Study on placental transmission of Pneumocystis carinii in mice using immunodeficient SCID mice as a new animal model.

Placental transmission of Pneumocystis carinii in mice was examined in 39 animals obtained by caesarean section from 17 pregnant SCID females experimentally infected with P. carinii. When examined with toluidine blue O, DAPI and immunofluorescent antibody stains, P. carinii was detected in the lungs of infected mothers but not in the lungs of caesarean section-derived neonates even after the neonates were treated with dexamethasone for 8 weeks. However, 13 neonates born to five infected females developed P. carinii pneumonia. These results indicate that P. carinii cannot be transmitted transplacentally in mice.     

Muscular dystrophy of mink: a new animal model.

Muscular dystrophies comprise an important group of inherited disorders of man. Although the disease has been studied extensively, little is known about the underlying primary pathomechanisms. Consequently, treatment of patients is difficult and prognosis is poor. An animal model of muscular dystrophy is a useful research tool for approaching the basic problems of pathogenesis in muscle diseases. An inherited progressive muscular dystrophy of mink which resembles the amyotonic forms of human muscular dystrophy is currently under study. Clinically, the earliest sign is progressive muscular weakness and atrophy. Muscle enzyme activities in serum are usually elevated to pathologic levels. Urinary creatine/creatinine ratio is elevated. Pathologic changes are limited to skeletal muscle and are typical of those seen in amyotonic forms of human muscular dystrophy. These changes include variation in diameter size of muscle fibers, centralized nuclei, floccular and hyaline degeneration of scattered muscle fibers, increase in connective tissue in endomysial and perimysial areas, and regenerative attempts. Both type I and type II muscle fibers are involved in the disease process. Genetic studies indicate an autosomal recessive mode of inheritance. Although the primary defect in muscular dystrophy is traditionally thought to reside in skeletal muscle, recent studies have produced theories of primary involvement of other tissues and organ systems. These theories are presented and relationships to the traditional theory are discussed.     

Deciphering phenotypic variance in different models of DNA-PKcs deficiency

DNA-PKcs deficiency has been studied in numerous animal models and cell culture systems. In previous studies of kinase inactivating mutations in cell culture systems, ablation of DNA-PK's catalytic activity results in a cell phenotype that is virtually indistinguishable from that ascribed to complete loss of the enzyme. However, a recent compelling study demonstrates a remarkably more severe phenotype in mice harboring a targeted disruption of DNA-PK's ATP binding site as compared to DNA-PKcs deficient mice. Here we investigate the mechanism for these divergent results. We find that kinase inactivating DNA-PKcs mutants markedly radiosensitize immortalized DNA-PKcs deficient cells, but have no substantial effects on transformed DNA-PKcs deficient cells. Since the non-homologous end joining mechanism likely functions similarly in all of these cell strains, it seems unlikely that kinase inactive DNA-PK could impair the end joining mechanism in some cell types, but not in others. In fact, we observed no significant differences in either episomal or chromosomal end joining assays in cells expressing kinase inactivated DNA-PKcs versus no DNA-PKcs. Several potential explanations could explain these data including a non-catalytic role for DNA-PKcs in promoting cell death, or alteration of gene expression by loss of DNA-PKcs as opposed to inhibition of its catalytic activity.Finally, controversy exists as to whether DNA-PKcs autophosphorylates or is the target of other PIKKs; we present data demonstrating that DNA-PK primarily autophosphorylates.     

Developing a new animal model of temporal lobe epilepsy

Epilepsy affects 1-2 % of the population. For 30 % of these patients, their syndrome will be refractory to medical treatment. To improve our understanding and treatment of the epilepsies, we need to develop clinically relevant animal models. As temporal lobe epilepsy is often preceded by prolonged febrile seizures and in our population associated with a focal cortical dysplasia, we hypothesised that an underlying predisposing anatomical lesion would predispose individuals to develop prolonged febrile seizures and that temporal lobe epilepsy would later develop. As predicted, all the lesioned animals developed prolonged febrile seizures, while all other control groups only showed simple febrile seizures. After a latent period, 86 % of the animals who had experienced a prolonged seizure developed spontaneously recurrent limbic seizures. We now need to understand the anatomical and electrophysiological changes underlying this new epilepsy model to try and develop more effective treatments for the condition.     

