Albumin-deficient rat mutant.

An analbuminemic colony was established from Sprague-Dawley rats. Analbuminemia was inherited as an autosomal recessive trait. The rates of growth and reproduction of the mutant rats were no different from those of normal rats. Biochemically, the mutant was characterized by an extraordinarily low serum albumin content and a hyperlipidemia. Total serum protein in the mutant rat was similar to that of control Sprague-Dawley rats, with increased globulin. Serum cholesterol was inversely correlated with a decrease in albumin; the correlation coefficient for ablumin was --.92. These mutant rats may serve as a model of human familial analbuminemia and may also be useful in elucidating the functional roles of albumin.

Evidence for the involvement of calpain in cataractogenesis in Shumiya cataract rat (SCR).

The Shumiya cataract rat (SCR) is a hereditary cataract model in which lens opacity appears spontaneously in the nuclear and perinuclear portions at 11-12 weeks of age. It was found that the proteolysis of some crystallins and cytoskeletal proteins is significantly enhanced in cataractous SCR lenses. The calcium concentrations in cataractous lenses rise markedly with age as compared with control lenses and the autolytic product of calpain is also detected in cataractous lenses. In order to provide direct evidence for the involvement of calpain in the proteolytic modification of lens proteins, we developed antibodies exclusively specific to the proteolytic products of some lens proteins produced by the action of calpain and analyzed their degradation during cataractogenesis in SCR by Western blotting and immunohistochemical staining. The results demonstrate that calpain participates in the proteolytic modification of lens proteins, at least alpha-crystallin (A and B chain), betaB1-crystallin, and alpha-fodrin. The proteolytic products formed by the action of calpain on these proteins are detected in cataractous lenses of SCR as young as 8 weeks of age and accumulate with age. It was also found that betaB1-crystallin, originally a soluble protein, is converted to an insoluble form by limited calpain proteolysis. The chaperon-like activity of alpha-crystallin from control lens is markedly reduced by calpain proteolysis in vitro, and alpha-crystallin in opaque lens that has already undergone proteolysis by calpain shows significantly reduced chaperon-like activity. Immunohistochemical studies reveal that the area where the calpain-mediated alpha-crystallin proteolysis is in progress coincides well with the area developing and destined to develop the opacification. These results strongly suggest that calpain may contribute to lens opacification during cataract formation in SCR.

Age-related alteration of brain gangliosides in senescence-accelerated mouse (SAM)-P/8.

The senescence-accelerated mouse (SAM)-P/8 was examined with respect to changes in the content and composition of brain gangliosides during aging from juvenile to senescence. The gangliosides were compared with those of control mice, senescence-accelerated resistant mouse (SAM)-R/1. The ganglioside contents in the whole brains of SAM-P/8 and -R/1 were at almost constant level from 0.5 to 6 months, but decreased thereafter until senescence to about 80% of the levels reached at the younger ages. Upon aging, the ganglioside compositions changed with an increase of GM1, and decreases of GD1a, GD1b and GT1b in both strains (GT1b greater than GD1a greater than GD1b). A minor component, GM3 was two to four fold higher in the molecular distributions of the whole brain gangliosides of SAM-P/8 than those of -R/1 at any age examined throughout the life span. The regional gangliosides in olfactory bulb, cerebral cortex, hippocampus, hypothalamus, cerebellum, corpora quadrigemina region, brain stem and medulla oblongata were compared between the two strains at the age of three months. The ganglioside contents in the brain stem and medulla oblongata were lower in SAM-P/8 than -R/1, but there was no significant difference between the two strains in the other regions. As a minor component, GM3 was found to occur in a higher concentration in SAM-P/8 than -R/1 in all brain regions examined, except in the olfactory bulb where GM3 was detected as a major component with no difference in the distribution level between the two strains.

Evaluation of carbon-11-labeled KF17837: a potential CNS adenosine A2a receptor ligand.

