot by Mallard Ducks (Anas platyrhynchos).

Lead fishing weights and gun shot have been identified as a widespread cause of poisoning of swans and other wildfowl (order Anseriformes). As part of the efforts to find environmentally acceptable substitutes for lead, this paper describes the results obtained from a trial which involved the feeding of zinc shot to groups of mallard (Anas platyrhynchos). Observations of the mallard during the experimental period, post mortem examination, histo-pathological examination and also chemical analysis of liver, kidney and feathers, revealed no ill effects to the dosed birds and indicated that zinc metal in shot from probably presents no detectable health threat to wild birds if ingested.

Dioxin and furan residues in wood mice (Apodemus sylvaticus) following a large scale polyvinyl chloride (PVC) fire.

A forest ecosystem was contaminated as a result of a fire involving 600 t of PVC. A wide range of 2,3,7,8-substituted dioxin and furan congeners were elevated (by up to 4-fold) on soil adjacent to the factory compared to a site 200 m from the factory perimeter. Livers of wood mice (Apodemus sylvaticus) caught on these areas were also analysed for dioxins and furans. Toxic equivalents (TEQs) were 9-fold higher in wood mice caught on the site 10 m from the factory perimeter compared with the site 200 m from the perimeter, with individual 2,3,7,8-substituted congeners being elevated by up to 30-fold. Wood mouse liver TEQs were found to be highly correlated with cadmium kidney concentrations, cadmium also being found at elevated concentrations at the accident site. There was also a significant positive correlation between wood mouse liver TEQs and relative liver weights (wet weights expressed as a percentage of total body weight). The results of this study are discussed in the wider context of dioxin contamination in the environment.

Toxicity and tissue distribution of pentachlorophenol and permethrin in pipistrelle bats experimentally exposed to treated timber.

The dependence of bats in Britain on houses as roosts may result in them being exposed to pesticides used in remedial timber treatments. Pentachlorophenol (PCP) and permethrin are used as a fungicide and an insecticide for timber treatment, respectively. The present study investigated toxicity and distribution in body tissues of these two pesticides in pipistrelle bats. Four groups of nine to ten bats were kept in separate outdoor flight enclosures and were provided with roost boxes treated with either PCP only, permethrin, PCP/permethrin mixture or solvent only (control). At the start of the experiment, mean (+/-SE) PCP and permethrin concentrations on the surface of wooden blocks that had been treated in the same way as roost boxes were 69.32+/-6.76 mg g(-1) (n=6) and 3.3+/-1.6 mg g(-1) (n=3), respectively. All bats exposed to PCP and PCP/permethrin treated boxes died within 24 and 120 h, respectively; nine out of the ten controls survived the 32 day experimental period (P<0.001; both groups compared with control). Bats exposed to permethrin treated boxes survived as well as controls. Mean (+/-SE) carcass PCP concentration (excluding deposits on fur) of bats exposed to PCP and PCP/permethrin treated boxes was 13.11+/-2.52 microg g(-1)BW (n=20). PCP burdens on fur were positively correlated with total weight of PCP in the carcass (P<0.001). PCP was present in fat depots, liver, kidney and the remainder of the body which, despite containing low PCP concentrations, was the main PCP reservoir (66.4+/-5.0% of carcass PCP load; n=20). Total PCP in the carcass was significantly correlated with lipid weight (P<0.005). Permethrin was not detectable in body washes and tissues of bats exposed to PCP/permethrin mixture or permethrin.

Organochlorines and mercury in pilot whale blubber consumed by Faroe islanders.

Some 22,000 pilot whales (Globicephala melaena) were taken in the Faroe Islands between 1970 and 1992. It is known that tissues from these animals are widely consumed by the islanders. The position of these animals at the apex of a direct marine food chain renders them liable to accumulate toxic chemicals, such as metals and organochlorines. Although the consumption of contaminating metals in pilot whale tissues has been studied, the significance of blubber as a dietary source of organochlorine compounds has not been fully considered. This study reports levels of organochlorine and mercury contamination in the blubber of pilot whales taken in two Faroese kills. Published estimates of pilot whale tissue consumption are used to evaluate dietary organochlorine intake in relation to established national and international guidelines and clinical studies conducted in the North American Great Lakes.

Organochlorine residues in fish oil dietary supplements: comparison with industrial grade oils.

The market for fish oils as dietary supplements is of global importance. Although it is widely recognised that lipophilic organic chemicals, particularly organochlorines, can accumulate in fish oils, dietary supplements are not routinely considered when estimating average daily intakes for these contaminants. This paper reports levels of organochlorine residues in 44 fish oils, collected from 15 countries between 1994 and 1995, including 38 purchased over the counter as dietary supplements. Despite controls on the use of persistent organochlorine substances, appreciable quantities are found in oils sold as dietary supplements. Levels are discussed in relation to the significance of fish oil dietary supplements as contributors to daily intake of PCBs and pesticide residues.