Sandy: a new mouse model for platelet storage pool deficiency

Sandy (sdy) is a mouse mutant with diluted pigmentation which recently arose in the DBA/2J strain. Genetic tests indicate it is caused by an autosomal recessive mutation on mouse Chromosome 13 near the cr and Xt genetic loci. This mutation is different genetically and hematologically from previously described mouse pigment mutations with storage pool deficiency (SPD). The sandy mutant has diluted pigmentation in both eyes and fur, is fully viable and has prolonged bleeding times. Platelet serotonin levels are extremely low although ATP dependent acidification activity of platelet organelles appears normal. Also, platelet dense granules are extremely reduced in number when analysed by electron microscopy of unfixed platelets. Platelets have abnormal uptake and flashing of the fluorescent dye mepacrine. Secretion of lysosomal enzymes from kidney and from thrombin-stimulated platelets is depressed 2- and 3-fold, and ceroid pigment is present in kidney. Sandy platelets have a reduced rate of aggregation induced by collagen. The sandy mutant has an unusually severe dense granule defect and thus may be an appropriate model for cases of human Hermansky-Pudlak syndrome with similarly extreme types of SPD. It represents the tenth example of a mouse mutant with simultaneous defects in melanosomes, lysosomes and/or platelet dense granules.     

Roles of salivary components in Streptococcus mutans colonization in a new animal model using NOD/SCID.e2f1-/- mice

Streptococcus mutans plays an important role in biofilm formation on the tooth surface and is the primary causative agent of dental caries. The binding of S. mutans to the salivary pellicle is of considerable etiologic significance and is important in biofilm development. Recently, we produced NOD/SCID.e2f1(-/-) mice that show hyposalivation, lower salivary antibody, and an extended life span compared to the parent strain: NOD.e2f1(-/-). In this study we used NOD/SCID.e2f1(-/-) 4 or 6 mice to determine the roles of several salivary components in S. mutans colonization in vivo. S. mutans colonization in NOD/SCID.e2f1(-/-) mice was significantly increased when mice were pre-treated with human saliva or commercial salivary components. Interestingly, pre-treatment with secretory IgA (sIgA) at physiological concentrations promoted significant colonization of S. mutans compared with sIgA at higher concentrations, or with human saliva or other components. Our data suggest the principal effects of specific sIgA on S. mutans occur during S. mutans colonization, where the appropriate concentration of specific sIgA may serve as an anti-microbial agent, agglutinin, or an adherence receptor to surface antigens. Further, specific sIgA supported biofilm formation when the mice were supplied 1% sucrose water and a non-sucrose diet. The data suggests that there are multiple effects exerted by sIgA in S. mutans colonization, with synergistic effects evident under the condition of sIgA and limited nutrients on colonization in NOD/SCID.e2f1(-/-) mice. This is a new animal model that can be used to assess prevention methods for dental biofilm-dependent diseases such as dental caries.     

Zebrafish as a new animal model for movement disorders

The zebrafish, long recognized as a model organism for the analysis of basic developmental processes, is now also emerging as an alternative animal model for human diseases. This review will first provide an overview of the particular characteristics of zebrafish in general and their dopaminergic nervous system in particular. We will then summarize all work undertaken so far to establish zebrafish as a new animal model for movement disorders and will finally emphasize its particular strength – amenability to high throughput in vivo drug screening.     

Gastroesophageal reflux disease in baboons (Papio sp.): a new animal model

BACKGROUND: Gastroesophageal reflux disease (GERD) is increasingly prevalent in the human population. Current animal models require surgical or other manipulation to produce symptoms. An animal model that exhibits spontaneous GERD would provide the opportunity for much-needed research examining the susceptibility, diagnosis, and treatment of GERD. METHODS: Eight baboons (Papio hamadryas sp.) were diagnosed with GERD histopathologically using biopsies or postmortem tissues. RESULTS: The disease was characterized by a spectrum of symptoms comparable with that found in the human population. Some subjects had no gross signs of clinical disease, but were diagnosed by histopathological examination. Almost all subjects presented with at least one clinical sign of the disease. Regurgitation was the most common. CONCLUSIONS: The baboon may be a superior animal model for GERD research because it is a naturally occurring model and is anatomically and physiologically similar to humans.     

