Peptidases play an important role in cataractogenesis: an immunohistochemical study on lenses derived from Shumiya cataract rats.

The role of proteolytic enzymes in Shumiya cataract rats in alterations to lens proteins during cataract formation was studied immunohistochemically using antibodies against exopeptidases, such as lysosomal dipeptidyl peptidase II (DPP II), cytosolic dipeptidyl peptidase III, and soluble and membrane-bound alanyl aminopeptidases, and against cytosolic endopeptidases such as mu- and m-calpains, and 20S proteasome. AlphaB-crystallin was detected as a proteolytic marker in the lenses. A constant immunoreactivity against all the antibodies employed was observed in the lens epithelium independent of the strain and age of the rats. A weak immunoreactivity against exo- and endopeptidases and an intense reactivity against alphaB-crystallin were observed in the lens fibres of control rats at all ages. The immunoreactivity of these peptidases in lens fibres increased with age in cataract rats, but that of alphaB-crystallin decreased. No reactivity against exo- and endopeptidases was seen in the perinuclear region of lenses of control rats at all ages or in Shumiya cataract rats at 8 and 10 weeks of age, but an intense reactivity against these peptidases was observed in the lens perinuclear region of lenses in cataract rats at 12 and 14 weeks of age. AlphaB-crystallin immunoreactivity was observed with ordered striations in the lens perinuclear region of all control rats whereas the striations in this area of cataract rat lens were disorganized. Membrane-bound alanyl aminopeptidase was detected feebly in the lens epithelium and fibres of both types of rat at all weeks of age. These findings indicate that exo- and endopeptidases, except for membrane-bound alanyl aminopeptidase, are expressed intensively and are age-dependent. Conversely, the amount of alphaB-crystallin decreased with age in lens fibres of cataract rats. Calpains (mu- and m-), 20S proteasome, dipeptidyl peptidases II and III and soluble alanyl aminopeptidase are thought to induce lens opacification kinetically during cataract formation in Shumiya cataract rats through the intracellular turnover of lens proteins.

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.

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.

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.

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.

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.

The beneficial effects of recombinant human insulin-like growth factor-I (IGF-I) on wound healing in severely wounded senescent mice.

The effects of recombinant insulin-like growth factor I (rIGF-I) on wound healing were tested using senescent and young BDF-1 mice, aged 108 weeks and 10 weeks, respectively. After inflicting a full thickness dermal burn encompassing 15% of the body surface, a skin incision, 2 cm in length, was made in the back. A silicone tube containing a piece of polyvinyl sponge was then implanted into a subcutaneous pocket in the flank to collect body fluid. An osmotic pump was buried in the abdominal subcutaneous tissue for the continuous infusion of rIGF-I, the control being treated with the solvent of IGF-I, physiological saline, only. The administration of IGF-I produced favorable effects on wound healing in the senescent mice, shown by enhanced tensile strength and an elevated concentration in the hydroxyproline of the polyvinyl sponge content. The IGF-I-treated severely wounded senescent mice healed better than their counterparts and their skeletal muscles contained more glutamine. Furthermore, they showed more enhanced cutaneous hypersensitivity towards dinitrofluorobenzene than the controls, suggesting an enhanced grade of cellular immunity. There were no conspicuous differences between the two groups of young mice. These data may suggest the beneficial effects of rIGF-I on wound healing, especially in geriatric surgery.

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.

Apoptosis in the aged dog brain.

Apoptosis similar to that seen in Alzheimer's disease patients was found in the brain of aged dogs by the TUNEL method of detecting in situ DNA fragmentation. Apoptosis was observed in both neurons and glial cells, and was morphologically characterized by round and swollen cytoplasm and aggregated nuclear chromatin, although these changes were slight. Neurons and astrocytes in the gray matter and oligodendrocytes in the white matter were affected. The number of ApopTag-positive brain cells increased slightly with age, but was not correlated to the number of senile plaques. A good correlation between the number of ApopTag-positive cells and the dementia index was clearly found. The present study indicates that brain cell apoptosis could account for dementia in aged dogs and suggested that aged dogs may be useful as a simplified animal model for Alzheimer's disease in man.

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).

Histopathological study of hereditary cataractous lenses in SCR strain rat.

A new cataractous rat strain, Shumiya Cataract Rat (SCR), which was derived from the cross-breeder formed between the spontaneous hypertensive rat and the Zucker fatty rat, was studied by light and electron microscopy. The earliest change was the occurrence of some displaced nuclei in the posterior cortical fibres at birth. By 2 weeks of age, mild dysplasia of the anterior suture, aggregated epithelial cells overlying the sutural area, and poorly differentiated epithelial cells at the bow area were observable. At 3 weeks, unusually dense lens fibres near dense epithelial cells were found at the equator, and some epithelial cells showed accellularity after 4 weeks. Swelling and liquefaction of the lens fibres appeared in the anterior cortex by this stage. By 6 weeks, these changes extended toward the equatorial region. At 7 weeks, a sutural gap occurred at the anterior polar area, and this gap reached a maximum at 8 weeks. At this time, swelling of the posterior extremities of the lens fibres appeared, showing a small placoid opacity of the posterior subcapsular region under a dissecting microscope. At 10 weeks, small amounts of liquefaction were observed in this region. Between 10.5 and 11 weeks, as the posterior suture of liquefaction were observed in this region. Between 10.5 and 11 weeks, as the posterior suture separated, opacification appeared around the separated sutural area and rapidly developed into a mature cataract. The mechanism of cataract formation in the SCR rat lens may be associated with the continuous occurrence of a small proportion of poorly-differentiated epithelial cells at the bow area and sutural dysplasia of the anterior lens fibres.