UNLABELLED: The 11C-labeled KF17837 ([7-methyl-11C](E)-8-(3,4-dimethoxystyryl)-1,3-dipropyl-7-methylxa nthine) was evaluated as a PET ligand for mapping adenosine A2a receptors in the central nervous system (CNS). METHODS: The regional brain distribution of [11C]KF17837 and the effect of adenosine antagonists on the distribution were measured in mice by the tissue sampling method. In rats, the regional brain uptake of [11C]KF17837 and the effect of carrier KF17837 was visualized by autoradiography. Imaging of the monkey brain with [11C]KF17837 was performed by PET. RESULTS: In mice, a high uptake of [11C]KF17837 was found in the striatum in which A2a receptors were highly enriched. The uptake was decreased by co-injection of carrier KF17837 or a xanthine-type A2a antagonist CSC but not by nonxanthine-type A2a antagonists ZM 241385 or SCH 58261, or an A1 antagonist KF15372. In the rat brain, [11C]KF17837 was accumulated higher in the striatum than in other brain regions, and the uptake was blocked by co-injection of carrier KF17837. In a monkey PET study, a high striatal uptake of radioactivity was observed. CONCLUSION: Carbon-11-KF17837 binds to adenosine A2a receptors in the striatum. However, the presence of an unknown but specific binding site for xanthine-type compounds also was suggested in the other brain regions. The results also suggested that the in vivo receptor-binding sites of xanthine-type ligands are slightly different from those of nonxanthine-type A2a antagonists.

Penetration of bilirubin into the brain in albumin-deficient and jaundiced rats (AJR) and Nagase analbuminemic rats (NAR).

An albumin-deficient and jaundiced strain of rats (AJR) was established by crossing Nagase analbuminemic rats (NAR) with jaundiced Gunn rats. AJR have no serum albumin and die with kernicterus within 3 weeks after birth. Their serum bilirubin level was 25 per cent (20 micrograms/ml) of that of Gunn rats at 1-2 weeks of age, while their brain bilirubin content was 1.2-2.7 times (4-5 micrograms/g brain) that of Gunn rats. Binding of bilirubin to NAR plasma proteins was examined. The bilirubin binding protein in NAR plasma was found to be lipoprotein, showing an association constant of 6.7 X 10(6) M-1. Bilirubin-transport into the brain of postnatal NAR was investigated by intravenous infusion of bilirubin with taurocholate. The brain bilirubin level of NAR infused with free bilirubin was 1.6 times that of normal rats. In NAR, the brain level on infusion of lipoprotein-bound bilirubin was similar to that on infusion of free bilirubin, but albumin-bound bilirubin scarcely entered the brain. These findings suggest that lipoprotein-bound bilirubin can diffuse across the blood-brain barrier into the brain.

Aminoguanidine-treatment results in the inhibition of lens opacification and calpain-mediated proteolysis in Shumiya cataract rats (SCR).

The Shumiya cataract rat (SCR) is a hereditary cataract model in which lens opacity appears spontaneously in the nuclear and perinuclear portions at 11-12 weeks of age. We found incidentally that the oral administration of aminoguanidine (AG), an inhibitor of inducible nitric oxide synthase (iNOS), strongly inhibits the development of lens opacification in SCR. Since our previous results strongly suggested that calpain-mediated proteolysis contributes to lens opacification during cataract formation in SCR, we examined the calpain-mediated proteolysis in AG-treated SCR lenses in detail. The results show that the calpain-mediated limited proteolysis of crystallins is also inhibited by AG-treatment. However, the administration of AG has no effect on the substrate susceptibility to calpain. On the other hand, the autolytic activation of calpain in AG-treated lenses is strongly inhibited, although AG itself does not inhibit calpain activity in vitro. Then, we analyzed the effect of AG-treatment on calcium concentrations in lens, and found that the elevation in calcium concentration that should occur prior to cataractogenesis in lenses is strongly suppressed by AG-treatment. These results strengthen our previous conclusion that calpain-mediated proteolysis plays a critical role in the development of lens opacification in SCR. Moreover, our results indicate that the inhibition of calpain-mediated proteolysis by AG-treatment is due to the suppression of calcium ion influx into the lens cells.

Age-related changes in radial-arm maze learning and basal forebrain cholinergic systems in senescence accelerated mice (SAM).