The rabbit as an animal model of hepatic lipase deficiency

A natural deficiency of hepatic lipase in rabbits has been exploited to gain insights into the physiological role of this enzyme in the metabolism of plasma lipoproteins. A comparison of human and rabbit lipoproteins revealed obvious species differences in both low-density lipoproteins (LDL) and high-density lipoproteins (HDL), with the rabbit lipoproteins being relatively enlarged, enriched in triacylglycerol and depleted of cholesteryl ester. To test whether these differences related to the low level of hepatic lipase in rabbits, whole plasma or the total lipoprotein fraction from rabbits was either kept at 4 degrees C or incubated at 37 degrees C for 7 h in (i) the absence of lipase, (ii) the presence of hepatic lipase and (iii) the presence of lipoprotein lipase. Following incubation, the lipoproteins were recovered and subjected to gel permeation chromatography to determine the distribution of lipoprotein components across the entire lipoprotein spectrum. An aliquot of the lipoproteins was subjected also to gradient gel electrophoresis to determine the particle size distribution of the LDL and HDL. Both hepatic lipase and lipoprotein lipase hydrolysed lipoprotein triacylglycerol and to a much lesser extent, also phospholipid. There were, however, obvious differences between the enzymes in terms of substrate specificity. In incubations containing hepatic lipase, there was a preferential hydrolysis of HDL triacylglycerol and a lesser hydrolysis of VLDL triacylglycerol. By contrast, lipoprotein lipase acted primarily on VLDL triacylglycerol. When more enzyme was added, both lipases also acted on LDL triacylglycerol, but in no experiment did lipoprotein lipase hydrolyse the triacylglycerol in HDL. Coincident with the hepatic lipase-induced hydrolysis of LDL and HDL triacylglycerol, there were marked reductions in the particle size of both lipoprotein fractions, which were now comparable to those of human LDL and HDL3, respectively.     

The C-terminal conserved domain of DNA-PKcs, missing in the SCID mouse, is required for kinase activity

DNA-PKcs, the catalytic subunit of DNA-dependent protein kinase (DNA-PK), has a phosphoinositol 3-kinase (PI 3-K) domain close to its C-terminus. Cell lines derived from the SCID mouse have been utilised as a model DNA-PKcs-defective system. The SCID mutation results in truncation of DNA-Pkcs at the extreme C-terminus leaving the PI 3-K domain intact. The mutated protein is expressed at low levels in most SCID cell lines, leaving open the question of whether the mutation abolishes kinase activity. Here, we show that a SCID cell line that expresses the mutant protein normally has dramatically impaired kinase activity. We estimate that the residual kinase activity typically present in SCID fibroblast cell lines is at least two orders of magnitude less than that found in control cells. Our results substantiate evidence that DNA-PKcs kinase activity is required for DSB rejoining and V(D)J recombination and show that the extreme C-terminal region of DNA-PKcs, present in PI 3-K-related protein kinases but absent in bona fide PI 3 lipid kinases, is required for DNA-PKcs to function as a protein kinase. We also show that expression of mutant DNA-PKcs protein confers a growth disadvantage, providing an explanation for the lack of DNA-PKcs expression in most SCID cell lines.     

Development and characterization of an animal model of carnitine deficiency.

