Increased seizure sensitivity, emotional defects and cognitive impairment in PHD finger protein 24 (Phf24)-null rats.

Phf24 is known as Gαi-interacting protein (GINIP) and is associated with the GABAB receptor. To study the function of Phf24 protein in the central nervous system (CNS), we have newly developed Phf24-null rats and investigated their behavioral phenotypes, especially changes in seizure sensitivity, emotional responses and cognitive functions. Phf24-null rats did not exhibit any spontaneous seizures. However, they showed a higher sensitivity to pentylenetetrazol (PTZ)- or pilocarpine-induced convulsive seizures. Phf24-null rats also showed an elevated susceptibility to kindling development with repeated PTZ treatments, suggesting that Phf24 acts as an inhibitory modulator in epileptogenesis. Although young Phf24-null rats showed normal gross behaviors, elevated spontaneous locomotor activity, especially in terms of the circadian dark period, emotional hyper-reactivity, reduced anxiety behaviors in the elevated plus-maze (EPM) test, and cognitive deficits in the Morris water maze test were explicitly observed at older age (20-week-old). The present results suggest that Phf24 is essential for proper functioning of the CNS, especially in preventing epileptogenesis and controlling emotional and cognitive functions.

Rat polyomavirus 2 infection in a colony of X-linked severe combined immunodeficiency rats in Japan.

Polyomaviruses (PyVs) infect a wide range of animals and provoke wasting diseases, particularly in immunosuppressed hosts. Recently, a novel Rattus norvegicus polyomavirus 2 (RatPyV2) has been identified in a colony of X-linked severe combined immunodeficiency (X-SCID) rats in the United States. Here, we describe the first report of the RatPyV2 infection in an X-SCID rat colony in Japan. The affected rats exhibited adult-onset wasting. Histologically, we observed large basophilic intranuclear inclusion bodies within the hyperplastic or dysplastic epithelial cells in the salivary glands, Harderian glands, extraorbital lacrimal glands, and in respiratory and reproductive tissues. Among these organs, the parotid salivary, Harderian, and extraorbital lacrimal glands were most obviously affected. In particular, the parotid salivary glands were the most severely and diffusely affected and atrophic lesions were prominent even at 1 month of age, which suggested that the parotid salivary glands would be highly susceptible to RatPyV2 in X-SCID rats. RatPyV2 inclusion bodies were also detected in the tail of the epididymis and deferent duct. Such reproductive lesions developed significantly in the later stage of breeding age, and therefore may be associated with the reduced fecundity observed in the infected X-SCID rats. We also established a simple, rapid, and non-invasive diagnostic method based on the Amp-FTA method, using buccal swabs for the detection of RatPyV2 in immunodeficient rats. Our findings contribute to the early detection and diagnosis of RatPyV2 infections.

Identification of Candidate Genes for Generalized Tonic-Clonic Seizures in Noda Epileptic Rat.

The Noda epileptic rat (NER) exhibits generalized tonic-clonic seizures (GTCS). A genetic linkage analysis identified two GTCS-associated loci, Ner1 on Chr 1 and Ner3 on Chr 5. The wild-type Ner1 and Ner3 alleles suppressed GTCS when combined in double-locus congenic lines, but not when present in single-locus congenic lines. Global expression analysis revealed that cholecystokinin B receptor (Cckbr) and suppressor of tumorigenicity 5 (St5), which map within Ner1, and PHD finger protein 24 (Phf24), which maps within Ner3, were significantly downregulated in NER. De novo BAC sequencing detected an insertion of an endogenous retrovirus sequence in intron 2 of the Phf24 gene in the NER genome, and PHF24 protein was almost absent in the NER brain. Phf24 encodes a Gαi-interacting protein involved in GABAB receptor signaling pathway. Based on these findings, we conclude that Cckbr, St5, and Phf24 are strong candidate genes for GTCS in NER.

Genetic profiling of two phenotypically distinct outbred rats derived from a colony of the Zucker fatty rats maintained at Tokyo Medical University.

