An ultrastructural study of the kidney of normal, copper poisoned and thiomolybdate-treated sheep

Histological, ultrastructural and kidney function techniques were used to assess changes in the kidney of sheep given either copper (Cu) or Cu and the Cu complexing agent thiomolybdate (TM), or TM alone. Kidney function was normal in sheep given Cu and TM together or TM alone. In these animals the cells lining cortical tubules accumulated Cu within numerous, large, electron-dense lysosomes. Sheep given Cu alone developed haemolysis, impaired kidney function and a variety of morphological defects including an increase in number of large lysosomes in cells of the cortical tubules. There was a breakdown of the glomerular endothelial lining and fusion of foot processes. Cells of the cortical tubules showed degeneration and necrosis and an increase in microbodies and rough endoplasmic reticulum. Cortical and medullary blood vessels were dilated, with evidence of breakdown of the endothelial lining. Copper appeared to injure kidney tissue at three sites, tubular epithelium, glomerular basal lamina and capillary blood vessels. Changes reported here are similar to the renal lesions in cadmium toxicity.     

Lysosomes in the pathogenesis of liver injury in chronic copper poisoned sheep: an ultrastructural and morphometric study

Chronic copper poisoning was induced in sheep by oral dosing with CuSO4. The distribution of copper between hepatocytes was unequal and, with increasing liver copper concentration, isolated hepatocytes packed with electron-dense lysosomes were seen. These cells underwent degeneration and necrosis. During the pre-haemolytic period, the concentration of Cu in the liver increased and the volume density, numerical density and mean volume of hepatocyte lysosomes increased in a linear fashion, indicating that there was proliferation as well as increase in the size of lysosomes. However, in animals killed during haemolysis, the numerical density had decreased but the volume density was little changed which indicates that lysosomal production may have diminished. It is postulated that the necrosis of hepatocytes packed with electron-dense lysosomes may be due to the accumulation of toxic amounts of copper in the cytosol, resulting from a reduced uptake of copper into the lysosomal system of these cells, and that the susceptibility of liver cells to Cu-induced damage may be increased if lysosome production is diminished.     

Absorption and excretion of organic compounds of copper by sheep

When sheep are injected subcutaneously with copper calcium edetate or copper oxyquinoline sulphonate there is a rapid increase in the concentration of copper in whole blood, serum and urine within the first 24 h. When sheep are injected with copper methionate the concentration of copper in whole blood and serum rises slowly over a period of about 10 days and there is no detectable increase in urinary copper. After the injection of each of the three compounds, there was a steady increase in the caeruloplasmin activity in serum over a period of 10 to 20 days, followed by a slow fall to pre-injection activity by 40 days. There was a marked increase in the beta-globulin fraction of serum 9 days after the injection of copper methionate but not after the other 2 compounds and the amounts of 2 copper containing proteins in liver were greater 60 days after the injection of copper methionate than after the injection of the other two compounds. The copper content of the 3 organic compounds is absorbed and excreted at different rates by sheep. The amounts of copper-containing protein produced in the liver also differ according to the organic component of the compound injected. The results suggest that the lower toxicity of copper injected as methionate compared with that injected as copper calcium edetate or copper oxyquinoline sulphonate is due to the slower absorption and transport of the copper to the liver and kidney.     

Experimental copper and heliotrope intoxication in sheep: morphological changes

Young Merino wethers were used to determine the effects of copper and heliotrope, fed together or separately, on the development of toxicity and the concentration of trace elements in the liver and kidney. In one experiment copper and heliotrope were given concurrently, in a second experiment heliotrope was fed for 12 weeks and copper administration commenced 8 weeks later. The 10 sheep fed heliotrope alone did not show signs of clinical illness but one died and was found to have severe liver damage. Eleven sheep were given copper alone and three developed the clinical signs and lesions of haemolysis. Fourteen sheep were given copper and heliotrope and 13 became ill. Of these, three developed haemolysis, eight became jaundiced and two became weak without developing jaundice. The concentrations of copper in the livers of control and heliotrope-treated sheep, were comparable. In the animals given copper alone, the concentration of copper in the liver was twice as high as that in controls and in those given heliotrope and copper, it was three times as high as in the liver of control sheep. Feeding heliotrope alone induced the histological changes of pyrrolizidine alkaloid toxicity in the liver, but this was not associated with an excessive accumulation of copper or the development of clinical illness. However, it did predispose the animals to the effects of a second toxin since giving heliotrope and copper concurrently, or giving copper subsequent to feeding heliotrope, markedly enhanced the toxicity of the two substances and caused an excessive accumulation of copper in the liver.     