Age-related changes in learning performance and the brain cholinergic system were studied in a senescence accelerated mice-prone series (SAM-P/8) and a senescence accelerated mice-resistant series (SAM-R/1, control) bred under specific pathogen-free conditions. In a radial-arm maze task, SAM-P/8 mice at 4 and 12 months of age showed virtually no significant impairment in working memory or reference memory compared with SAM-R/1 mice at the same age, although they needed more time to complete a trial than SAM-R/1. In contrast, in a passive avoidance task, SAM-P/8 showed a marked age-accelerated deficit in acquisition performance relative to SAM-R/1. Also, SAM-P/8 showed an age-accelerated decrease in locomotion and rearing in an open-field box. At the end of these behavioral tasks, neurochemical analyses showed that there were no differences in the concentrations of acetylcholine (ACh) in the cortex, hippocampus, striatum, midbrain, or cerebellum between SAM-P/8 and SAM-R/1. Although SAM-P/8 mice did not demonstrate any age-accelerated decline in radial-arm maze performance, they showed a normal age-related decline particularly in working memory, equal to that observed in SAM-R/1. Also, ACh levels in the aged groups of SAM-P/8 showed a significant decrease related to normal aging in the hippocampus and striatum, and a slight decrease in the cortex compared to the young group of the same strain. Thus, we found that SAM-P/8 show dissociative effects of aging in spatial learning and passive avoidance performance.

A PET study of adenosine A1 receptor in anesthetized monkey brain.

We demonstrated the distribution of adenosine A1 receptors in the anesthetized monkey brain with positron emission tomography (PET) using [(11)C]KF15372 ([1-propyl-(11)C]8-dicyclopropylmethyl-1, 3-dipropylxanthine). [(11)C]KF15372 was injected intravenously. The regional standardized uptake values and the distribution volume were calculated. We also investigated the effect of carrier on the uptake and regional brain distribution of [(11)C]KF15372. The use of [(11)C]KF15372 with dynamic PET scanning could be an appropriate method to analyze the regional binding potential of adenosine A1 receptors in living brain.

Involvement of inducible nitric oxide synthase in cataract formation in Shumiya cataract rat (SCR).

PURPOSE: Our previous results showed that the oral administration of aminoguanidine (AG), an inhibitor of inducible nitric oxide synthase (iNOS), strongly suppresses lens opacification in Shumiya cataract rat (SCR). Therefore, we examine whether iNOS is upregulated and involved in cataract formation in SCR. METHODS: The expressions of iNOS mRNA and iNOS protein in SCR lenses were examined by RT-PCR and Western blotting, respectively. Calpain-mediated alphaB-crystallin proteolysis was analyzed by Western blotting using antibody specific to the calpain-generated fragment of alphaB-crystallin. Lens opacification was analyzed using computerized image analysis software connected to the Anterior Eye Segment Analysis System (EAS-1000, Nidek). Calcium contents in lenses were measured by atomic absorption spectrophotometry. RESULTS: High levels of iNOS mRNA and iNOS protein are expressed in cataractous lenses compared with normal lenses. The increases in their expression are markedly suppressed by the oral administration of AG, which acts to prevent lens opacification. The induction of iNOS protein is observed before the elevation in calcium content and the acceleration of calpain-mediated proteolysis, both of which are closely related to the development of lens opacification. CONCLUSIONS: These findings strongly suggest that iNOS is involved in cataract formation in SCR. The induction of iNOS occurs prior to the elevation of calcium content and its induction is inhibited by AG-treatment. Considering our previous result that the elevation of calcium content is also prevented by AG-treatment, it is conceivable that upregulation of iNOS causes calcium influx into lens cells and the subsequent activation of calpain.

Establishment and characteristics of four sub-strains of F344 rats reared on various low protein and low energy diets.

Four sub-strains, reared by sib-mating and having for their origin the F344/DuCrj strain of rats, were established by feeding with different levels of low protein and low energy diets, and their characteristics investigated. The amounts of crude protein (CP) and digestible energy (DE) in the four diets were 17.6%-3.0 kcal, 10.5%-2.5 kcal, 8.4%-2.0 kcal, and 10.5%-2.5 kcal, respectively, and the four sub-strains established here were provisionally designated as F344/Tig1, F344/Tig2, F344/Tig3 and F344/Tig4, respectively. Intakes of nitrogen-corrected metabolizable energy (MEn) did not differ, and a large intake of digestible crude protein (DCP) was observed in F344/Tig1 rats. The body weight of rats provided with lower-nutrient diets showed a tendency to decrease until the F2 generation, but no change among the generations was seen subsequently, and the same compiled differences in protein content were maintained. Similar transitions were observed in the lifetime rearing test. Though F344/Tig3 rats, which were reared on minimum nutrients, showed a tendency to delayed puberty, we were easily able to breed four pairs in every generation using procedures similar to those used for other strains of rats. There were no differences among the F344/Tig1 to -3 strains of rats in body length, digestive tract length, or organ weight per body weight, and all the rats had a normal range of biochemical values. But the F344/Tig4 showed a high glutamic-oxaloacetic transaminase (GOT), and a tendency to decreased liver function and a shorter lifespan. These sub-strains of F344 rats clarified differences in fatty acid compositions, such as docosahexaenoic acid (DHA) in serum, liver and the brain. The rats were intended to be useful animal models for the study of nutritional environments and their influence on the memory and learning.