The Zucker fatty (ZF) rat is an outbred rat and a well-known model of obesity without diabetes, harboring a missense mutation (fatty, abbreviated as fa) in the leptin receptor gene (Lepr). Slc:Zucker (Slc:ZF) outbred rats exhibit obesity while Hos:ZFDM-Leprfa (Hos:ZFDM) outbred rats exhibit obesity and type 2 diabetes. Both outbred rats have been derived from an outbred ZF rat colony maintained at Tokyo Medical University. So far, genetic profiles of these outbred rats remain unknown. Here, we applied a simple genotyping method using Ampdirect reagents and FTA cards (Amp-FTA) in combination with simple sequence length polymorphisms (SSLP) markers to determine genetic profiles of Slc:ZF and Hos:ZFDM rats. Among 27 SSLP marker loci, 24 loci (89%) were fixed for specific allele at each locus in Slc:ZF rats and 26 loci (96%) were fixed in Hos:ZFDM rats, respectively. This indicates the low genetic heterogeneity in both colonies of outbred rats. Nine loci (33%) showed different alleles between the two outbred rats, suggesting considerably different genetic profiles between the two outbred rats in spite of the same origin. Additional analysis using 72 SSLP markers further supported these results and clarified the profiles in detail. This study revealed that genetic profiles of the Slc:ZF and Hos:ZFDM outbred rats are different for about 30% of the SSLP marker loci, which is the underlying basis for the phenotypic difference between the two outbred rats.

Genetic Relatedness of WNIN and WNIN/Ob with Major Rat Strains in Biomedical Research.

WNIN (Wistar/NIN) is an inbred rat strain maintained at National Institute of Nutrition (NIN) for more than 90 years, and WNIN/Ob is an obese mutant originated from it. To determine their genetic relatedness with major rat strains in biomedical research, they were genotyped at various marker loci. The recently identified markers for albino and hooded mutations which clustered all the known albino rats into a single lineage also included WNIN and WNIN/Ob rats. Genotyping using microsatellite DNA markers and phylogenetic analysis with 49 different rat strains suggested that WNIN shares a common ancestor with many Wistar originated strains. Fst estimates and Fischer's exact test suggest that WNIN rats differed significantly from all other strains tested. WNIN/Ob though shows hyper-leptinemia, like Zucker fatty rat, did not share the Zucker fatty rat mutation. The above analyses suggest WNIN as a highly differentiated rat strain and WNIN/Ob a novel obese mutant evolved from it.

Slc:Wistar outbred rats show close genetic similarity with F344 inbred rats.

Although Slc:Wistar rats are used widely in biomedical research as outbred rats, close similarities in growth curves, survival rates, and immunological and biochemical phenotypes have been reported between Slc:Wistar and F344 inbred rats. We reported previously that nine genetic variations that were fixed in Slc:Wistar rats had identical genotypes in F344 rats. Here, we examined the genetic characteristics of Slc:Wistar rats using 27 simple-sequence length polymorphism (SSLP) markers and compared them with other Wistar stocks available in Japan and with some F344 strains. Among 27 SSLP loci, 23 (85%) were fixed in the Slc:Wistar rats, which was the highest among the other Wistar stocks. The 23 fixed loci shared identical genotypes with corresponding loci in F344 rats. Further, the predominant allele types in the unfixed loci had allele frequencies as high as 80%, and these alleles were identical in the F344 rats. When the nine genetic variations reported previously are added, a total of 32 (89%) out of the 36 loci examined were fixed and identical in the Slc:Wistar and F344 rat genomes. These findings indicate the low genetic variation in Slc:Wistar rats and the high genetic similarity between the Slc:Wistar and F344 inbred rats. This study demonstrates the importance of characterizing outbred rats and the need to pay ample attention to the genetic characteristics the Slc:Wistar rats for their proper use.

[A new radiographic technique for detecting avulsion fracture of the lateral malleolus in children].

The objectives of this study were to determine the usefulness of a newly-developed distal fibular axial view radiography modified method for depicting avulsion fracture of the lateral malleolus in children from a functional anatomy viewpoint. Conventional radiography was applied to sixty-nine avulsion fracture suspected ankles of 67 children. Average age and standard deviation at injury were 8.00 and 1.46, respectively. We compared the ability of the modified method to detect avulsion fractures of the lateral malleolus with those could not be depicted using the conventional method, and noted that 42 avulsion fractures (60.9%) could be depicted using the conventional method. We applied the modified method to 27 joints that the conventional method had diagnosed as normal. Of these, the modified method detected 13 avulsion fractures (48.1%). In conclusion, the modified radiography method made it possible to depict avulsion fracture of the lateral malleolus in cases that resisted detection by the conventional method.

Origins of albino and hooded rats: implications from molecular genetic analysis across modern laboratory rat strains.

