Species differences in N-glucuronidation

Glucuronidation of amines has been shown to exhibit species differences in vitro and in vivo. Substrates for N-glucuronidation can be classified according to the chemical structures of the resulting glucuronides into two groups: compounds that form non-quaternary N-conjugates, and those that form the quaternary counterparts. For compounds of the former class-such as sulfonamides, arylamines, and alicyclic, cyclic, and heterocyclic amines-species differences appear to be less striking and are of a quantitative nature. No one common laboratory animal species used routinely in metabolism research (e.g. rat, mouse, dog, non-human primate, rabbit, and guinea pig) has been shown to be deficient in N-glucuronidation when all of the substrates studied and reported are taken into consideration. The ability of a species to form N-glucuronides is compound-dependent, although rabbit and guinea pig appear to exhibit the highest capacity for this bioconjugation among preclinical species. For tertiary amines, most notably the tricyclic antidepressant and antihistamine drugs, N-glucuronidation is commonly observed in non-human primates and man. There are examples, however, of quaternary glucuronidation occurring in lower animal species. In exploring species differences in amine conjugation in vivo, it is noted that the apparent absence of N-glucuronides in animal urine may not reflect the inability of that species to form such conjugates, since the N-glucuronides may be excreted in bile. Problems such as degradation or low recoveries commonly encountered in isolation and identification of in vivo metabolites further complicate the interpretation of data. Because of the wide range of pKa values exhibited by various classes of amines, caution also should be exercised for in vitro studies since incubation conditions for N-glucuronidation often are substrate- and species-dependent. Explanations for the species differences observed in N-glucuronidation appear to be emerging as rapid advances are made in the understanding of the glucuronosyltransferases at the molecular level. More information, however, remains to be gathered from the glucuronosyltransferase genes of animal species other than humans before a better understanding of species differences in N-glucuronidation can be achieved.     

Inhibition of mammalian protoporphyrinogen oxidase by acifluorfen

We have studied the kinetics of the inhibition of mitochondrial protoporphyrinogen oxidase (PPO) from liver and placenta of 3 mammalian species by the diphenyl ether herbicide acifluorfen (AF). AF competitively inhibited PPO from human liver and placenta, mouse liver and pig placenta with respect to its substrate protoporphyrinogen. In contrast, mixed-type inhibition was shown for pig liver. The differing results shown in pig liver may point to structural differences in PPO derived from different species and tissues. We have also compared the effects of AF on the function of PPO in human lymphoblasts from normal subjects and those with variegate porphyria, an inherited disorder of PPO. Competitive inhibition was shown for both and there were no significant differences in the values of Ks or Ki.     

Test of Stice''s dual pathway model: dietary restraint and negative affect as mediators of bulimic behavior

Many investigators have used animal models to clarify the role of the human anterior cruciate ligament (ACL). Because none of these models are anatomically and biomechanically identical to the human ACL, there exists a need for an objective comparison of these models. To do this, we used a universal force-moment sensor to measure and compare the in situ forces, including magnitude and direction, of the ACL and the anteromedial (AM) and posterolateral (PL) bundles of human, pig, goat, and sheep knees. An Instron was used to apply 50 and 100 N anterior tibial loads at 90 degrees of knee flexion, while a universal force-moment sensor was used to measure the forces applied by the ACL to the tibia, the in situ force of the ACL. We found significant differences between the magnitude of force experienced by the goat and sheep ACL and AM and PL bundles when compared with the human ACL and AM and PL bundles. Also, the direction of the in situ force in the ACL and AM bundles of the goat and sheep were different from the human. The pig knee differed from the human only in the magnitude and direction of the in situ force in the PL bundle in response under anterior tibial loading. A tally of the significant differences between the animal models and the human knees indicates that goat and sheep knees may have limitations in modeling the human ACL, while the pig knee may be the preferred model for experimental studies.     