Alteration of lens disulfide bonds in newly developed hereditary cataract rat.

A newly developed hereditary cataract rat named Shumiya Cataract Rat (SCR) is introduced. The lens opacity manifests spontaneously in the nuclear and perinuclear portions of the lens in this model at 11 weeks of age. Raman optical dissection studies reveal that lens protein disulfide bonds are considerably decreased in the SCR opaque lens nucleus. This tendency is also detected in the precataractous stage. The simultaneous increase of lens protein sulfhydryl groups is observed in the affected lens nucleus. The glutathione disulfide (oxidized glutathione) level, on the contrary, is elevated in the SCR opaque lens, whereas the glutathione sulfhydryl (reduced glutathione) level is decreased. It is suggested that the impaired formation of protein disulfide bonds is related to lens opacification in a hereditary manner.

Lens reconstruction after mature cataract in SCR rat.

PURPOSE: To conduct a long-term observation study of SCR rats that had developed a mature cataract at 11 weeks of age at 3-month intervals until the rats were 12 months old. METHODS: Lenses of 15 rats were examined with both light and electron microscopes. RESULTS: At 12 weeks, opacity was observed in the perinuclear zone and the cortical intermediate layer. Liquefaction of the posterior subcapsular area and regression of cortical superficial fibers were also observed at this stage. Epithelial cells at the anterior polar area were multilayered. At 12 months, the lens recovered as a result of the regenerated lens fibers in the intermediate layer and the cortical superficial layer, although the opacity remained in the perinuclear zone. The multilayered cellular structure in the center of the epithelium returned to its original monolayer form. However, the equatorial epithelial cells became vacuolated and swollen with age, showing regression from the bow region. CONCLUSIONS: These results suggest that the decrease of opacity in SCR rats is merely a temporary phenomenon that reflects the differentiating and metabolizing functions of the epithelial cells. With initiation of epithelial regression, the regeneration of the lens fibers ceased, suggesting that further decrease in opacity was no longer possible.

Establishment of the hereditary cataract rat strain (SCR) and genetic analysis.

The Shumiya cataract rat (SCR) and normal control rat strains of NC1 and NC2 were established. Mean cataract appearance in adult SCR rats was 66.7%, and embryo death rate was 25%. Genetic analysis of cataract formation in the SCR was studied by breeding experiments with strains such as NC1, BUF, ACI, AD3, and normal SCR rats. No sex-based differences in cataract appearance were observed in any of the progeny. These results confirmed our hypotheses that two autosomal genes, a recessive cataract gene (ctr1), and a normal allele of a dominant cataract gene with a recessive lethal trait (Ctr2(1)) for provisional designations participated in cataract genesis in SCR (ctr1/ctr1, Ctr2(1)/Ctr2) rats. One recessive cataract gene was maintained in normal SCR, NC1 and NC2 (ctr1/ctr1, Ctr2/Ctr2) rats. The same recessive cataract gene was retained in the albumin-deficient brown hooded rat strain AD3 (ctr1/ctr1, Ctr2/Ctr2).

Establishment and characteristics of three analbuminemic congenic strains of rats.

We have established three analbuminemic congenic strains of rats (ACI-alb, F344-alb, and SHR-alb) by repeated backcrossing with a progeny test or intercrossing. Some coat color and biochemical marker genes of each congenic strain agreed with those of the background inbred strain of rats, except for the alb gene locus. These established congenic strains were maintained by cross-intercrossing. Body weights, organ weights and serum lipid concentrations of each strain were measured up to 30 weeks of age. Body weights of ACI-alb congenic strains (alb/alb and alb/+) were similar to those of the original ACI(+/+) strain, but those of F344-alb and SHR-alb were heavier in the order of +/+, alb/+ and alb/alb. The liver and adrenal weights of all strains were higher in the order of alb/alb, alb/+ and +/+. Serum lipid concentrations were also higher in the same order. These three analbuminemic congenic strains originating from different inbred strains should be useful in studies of carcinogenesis and genetically modified mechanisms of albumin functions.

Mapping of the hooded, Gc protein, and albumin gene loci in linkage group VI of the laboratory rat.