Mammals cover their carnitine needs by diet and biosynthesis. The last step of carnitine biosynthesis is the conversion of butyrobetaine to carnitine by butyrobetaine hydroxylase. We investigated the effect of N-trimethyl-hydrazine-3-propionate (THP), a butyrobetaine analogue, on butyrobetaine hydroxylase kinetics, and carnitine biosynthesis and body homeostasis in rats fed a casein-based or a vegetarian diet. The K(m )of butyrobetaine hydroxylase purified from rat liver was 41 +/- 9 micromol x L(-1) for butyrobetaine and 37 +/- 5 micromol x L(-1) for THP, and THP was a competitive inhibitor of butyrobetaine hydroxylase (K(i) 16 +/- 2 micromol x L(-1)). In rats fed a vegetarian diet, renal excretion of total carnitine was increased by THP (20 mg.100 g(-1) x day(-1) for three weeks), averaging 96 +/- 36 and 5.3 +/- 1.2 micromol x day(-1) in THP-treated and control rats, respectively. After three weeks of treatment, the total carnitine plasma concentration (8.8 +/- 2.1 versus 52.8 +/- 11.4 micromol x L(-1)) and tissue levels were decreased in THP-treated rats (liver 0.19 +/- 0.03 versus 0.59 +/- 0.08 and muscle 0.24 +/- 0.04 versus 1.07 +/- 0.13 micromol x g(-1)). Carnitine biosynthesis was blocked in THP-treated rats (-0.22 +/- 0.13 versus 0.57 +/- 0.21 micromol x 100 g(-1) x day(-1)). Similar results were obtained in rats treated with the casein-based diet. THP inhibited carnitine transport by rat renal brush-border membrane vesicles competitively (K(i) 41 +/- 3 micromol x L(-1)). Palmitate metabolism in vivo was impaired in THP-treated rats and the livers showed mixed steatosis. Steady-state mRNA levels of the carnitine transporter rat OCTN2 were increased in THP-treated rats in skeletal muscle and small intestine. In conclusion, THP inhibits butyrobetaine hydroxylase competitively, blocks carnitine biosynthesis in vivo and interacts competitively with renal carnitine reabsorption. THP-treated rats develop systemic carnitine deficiency over three weeks and can therefore serve as an animal model for human carnitine deficiency.     

An animal model of systemic carnitine deficiency produced by haemodialysis of sheep

1. Sheep, which had previously been surgically prepared with cannulae in various vessels to monitor substrate and metabolite exchanges across all the major organs, were connected to a haemodialysis machine and their blood was dialysed at an average rate of 6.23 ml/min/kg body weight. 2. Dialysis for 4 hr reduced the blood free carnitine concentrations to approx. 50% of the initial values and the concentrations returned to the initial values after 18 hr recovery. 3. Carnitine balance studies showed that approx. twice the amount of carnitine lost from the blood during dialysis passed into the dialysate indicating that carnitine was also lost from the extracellular fluid. 4. The average blood concentration of short-chain acylcarnitines did not vary significantly during dialysis or during the recovery phase. However, an output of short-chain acylcarnitines by the liver at 3 and 18 hr recovery and an uptake by the hind-body at 18 hr recovery was observed. 5. These results suggest that haemodialysis of sheep provides a useful model of systemic carnitine deficiency and suggest that treatment with acetylcarnitine or propionylcarnitine could be an efficient means of supplying carnitine in carnitine replacement therapy.     

In vivo microdialysis in an animal model of neurological disease: thiamine deficiency (Wernicke) encephalopathy

In vivo microdialysis allows for the constant monitoring of brain neurotransmitters in the extracellular fluid of awake and freely moving animals. Considerations including factors affecting probe recoveries, the blood-brain barrier, and tissue reactions to probe implantation are discussed in this paper. Details of the application of in vivo microdialysis to an animal model of encephalopathy are then presented. Thiamine deficiency encephalopathy is an animal model of Wernicke encephalopathy, a neurological disorder observed in alcoholics and in patients with severely compromised nutrition. Regionally selective neuronal cell death is observed in both patients and animals with thiamine deficiency (TD). Various thalamic nuclei suffer significant TD-induced cell death, and NMDA receptor-mediated glutamate excitotoxicity has been proposed as an underlying causative factor. A detailed methodology for the examination of the role of glutamate excitotoxicity using in vivo microdialysis in the neuronal cell death due to thiamine deficiency is presented.     