Variations in sheep serum conglutinating complement activity and pathway involved in reactivity with sheep erythrocytes sensitized by rabbit antibody

Based on conglutination tests with the sheep E-rabbit A indicator system, three types of sheep sera were encountered. Type 1 sera failed to directly conglutinate or sensitize sheep E-rabbit A for conglutination by bovine conglutinin. Type 2 sera also failed to directly conglutinate sheep E-rabbit A but sensitized the indicator for conglutination by bovine conglutinin. Type 3 sera both directly conglutinated and sensitized sheep E-rabbit A for conglutination. Changes in serum type were induced in sheep by venepuncture (type 1 to type 2) or venepuncture and an intraperitoneal injection of yeast cells (type 2 to type 3). Direct conglutinating activity of type 3 sera was inhibited by heating serum at 50 degrees C for 30 min and was not restored by alternative activation pathway factor B. Chelation of Ca2+ in type 2 and 3 sera blocked sensitization of sheep E-rabbit A for conglutination by bovine conglutinin, indicating that the classical activation pathway was involved.     

The elicitation of carboxylesterase activity in antibodies by reactive immunization with labile organophosphorus antigens: a role for flexibility

For the creation of powerfully catalytic antibodies, the technique of reactive immunization solves the problem inherent in immunization with transition-state analogs (TSAs), namely, that many interesting target reactions are multistep reactions, with multiple transition states, and thus in general, no single analog can adequately simulate all the transition states along the reaction path. In contrast, immunization with chemically reactive antigens such as phosphonylating agents, which phosphonylate B-cells during the immune response, produces antibodies that have been "trained" to recognize, bind, and stabilize all the actual transition states involved in the phosphonylation reaction. Therefore, catalytic antibodies have been selected by the immune system on the basis of their capacity to stabilize any number of transition states that occur during the target reaction. Somewhat surprisingly, phosphonolysis catalysts generated in this way commonly also catalyze esterolysis reactions. Esterolysis reactions should pass through transition states with a roughly tetrahedral disposition of ligands about a central carbon atom, while phosphonolysis reactions should pass through transition states with a roughly trigonal-bipyramidal disposition of ligands about a central phosphorus atom. These two divergent transition-state geometries suggest that the same active site should have difficulty recognizing and stabilizing both kinds of transition state. The observations thus indicate a puzzling form of "cross-reactivity" toward transition states. A possible explanation arises from evidence that at least some nucleophilic displacements at phosphorus do not pass through a trigonal-bipyramidal adduct, with a bond-formation transition state preceding it and a bond-fission transition state succeeding it. Instead a single transition state is traversed in which both bond-formation and bond-fission occur simultaneously. Such a concerted-reaction transition state should have two weak, partial bonds to phosphorus, one for formation of the nucleophile-P bond and one for fission of the P-leaving group bond. In a stepwise reaction through an intermediate, only one bond is partial and weak in each of the two transition states. The concerted-reaction transition state, with two weak bonds to phosphorus, may be more easily compressed, expanded, and otherwise distorted because of the lower force constants associated with partial bonds; particularly distortions of angles involving the two partial bonds should require relatively low energies. This may lend a high level of flexibility to phosphonolysis transition states, allowing them to be accommodated within an active site (or a range of active sites) with strong catalytic stabilization. Included among these active sites may be a majority that can also stabilize esterolysis transition states. Indeed many of the target esterolysis reactions studied to date may occur through a single concerted-reaction transition state rather than through separate transition states before and after a tetrahedral intermediate. Thus, the esterolysis transition states may also be highly flexible. Finally, flexibility present in germline antibodies may be specifically preserved in reactive immunization. The high flexibility of both kinds of ligands and of the antibody combining site may then account for the catalytic "cross-reactivity" of these antibodies.     