Crosses to determine the position of the three gene loci, h, Gc, and Alb, in the sixth linkage group of the rat used three strains, the TM strain, the ACI-alb analbuminemic congenic strain, and the abh-alb tester strain established by crossing the abh coat color tester strain and analbuminemic rats. Their genotypes were [C/C, h/h, GcB/GcB, Alb/Alb], [C/C, hi/hi, GcA/GcA, alb/alb] and [C/C, h/h, GcA/GcA, alb/alb], respectively. Determination of genotypes was performed by coat color and polyacrylamide gel electrophoresis (PAGE of serum protein for the Gc and albumin genes. The positions of the three gene loci in the VI linkage group were calculated from the recombination values from the phenotypes of progenies. According to this data, the three gene loci were in h-Gc-Alb tandem and the distances were 15.5 +/- 1.0% in h-Gc, 15.8 +/- 1.0% in h-Alb, and 0.32 +/- 0.16% in Gc-Alb. These data confirmed the relationship among the Gc, Alb, and Afp genes in the rat as well as in humans.

Treatment and improvement of the breeding system of the albumin-deficient and jaundiced strain of rats.

The AJR (alb/alb, j/j) strain is semi-lethal and the rats die at about two weeks of age. Cross breeding with a pari of ACJ (alb/alb, j/+) is required for maintenance and production. We studied the treatment of AJRs for improvement of the breeding system. AJRs with jaundice on the skin at about 7 days of age were intraperitoneally administered 1 ml/rat of normal rat serum every few days and irradiated with visible light for 14 hours a day in an irradiation box. This treatment allowed us to obtain mature AJRs. The treatment was effective when applied for short periods of 1-4 weeks of age. The frequency of maturation of the treated AJRs was about 27% and the best mating combination was a female ACJ and male AJR. The treatment of AJRs, made the progeny test for the selection of ACJs for breeding pairs of ACJ X ACR unnecessary and increased the frequency of appearance of AJRs to 50%.

Linkage of the analbuminemia locus (alb) and the hooded locus in the rat, Rattus norvegicus.

A linkage study of the analbuminemia (alb) with some coat color loci (a,b,c,h) has been carried out by crossing the analbuminemic random-bred mutant NAR strain with the abh tester strain of rats, the Agouti-Irish inbred ACI strain and the Agouti-Nonhooded inbred IS strain. The analbuminemia (alb) locus is not linked to a, b, and c loci but linked to hooded locus from F2 and backcross progeny of the F1 hybrids of abh X NAR, ACI X NAR and IS X NAR to NAR. Recombination value between alb and h was 16.4 +/- 2.3 per cent from the data of F2 and backcross progeny of (ACI X NAR) F1 and (IS X NAR) F1 to NAR. This is the third locus on the sixth linkage group of the rat.

Establishment of an albumin-deficient and jaundiced strain of rats.

An albumin deficient and jaundiced rat (AJR) strain was established from crosses between Albumin-Deficient rats (NAR) and jaundiced Gunn rats. AJRs have a double homozygous mutant trait (alb/alb, j/j), and systemic jaundice and various neurological signs are observed 5--7 days after birth, kernicterus occurs and they die within three weeks after birth. This strain of rats may serve as a model of human kernicterus and also be useful in elucidating the mechanism of bilirubin metabolism.

Reproductive activity of analbuminemic rats.

Long-term feeding and breeding in a conventional environment have been investigated in analbuminemic rats, a mutant strain established from Sprague-Dawley rats. Analbuminemic rats kept alive for over two years and maintained normal reproductive performance for over one year. The rotational system of one male and four females was better reproductive efficiency than the continuous pairing system of one male and one female. The litter size and weaning rate in analbuminemic rats were similar to those in Sprague-Dawley rats. Although we tried to establish a caesarean delivered SPF colony of analbuminemic rats, no changes in reproductive performance were recognized.

Establishment of an albumin-deficient and fatty strain of rats.

An albumin-deficient and fatty strain of rats (AFR) was established from crosses between albumin-deficient rats (NAR) and fatty Zucker rats. AFRs have double homozygous mutant genes (alb/alb, fa/fa). The AFRs are heavier than the fatty Zucker rats and they have enlarged livers and adrenal glands. In addition, AFRs show more severe hyperlipidemia than fatty Zucker rats. This strain of rats may serve as a model of human obesity and hyperlipidemia.