Comparison of human, primate, and canine femora: implications for biomaterials testing in total hip replacement

The canine model remains an animal of choice for determining the efficacy and safety of various materials and designs used in human total hip replacement (THR). The primate also is used in orthopedic-related research for studying limb anatomy, gait, and age-related bone loss. In order to better understand the appropriateness of these animal models for human THR, external morphologies of thirty-three adult Caucasian human, sixteen adult chimpanzee, and forty-two adult greyhound femora were compared using osteometric methods. Measured parameters included anteversion angle, cervico-diaphyseal angle, femoral head offset in the frontal plane, and anterior bow profiles along the femoral diaphysis. Although some of the measured parameters were approximately similar between species (e.g., mean cervico-diaphyseal angle of humans and chimpanzees), the majority demonstrated morphologic differences that may be biomechanically significant for interpreting stress transfer across the hip (e.g., mean anteversion angle and mean normalized femoral head offset between species). Additionally, age-related changes in proximal femoral morphology and gait pattern, as well as species-related differences in local muscle and inertial forces, may result in notably different loading conditions across the hip joint of each species. Therefore, discretion must be exercised when evaluating canine or primate THR materials and designs for potential use in the human hip.     

Determination of cholesterol absorption in man by intestinal perfusion

Gel filtration was used to measure drug interaction with protein-bound bilirubin in 0.5 ml samples of Gunn rat serum, human serum and fraction V human serum albumin. Using sulfadimethoxine as a prototype differences in displacement were found in all 3 sera. The differences between human and rat serum were related to the binding characteristics of sulfadimethoxine whereas the differences between human serum and fraction V human serum albumin were attributed to displacement of bilirubin from albumin to other proteins in serum. Gel filtration permitted the use of small samples with bilirubin-albumin ratios less than 1.0 and provided data that were used for analysis of drug displacement of bilirubin using principles of drug-receptor theory. Ten of 14 drugs found to alter serum bilirubin concentrations in icteric Gunn rats had measurable effects on protein binding of bilirubin.     

Intracerebral Whipple''s disease diagnosed by stereotactic biopsy: a case report and review of the literature

We studied the effects of bile stasis in a guinea pig model of pigment gallstone. The common bile ducts of guinea pigs were partially ligated, and the guinea pigs killed one or two weeks later. Biliary sludge or stones were examined with the Fourier transform infrared spectroscopy and the scanning electromicroscopy. The bile was analyzed for pH, free calcium, bile acids and bilirubin fractions, and the activities of both bacterial and endogenous beta-glucuronidase. After bile duct ligation, calcium bilirubinate precipitates or stones formed in all except one of the animals studied. The bile pH and the proportion of unconjugated bilirubin rose after bile duct ligation, with a concomitant fall of bilirubin monoglucuronide. The activity of bacterial beta-glucuronidase decreased after ligation, while the activity of endogenous beta-glucuronidase rose at week 2. Our results imply that precipitation of calcium bilirubinate in this animal model was induced by an increased bile pH and the nonenzymatic hydrolysis of conjugated bilirubin.     

Interspecies differences in enzymes reacting with organophosphates and their inhibition by paraoxon in vitro

1 Inhibition of cholinesterases (ChE) and carboxylesterases (CaE) by paraoxon (Px) was studied in vitro in the serum, liver, lung and muscle of mouse, guinea pig, rabbit and man (serum only). Moreover, the role of Px hydrolyzing enzyme (Pxase) in the detoxification of Px was studied by inhibiting its activity with EDTA. 2 The ChE and CaE activities as well as their sensitivity to Px varied in different tissues and species. The ChEs were more sensitive than CaEs to Px except in the liver. The CaE activity in human and rabbit sera was low and resistant to Px, indicating that it may have a minor importance for the binding of Px. 3 The Px-inhibited ChEs were spontaneously reactivated in the mouse and rabbit sera during 24 h. In mouse, also the CaE activity was recovered. The presence of EDTA in the incubation medium prevented this reactivation indicating that Pxase takes part in the reactivation process. 4 In rabbit, the serum Pxase activity was very high suggesting a good Px detoxifying capacity of the rabbit serum. 5 The results show that amounts and sensitivities of esterases to OPs in rodents may markedly differ from that in man. Possible species-related differences in the affinity of ChEs and CaEs for OPs and the OP hydrolyzing activity should be taken into the consideration, when animal data are extrapolated to man.     