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.

[Age-related non-tumorous lesions in SPF C57BL/6 mice with special reference to amyloidosis].

Non-tumorous pathological changes in C57BL/6 CrSlc mice, which were reared under a barrier system and died spontaneously, were examined. At 3 months intervals 125 to 209 mice were purchased at 4 weeks of age and raised for the supply of aged animals. A large portion of the mice were used for various experiments between 3 and 30 months of age, while not a small number died spontaneously and were autopsied. The major non-neoplastic lesion was amyloidosis, with incidence of 55.5% and 74.4% for the autopsied female and male, respectively. The organs involved were the liver, kidneys, spleen, adrenal glands, ileum, heart and lungs. Skin ulceration and its scar, cerebral vascular calcification, glomerulosclerosis and sepsis in both sexes, distension of the seminal vesicles in males, fibroblast growth of the adrenal glands in females were commonly found. Incidence of spontaneous neoplastic lesions was 69.7% and 55.1% for the female and male, respectively.

[Age-related incidence of spontaneous tumors in SPF C57BL/6 and BDF1 mice].

Incidence of spontaneous tumors in C57BL/6 NCrj (CR), C5BL/6 CrSlc (SL) and B 6 Crj x DBA/2 NCrj F1 (BD) mice, which were reared under a barrier system and died natural death, were examined. Cohorts of mice in 200 to 300 each were purchased at 4 weeks of age and raised under SPF conditions. A large portion of the mice were used for various experiments between 3 and 30 months old while not a small number died before use and were autopsied. Median survival periods of the female and male were estimated at 697 and 680 days for CR, 764 and 806 days for SL and 866 and 929 days for BD, respectively. Incidence of spontaneous neoplastic lesions in the autopsied animals were 77.4% and 79.2% of 535 female and 590 male CR, 69.7% and 55.1% of 502 female and 463 male SL, and 75.8% and 78.0% of 298 female and 346 male BD, respectively. In CR, histiocytic sarcoma was the most predominant tumor, accounting for 72.1% of all tumors. In SL, malignant lymphoma was the most prevailing, forming 62.3%, and, in male BD, hepatocellular carcinoma was the most frequent, accounting for 41.8% of all tumors.

High susceptibility of analbuminemic rats to neurogenic tumor induction by transplacental administration of N-ethyl-N-nitrosourea.

The susceptibilities of Nagase analbuminemic rats (NAR) and control Sprague-Dawley rats (SDR) to N-ethyl-N-nitrosourea (ENU) were compared. In Experiment I, the rats were given daily subcutaneous injections of 10 mg/kg of ENU for a week from 4 weeks of age. In Experiment II, mother rats were given a single subcutaneous injection of 60 mg/kg of ENU on day 17 of pregnancy and tumor development in their offspring was examined. In Experiment I, the incidence of neurogenic tumors was slightly, but not significantly, higher in NAR than in control rats. In Experiment II, the incidence of total tumors including neurogenic tumors was significantly higher in NAR (40/43, 93.0%) than in SDR (13/61, 21.3%). NAR showed particularly high susceptibility to induction of neurogenic tumors (34/43, 79.1%) and renal tumors (15/43, 34.9%). In an attempt to elucidate the underlying mechanisms of the increased susceptibility of NAR to ENU, O6-ethylguanine, a major premutagenic ethylated DNA adduct, was quantitated in fetal brain DNA of NAR and SDR after a pulse exposure to 60 mg/kg ENU. No significant difference in the initial formation or subsequent repair of O6-ethylguanine was observed in the two strains, indicating that abnormality at some later stage(s) of chemical carcinogenesis may lead to the increased susceptibility of NAR to induction of neurogenic tumors.

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.

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.

Identification of a protein increasing in serum of Nagase analbuminemic rats bearing intestinal tumors as an isotype of T-kininogen.

Increase in an unidentified protein was observed in serum of Nagase analbuminemic rats (NAR) bearing intestinal tumors induced by azoxymethane. This protein seemed to be a polymer of a protein of 73 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and so was tentatively named 73K-protein. The serum concentration of 73K-protein in NAR bearing intestinal tumors was 11.9 +/- 2.2 mg/ml (mean +/- SD, n = 5), whereas that in control NAR was 2.0 +/- 0.2 mg/ml. Increase of the serum 73K-protein level was also observed in Sprague-Dawley rats bearing intestinal tumors, skin tumors, subcutaneous sarcomas, or mammary tumors and in ACI rats bearing urinary bladder tumors. On double immunodiffusion analysis, the 73K-protein was not detected in mouse, guinea pig, pig, horse, or human serum. A cDNA clone bearing the sequence encoding 73K-protein was isolated from a cDNA library constructed from rat liver mRNA. The nucleotide sequence of the 73K-protein showed 98.8% and 96.9% homologies with the sequences of the 3'-proximal domains of the cDNAs for TI- and TII-kininogen, respectively. Therefore, the 73K-protein was concluded to be an isotype of T-kininogen.

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.