Taste potentiation of poisoned odor by temporal contiguity

The synergistic interaction between odor and taste in flavor-toxicosis conditioning was tested in two experiments. The temporal interval between a 2-min odor and a 2-min taste was varied for thirsty rats licking at a water spout. In the first experiment, taste was presented at time zero, and odor was presented at -10, -1, 0, 1 and 10 min to independent groups in a simple compartment. In the second experiment, taste was presented at 0, and odor was presented at -5, -2, and 0 min in a "wind tunnel" apparatus. The results indicated that odor alone is an ineffective conditioned stimulus for a toxic unconditioned stimulus under our conditions, simultaneous (0-min) presentation of odor with taste potentiates the odor component so that it becomes more effective than the taste component, a 2-min interval between odor and taste attenuates potentiation, and a 5-min interval disrupts the effect, and the interaction in asymmetrical, that is, odor has no such systematic effect on the conditioning of taste.     

Brain changes in a dog poisoned by the insecticide dieldrin

Focal cerebro-cortical changes, broadly classified as pale infarction, were found to coincide with convulsions in a dog poisoned by dieldrin. A primary relationship between the lesions and the mode of action of the chemical is proposed.     

Small-intestinal myoelectric activity in sheep: rebound excitation versus phase-3-like activity revealed by hexamethonium and atropine administration

Postinhibitory or rebound excitation (RE) is a known phenomenon in gastrointestinal motility, but its precise character and triggering mechanisms have not been defined so far. This study was thus devoted to analyzing its occurrence following hexamethonium (Hx) and atropine (At) administration during various phases of the migrating myoelectric or motor complex (MMC) in fasted, non-fasted, and fed sheep and to determine the nature of RE in comparison with phase 3 of the small-intestinal MMC. In the course of chronic experiments, various doses of Hx and At evoked RE alternating with phase-3-like activity (not the organized phase 3 of the MMC or its fragments) with periods and intensities related to the drug dose. In non-fasted sheep these changes were less pronounced and more delayed, while after feeding no excitatory response was observed. When the drug was given during phase 1 of the MMC, RE did not occur or was greatly reduced and delayed. Hx triggered RE mostly in the duodenum and At mostly in the jejunum. Rather, no RE was observed in the ileum. It is concluded that Hx and At inhibit small-intestinal motility and evoke RE and phase-3-like activity as a secondary stimulatory response in conscious sheep.     

Pharmacokinetics and biological activity of kinetensin in conscious sheep

Kinetensin is a nonapeptide, originally isolated from pepsin-treated plasma, that shares some sequence homology with the C-teminal end of neurotensin. The present study was designed to determine, by infusing kinetensin to conscious sheep, the pharmacokinetics and a neurotensin-like biological activity (pancreatic polypeptide response) of kinetensin. Kinetensin was rapidly metabolized, approximately 200-fold more rapidly than neurotensin [1–13]. The majority of the metabolism occurred in the circulation as demonstrated bothin vivo andin vitro. The lung and gut cleared kinetensin also. Inhibition of converting enzyme, present in highest concentration in the lung, abolished lung clearance but was without effect on kinetensin metabolism by the gut or in the general circulation. Arterial infusion of kinetensin which achieved high blood kinetensin levels at the pancreas did not increase plasma pancreatic polypeptide. We conclude that the extremely rapid degradation of exogenous kinetensin, together with the lack of biological activity, makes it unlikely that kinetensin plays a role as a circulating regulatory peptide. Nevertheless, since the putative kinetensin substrate circulates at μM concentrations, it is feasible that kinetensin is generated and metabolized at the target organ.     