A fluorescence double-quenching study of native lipoproteins in an animal model of manganese deficiency

Iodide and acrylamide were applied simultaneously in a doublequenching experiment to compare acrylamide quenching constants for internal and external fluorophores of high-density lipoproteins (HDL₁ and HDL₂) from manganese-adequate (MnA) and deficient (MnD) rats, free of the electrostatic effects associated with iodide. In MnA HDL₁ compared to MnD HDL₁, the acrylamide quenching constant for external fluorophores was different (P < 0.1). In MnA HDL₂, there were two populations of fluorophores accessible to acrylamide, whereas in MnD HDL₂, all fluorophores were accessible to both quenchers. We concluded that there were structural (local environmental) differences, possibly charge-related, around the external fluorophores, and a slightly larger population of buried fluorophores in the MnD HDL₁ compared with MnA HDL₁. In MnA HDL₂, one-third of the fluorophores were accessible to iodide, and all external and internal fluorophores were accessible to acrylamide, whereas in MnD HDL₂, all fluorophores were accessible to both quenchers.     

Mycobacterium marinum produces long-term chronic infections in medaka: a new animal model for studying human tuberculosis

Human infection by Mycobacterium tuberculosis is endemic, with approximately 2 billion infected and is the most common cause of adult death due to an infectious agent. Because of the slow growth rate of M. tuberculosis and risk to researchers, other species of Mycobacterium have been employed as alternative model systems to study human tuberculosis (TB). Mycobacterium marinum may be a good surrogate pathogen, conferring TB-like chronic infections in some fish. Medaka (Oryzias latipes) has been established for over five decades as a laboratory fish model for toxicology, genotoxicity, teratogenesis, carcinogenesis, classical genetics and embryology. We are investigating if medaka might also serve as a host for M. marinum in order to model human TB. We show that both acute and chronic infections are inducible in a dose dependent manner. Colonization of target organs and systemic granuloma formation has been demonstrated through the use of histology. M. marinum expressing green fluorescent protein (Gfp) was used to monitor bacterial colonization of these organs in fresh tissues as well as in intact animals. Moreover, we have employed the See-Through fish line, a variety of medaka devoid of major pigments, to monitor real-time disease progression, in living animals. We have also compared the susceptibility of another prominent fish model, zebrafish (Danio rerio), to our medaka-M. marinum model. We determined the course of infections in zebrafish is significantly more severe than in medaka. Together, these results indicate that the medaka-M. marinum model provides unique advantages for studying chronic mycobacteriosis.     

DNA-PKcs deficiency leads to persistence of oxidatively induced clustered DNA lesions in human tumor cells

DNA-dependent protein kinase (DNA-PK) is a key non-homologous-end-joining (NHEJ) nuclear serine/threonine protein kinase involved in various DNA metabolic and damage signaling pathways contributing to the maintenance of genomic stability and prevention of cancer. To examine the role of DNA-PK in processing of non-DSB clustered DNA damage, we have used three models of DNA-PK deficiency, i.e., chemical inactivation of its kinase activity by the novel inhibitors IC86621 and NU7026, knockdown and complete absence of the protein in human breast cancer (MCF-7) and glioblastoma cell lines (MO59-J/K). A compromised DNA-PK repair pathway led to the accumulation of clustered DNA lesions induced by γ-rays. Tumor cells lacking protein expression or with inhibited kinase activity showed a marked decrease in their ability to process oxidatively induced non-DSB clustered DNA lesions measured using a modified version of pulsed-field gel electrophoresis or single-cell gel electrophoresis (comet assay). In all cases, DNA-PK inactivation led to a higher level of lesion persistence even after 24–72 h of repair. We suggest a model in which DNA-PK deficiency affects the processing of these clusters first by compromising base excision repair and second by the presence of catalytically inactive DNA-PK inhibiting the efficient processing of these lesions owing to the failure of DNA-PK to disassociate from the DNA ends. The information rendered will be important for understanding not only cancer etiology in the presence of an NHEJ deficiency but also cancer treatments based on the induction of oxidative stress and inhibition of cluster repair.