The effect of wound dressings on diagnostic ultrasound imaging

The 5-HT1D receptor is a potential target of anti-migraine drugs, and here its genes were cloned from chimpanzee, gorilla and rhesus monkey, via polymerase chain reactions with their genomic DNAs and the primers designed from the 5' and 3' untranslated regions of the human receptor. Direct sequencing of the polymerase chain reaction (PCR) products revealed high degrees of identity between their deduced amino acid sequences (the chimpanzee, gorilla and rhesus monkey) and that of human, differing by two, four and 11 residues, respectively. The binding properties of the receptors, as expressed in human embryonic kidney 293 cells, were compared to those obtained with the human and guinea pig receptors, the latter differing by 33 residues from the human receptor. Standard serotonergic ligands including several indoles, ergots and methiothepin bound all the cloned primate and guinea pig receptors with comparable, low nanomolar affinities, leading to high correlation coefficients among their Ki values. R(+)-8-Hydroxydipropylaminotetralin, on the other hand, bound the human receptor with the affinity higher than those for the primates and guinea pig receptors. This indicates that certain chemical templates may differentiate the molecular divergences among the 5-HT1D receptors of various animal species, and the use of the non-human primates will be beneficial for pharmacological characterizations, more relevant to the human receptor, of future novel ligands for the 5-HT1D receptor, which are potential anti-migraine drugs.     

Nicotine induced c-fos expression in the striatum is mediated mostly by dopamine D1 receptor and is dependent on NMDA stimulation

Serum humoral immune response to Cryptosporidium parvum was evaluated in six species: mouse, rabbit, lamb, calf, pig and man. Electrophoretic and immunoblot analysis showed that specific animal antibody response appeared between Day 4 and Day 15 post inoculation. The two main target antigens had apparent molecular weights of 15-17 and 23 kDa. They were recognised by each species studied. Serum IgA intensively recognised the 15-17 kDa antigen, except in rabbit. This study demonstrates that these two antigens are consistent targets of humoral immune response and can therefore be of great interest in studies of therapy/prophylaxis.     

Presence of cross-reacting thymus and brain antigens in the cerebral cortex

Rabbit antisera against antigens of mouse, rabbit, guinea pig and human whole brain cross-reacted in the cytotoxic test with the lymphocytes of the thymus, lymph nodes and the spleen of these animal species. Mouse thymocytes were most sensitive to the antibrain sera (cytotoxic index -- 63--100 per cent); cells from other mouse lymphoid organs and lymphocytes of rabbit, guinea pig and man were more resistant. Bone marrow lymphocytes were not damaged by any of these sera. The antigens which induced the cytotoxic properties of the sera were found only in the human cerebral cortex, but not in the white matter of the brain stem.     

The influence of various aminoglycoside preparations on bilirubin/albumin binding