Hypothalamic multiunit activity and LH secretion in conscious sheep

Summary The relationship between the pulsatile secretion of luteinizing hormone (LH) and multiunit activity (MUA) in the median eminence and retrochiasmatic area (RCh-ME) of the hypothalamus was investigated within 13 conscious, ovariectomized ewes during the anoestrous season. Eight of the ewes had been treated with oestradiol to reduce their LH pulse frequency. To test whether the activity had been recorded from sites involved in the control of GnRH release, we electrically stimulated the sites studied with the recording electrode and used the LH responses to classify the animals retrospectively for analysis of the MUA data. Following stimulation, LH secretion was either stimulated (Group STIM, n=5 ewes), inhibited (Group INHIB, n=4) or showed no response (Group NR, n=4). Statistical analysis of the MUA data revealed that the onset of LH pulses was associated with an increase in cell activity in STIM ewes and a decrease in activity in both Group INHIB and Group NR ewes. Histological examination revealed that the electrodes were located near the midline in Group STIM ewes, but 1.25 or 2 mm lateral of the midline in Groups NR and INHIB, respectively. We concluded that the MUA in the RCh-ME probably reflects the activity of cells with fibres or terminals involved in the control of GnRH release. Activity which increased at the onset of LH pulses was detected in medial areas of the RCh-ME and may reflect the activity of GnRH neurones. The activity which decreased at the onset of LH pulses in lateral areas of the RCh-ME suggests that both stimulatory and inhibitory inputs may be involved in the release of GnRH pulses.     

Correlation of zinc and copper values in the blood serum of Makuii sheep

In autumn 2009, 87 blood samples (28 male and 59 female) were taken, via jugular vein puncture using a heparinised venoject, from Makuii sheep in a preserved herd. The samples were divided into four age groups. Blood samples were transferred to a laboratory on ice, and the serum samples were separated from whole blood by centrifugation. Concentration of zinc and copper was measured in the serum by atomic absorption spectrophotometry. The mean?±?SE of zinc and copper was 8.34?±?1.07 and 12.32?±?0.28?μmol/l, respectively. Zinc values were less than the normal expected range (P?<?0.05), but the copper value was normal in serum samples. It is possible that there is zinc deficiency in the herd in this part of Iran.     

Comparing serum and hepatic concentrations of iron, copper, and cobalt in healthy sheep and sheep with chronic fasciolosis

Fasciolosis is one of the most important parasitic diseases of ruminants. This parasite causes liver dysfunction which may contribute to albumin synthesis and copper transferring to the liver and also reduces the elemental storage. Due to these reasons and to determine the effects of fasciolosis on serum and liver iron, copper, and cobalt status, the present study was conducted on 460 slaughtered sheep. During antemortem examination and after giving an identification number, blood samples were taken, and age, sex, and origin of animals were recorded. Visual inspection and palpation were performed on the liver to determine Fasciola infestation; then, liver samples were taken from 100 sheep (50 infested and 50 healthy ones), and the concentration of the mentioned minerals was measured by atomic absorption spectrophotometry. The results indicated that liver iron, copper, and cobalt concentrations in healthy sheep (74.47, 32.95, and 0.13 ppm, respectively) were significantly higher than those in sheep with fasciolosis (36.78, 11.22, and 0.05 ppm, respectively) (P?<?0.05). These findings may explain the role of hepatic injuries caused by fasciolosis on mineral status and also the role of minerals on parasite persistency.     

Studies on the relationship between haemoglobin, copper, ceruloplasmin, iron, and superoxide dismutase activity in blood of Iranian fat-tailed sheep

The relationship between haemoglobin, copper, ceruloplasmin, iron, and superoxide dismutase (SOD) activity in blood of Iranian fat-tailed sheep was studied. Blood samples were taken from the jugular vein of 128 clinically healthy Iranian fat-tailed sheep grouped according to their age (<6, 6–12, 12–24, 24–36 and >36 months) and sex. Age had a significant effect on blood concentrations of haemoglobin, ceruloplasmin and SOD activity (P<0.05). Sex had no significant effect on blood concentrations of haemoglobin, copper, iron, ceruloplasmin and SOD. There was no significant correlation between the concentrations of haemoglobin, copper, iron, ceruloplasmin and SOD. The identification of regulatory mechanisms of copper metabolism in Iranian fat-tailed sheep will require further investigations.