The effect of antibiotics of aminoglycoside structure on the albumin binding of bilirubin has been tested in homozygous (jaundiced) Gunn rats aged 3-5 days. The following drugs were investigated: different preparations of gentamycin, kanamycin, tobramycin and sisomicin. The animals received 50-75% of the LD50 of heterozygous (non-jaundiced) Gunn rats. Mortality, weight gain and changes in the plasma bilirubin concentration were recorded. It was found that the displacement of bilirubin from albumin is caused by the different stabilizers used and not by the antibiotic itself. With the exception of lyophilized preparations of gentamycin for intrathecal application all vials contain different amounts of these preservatives. Special preparations used during the newborn period contain relatively more of these stabilizers. The toxicity of the additives has already a negative influence on the LD50 for heterozygous Gunn rats when the low dosed Refobacin and Sulmicin vials are given. For Refobacin (production 1973/74) the tolerance is reduced by nearly 50%. The toxicity caused by the stabilizer alone is even more marked when given to homozygous (jaundiced) Gunn rats. It becomes evident that benzylalcohol is the substance responsible for the displacement of bilirubin from albumin. The serum concentration of bilirubin decreases for 3-24 hrs depending on the doses given to the animal. This offers the opportunity to measure the competitive displacement of bilirubin easily and exactly. The free unbound, unconjugated bilirubin tends to diffuse into the lipid of the brain with resultant kernicterus. This was shown in histochemical preparations of the cerebellum of young homozygous Gunn rats. Using enzyme reactions for lactic acid dehydrogenase and NADH2-tetrazolium reductase the cytotoxic effect of bilirubin on PURKINJE cells could be demonstrated. The effect of the stabilizers used in the other antibiotic drugs tested can be neglected under clinical conditions. Finally the steepness and duration of the decrease of plasma bilirubin after injection of the dangerous stabilizers was studied in animals of different age (3-5 days; 3-4 weeks). Different results observed can be explained by the more rapid metabolism of benzoates in older animals. However, it remains an open question at what age Gunn rats reflect most precisely the human situation in premature and newborn babies.     

Inhibition of major histocompatibility complex class I antigen presentation in pig and primate cells by herpes simplex virus type 1 and 2 ICP47

Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) express an immediate-early protein, ICP47, that effectively inhibits the human transporter associated with antigen presentation (TAP), blocking major histocompatibility complex (MHC) class I antigen presentation to CD8+ T cells. Previous work indicated that the mouse TAP is relatively resistant to inhibition by the HSV-1 and HSV-2 ICP47 proteins (ICP47-1 and ICP47-2) and that mouse cells infected with HSV-1 are lysed by anti-HSV CD8+ cytotoxic T lymphocytes (CTL). Therefore, mice are apparently not suitable animals in which to study the in vivo effects of ICP47. In order to find an animal model, we introduced ICP47-1 and ICP47-2 into cells from various animal species-mice, rats, guinea pigs, rabbits, dogs, pigs, cows, monkeys, and humans-and measured TAP activity in the cells. Both proteins were unable to inhibit TAP in mouse, rat, guinea pig, and rabbit cells. In contrast, ICP47-1 and ICP47-2 inhibited TAP in pig, dog, cow, and monkey cells, and the TAP in pig and dog fibroblasts was often more sensitive to both proteins than TAP in human fibroblasts. These results were extended by measuring CD8+-T-cell recognition (CTL lysis) of cells from various species. Cells were infected with recombinant HSV-1 constructed to express murine MHC class I proteins so that the cells would be recognized and lysed by well-characterized murine anti-HSV CTL unless antigen presentation was blocked by ICP47. Anti-HSV CD8+ CTL effectively lysed pig and primate cells infected with a recombinant HSV-1 ICP47- mutant but were unable to lyse pig or primate cells infected with a recombinant HSV-1 that expressed ICP47. Therefore, pigs, dogs, and monkeys may be useful animal models in which to test the effects of ICP47 on HSV pathogenesis or the use of ICP47 as a selective immunosuppressive agent.     

Child psychiatry perspectives. After the "civilized" divorce

The sorption of bile acids, bilirubin, bilirubin glucuronates and cholesterol on ion-exchange resin is described. After some successful attempts of sorption of these substances from human and animal bile in vitro, this exchanger has been used in human therapy. Positive results obtained showed the usefulness of the method. The purpose of the experiments was to study the usefulness of ion-exchangers for sorption of bile components of bile acids, bilirubin, bilirubin glucuronates and cholesterol. First sorption was carried out on human and animal bile, on Zerolit-FS-ip, in cycles (OH-) and (Cl-), by passing through glass columns downwards and some of them by mixing the bile with ion-exchange resin. Zerolit FS-ip is an organic sorbent, a scavenger, a strongly basic macroporous, anion exchanger. Positive results of these sorptions indicated the possibility of using this method in therapy of disturbed lipid conditions.     

Regulation of skeletal muscle perfusion during exercise

OBJECTIVE: To compare the composition and mechanical properties of the newly developed bladder acellular matrix graft (BAMG) with the normal urinary bladder in rat, pig and human. MATERIALS AND METHODS: Rat, pig and human urinary bladders were harvested and divided into control and experimental groups. For the latter, BAMGs were prepared, and light and transmission electron microscopic studies performed. Strips from the normal bladders and the BAMGs (10 in each group) were tested under tension, and the ultimate tensile strength, maximum strain, and elastic modulus were determined from stress/strain curves. RESULTS: Both types I and III collagen, as well as elastic fibres, were observed as major components of the matrix scaffold. There were more collagen type I fibres in the rat than in the pig and human BAMGs, whereas the pig, and particularly the human, both showed higher levels of type III collagen and elastic fibres. These different matrix scaffold patterns were confirmed by electron microscopy. Results from biomechanical testing showed no significant differences for strength, strain or elastic modulus between BAMG and control bladder strips, except in the rat where the maximum strain values were significantly lower. CONCLUSION: There are variations in the acellular matrix structure with similar biomechanical properties between the BAMG and the normal urinary bladder in three different species. These results may underscore the potential of the BAMG. Furthermore, this in vitro model provides a suitable method to study the mechanical properties of the urinary bladder and may serve as a diagnostic tool for various investigations.     

Human beta-glucuronidase. I. Recognition and uptake by animal fibroblasts suggests animal models for enzyme replacement studies

As part of an effort to develop an animal model for studies of uptake of human beta-glucuronidase, fibroblasts were established from primary explants of connective tissue from nine different animal species, and examined for their ability to take up human platelet beta-glucuronidase. Endogenous fibroblast beta-glucuronidase was inactivated by heating extracts to 65 degrees for 30 min. Human beta-glucuronidase was stable to this treatment. Uptake of human beta-glucuronidase by animal fibroblasts was measured as heat-stable beta-glucuronidase present in fibroblasts after exposure to partially purified human platelet beta-glucuronidase for 48 hr. Althought all animal fibroblasts examined exhibited some uptake capacity for human beta-glucuronidase, the uptake capacity of different animal fibroblasts varied over a 10-fold range. The uptake capacity of bovine fibroblasts was at least 80% that of human fibroblasts. Rat and hamster fibroblasts showed about half the uptake capacity of human fibroblasts. The rat fibroblasts resembled the human fibroblasts in the kinetics of uptake of hihg uptake (platelet) enzyme, poor uptake of human placental enzyme, and lack of appreciable turnover of enzyme taken up over 4 days. Heating extracts of rat organs containing added human beta-glucuronidase at 65 degrees selectively inactivated rat enzyme.     

Insulin receptors are widely distributed in human brain and bind human and porcine insulin with equal affinity

Insulin receptors differing structurally from those in other tissues have been demonstrated in brain from many species. Subtle differences in binding properties have been reported between insulin receptors in brain and other tissues, including differences in affinity of pig brain receptors for human and porcine insulin. Insulin binding has been demonstrated in human cerebral cortex, but insulin binding has not been characterized in other areas of human brain. We have studied the binding of 125I labelled human insulin, and its displacement by unlabelled human and porcine insulin, in homogenates prepared from human hypothalamus, cerebral cortex and cerebellum obtained post-mortem from eight non-diabetic subjects. Specific binding was demonstrated in all brain regions studied, and displacement curves obtained with unlabelled human and porcine insulin were identical. By contrast, unlabelled insulin-like growth factor-1 did not significantly displace 125I labelled human insulin over the same concentration range. We therefore conclude that insulin receptors are widely distributed in human brain and do not differ in their affinity for human and porcine insulin.     

Pathogenic micro-organisms in slaughterhouse sludge--a survey

During slaughtering of animals and subsequent meat processing the process water used becomes polluted with organic matter of animal origin (i.e. protein and fat). This organic sludge is, in principle, a product suitable for animal feeding. To investigate the microbiological contamination level of sludge, raw sludge was collected at pig (n = 8) and poultry (n = 5) slaughterhouses. Both flocculated and aerobically activated sludge was monitored. Slaughterhouse sludge was heavily contaminated with Enterobacteriaceae (6.3-10.0 in log10 N/gram dry matter) and enterococci (4.6-7.9). Clostridia were present in sludge at a level of 3.1-5.8 (in log10 N/g DM). Salmonella was present in the sludge from all slaughterhouses examined. Yersinia enterocolitica serotypes O:3 and O:9 were found in sludge from seven out of thirteen slaughterhouses. The prevalence of Campylobacter jejuni/coli was higher in flocculated poultry sludge than in both flocculated pig sludge and aerobically activated pig sludge. Obviously, decontamination of the sludge is mandatory when it is to be applied as a feed constituent, to prevent bacterial cycles from occurring in livestock, as well as the spread of human pathogenic zoonoses like campylobacter, salmonella and yersinia, to minimize loss of protein quality by the microbial breakdown of amino acids and the formation of possible toxic metabolites in sludge during storage.     

Studies on the functional ability of swine liver perfused with human blood in machine recirculation attempt. Experiment design, elimination of indocyanine green, galactose, bilirubin and ammonia, synthesis of urea, bile secretion

In 11 machine experiments of recirculation the abilities of survival and functioning of livers of dwarf pigs perfused with human preserved blood were tested with the help of the eliminations of indocyanine green, galactose, bilirubin and ammonia from a closed circulation. Further urea-nitrogen were measured as indicator of the urea synthesis and bile secretion. The perfusion of the animal livers was carried out in 9 cases only through the portal vein. The values of the blood flow were between 0.4 and 1.0ml pro g liver tissue and minute in portal pressure values about 15 cm water column. Two recirculations were carried out with additional flow through the hepatic artery with about 1/5 of the whole blood flow. The results of the experiments described prove a pretty good functional performance of the perfused with human blood livers of dwarf pigs during several hours. The different animal livers, however, showed at the beginning as well as during the further course of the perfusion clear differences concerning their functional capacity. Decrease of the blood flow values below 0.5 ml pro g liver tissue lead to functional loss. The additional flow through the hepatic artery yielded no better results, compared with the only perfusion of the animal liver through the portal vein.     

Intact pancreatic islet function despite humoral xenorecognition in the pig-to-monkey combination

BACKGROUND: The aim of this study was to analyze humoral xenoreactivity of various Old World primate species sera against pig islets and the effects of these sera on pig islet viability and function after culture. METHODS: Freshly isolated or cultured adult pig islets were analyzed by immunohistology or by cytofluorimetry for Old World primate xenoreactive natural antibody (XNA) binding and complement deposition. Complement-mediated cytotoxicity was evaluated by 51Cr release assays. After 4 days of culture in 50% sera from Old World primates, the morphology and in vitro metabolic function of pig islets were also analyzed. RESULTS: Chimpanzee, Macaca mulatta (rhesus), or baboon XNA binding was detectable only on intra-islet endothelial cells (ECs). Incubation of pig islets with sera from all Old World primate species tested showed C3 and C4 deposition on ECs and on some surrounding endocrine cells. However, membrane attack complex (MAC) showed a pattern of positivity similar to XNA binding, i.e., restricted to ECs only. No deposition of factor B was detected. Although complement cascade was activated, no cytotoxicity was observed after incubation of islets with chimpanzee serum, whereas between 10% and 35% 51Cr specific release was obtained with rhesus, baboon, or Macaca fascicularis sera. Despite this cytotoxic effect, purified pig islets showed a normal morphology and a well-preserved insulin release in response to an acute glucose stimulus, after prolonged culture with 50% serum obtained from all primate species considered. CONCLUSIONS: Despite the fact that pig beta-cell function was not affected by the serum of any of the primate species tested, some of them yielded significant lysis of islet cells, presumably as a result of a cytotoxic effect on intra-islet ECs. These data show that Old World primate sera from different species do not have equivalent effect on pig islets; these differences should be taken into account in preclinical trials of pig islet xenotransplantation.