Albino and hooded (or piebald) rats are one of the most frequently used laboratory animals for the past 150 years. Despite this fact, the origin of the albino mutation as well as the genetic basis of the hooded phenotype remained unclear. Recently, the albino mutation has been identified as the Arg299His missense mutation in the Tyrosinase gene and the hooded (H) locus has been mapped to the ∼460-kb region in which only the Kit gene exists. Here, we surveyed 172 laboratory rat strains for the albino mutation and the hooded (h) mutation that we identified by positional cloning approach to investigate possible genetic roots and relationships of albino and hooded rats. All of 117 existing laboratory albino rats shared the same albino missense mutation, indicating they had only one single ancestor. Genetic fine mapping followed by de novo sequencing of BAC inserts covering the H locus revealed that an endogenous retrovirus (ERV) element was inserted into the first intron of the Kit gene where the hooded allele maps. A solitary long terminal repeat (LTR) was found at the same position to the ERV insertion in another allele of the H locus, which causes the so called Irish (h(i)) phenotype. The ERV and the solitary LTR insertions were completely associated with the hooded and Irish coat patterns, respectively, across all colored rat strains examined. Interestingly, all 117 albino rat strains shared the ERV insertion without any exception, which strongly suggests that the albino mutation had originally occurred in hooded rats.

Role of novel rat-specific Fc receptor in macrophage activation associated with crescentic glomerulonephritis.

Crescentic glomerulonephritis (Crgn) is a complex disease where the initial insult is often the glomerular deposition of antibodies against intrinsic or deposited antigens in the glomerulus. The role of Fc receptors in the induction and progression of Crgn is increasingly recognized, and our previous studies have shown that copy number variation in Fcgr3 partially explains the genetic susceptibility of the Wistar-Kyoto (WKY) rat to nephrotoxic nephritis, a rat model of Crgn. The Fcgr3-related sequence (Fcgr3-rs) is a novel rat-specific Fc receptor with a cytoplasmic domain 6 amino acids longer than its paralogue, Fcgr3. The Fcgr3-rs gene is deleted from the WKY rat genome, and this deletion is associated with enhanced macrophage activity in this strain. Here, we investigated the mechanism by which the deletion of Fcgr3-rs in the WKY strain leads to increased macrophage activation. By lentivirus-mediated gene delivery, we generated stably transduced U937 cells expressing either Fcgr3-rs or Fcgr3. In these cells, which lack endogenous Fcgr3 receptors, we show that Fcgr3-rs interacts with the common Fc-γ chain but that Fc receptor-mediated phagocytosis and signaling are defective. Furthermore, in primary macrophages, expression of Fcgr3-rs inhibits Fc receptor-mediated functions, because WKY bone marrow-derived macrophages transduced with Fcgr3-rs had significantly reduced phagocytic activity. This inhibitory effect on phagocytosis was mediated by the novel cytoplasmic domain of Fcgr3-rs. These results suggest that Fcgr3-rs may act to inhibit Fcgr3-mediated signaling and phagocytosis and could be considered as a novel mechanism in the modulation of Fc receptor-mediated cell activation in autoimmune diseases.

[Attitude survey of radiological technologists toward qualification acquisition of licentiate and degrees in Mie prefecture].

We conducted a questionnaire consciousness survey concerning qualification acquisition of licentiate and degrees among 406 radiological technologists working for medical facility in Mie Prefecture. We employed a mail-back questionnaire method. Then we analyzed relationships between progressive purpose and gender, age, and membership of the Japanese Society of Radiological Technology (JSRT). Results derived note the most common licentiate the participants wanted to acquire was the license concerning emergency medical care. On the other hand, they had limited interest in radiation therapy, nuclear medicine areas and the working environment measurement expert class I. In conclusion, the results of this study revealed the present condition that the necessity of qualification acquisition of licentiate is not recognized incorrectly. In addition, there was a mountain of issues for working radiological technologists to attend graduate school to get degrees. It is important to utilize the results of this study to enlighten their consciousness for limited interest licentiate, and interest them in attending graduate schools.

[An investigation of the effect of gamma rays emitted by patients undergoing radionuclide bone scintigraphy in computed radiography during X-ray mammography examination].

For convenience of outpatients, mammographies of outpatients are often taken after the injection of a radionuclide. In this study, we investigated the effects of gamma rays emitted by a patient onto imaging plates (IPs). We used a flat container filled with (99m)Tc solution as a planar source to irradiate gamma rays onto IPs. We changed irradiation times on each IP, and took radiographies of an ACR-specified 156 model phantom and AGH-D210F phantom. We evaluated radiography images, using visual evaluation, and profile curves, histograms, and CNR and RMS granularities analyses. The results indicated that the depiction ability of a fibrous part began to fall when the irradiation time exceeded 3 minutes. With an increase in irradiation time, an increase in pixel value and RMS granularity value and a decrease in CNR value were observed. In conclusion, IP exposed by gamma rays influenced the evaluation of phantom images.

A novel middle-wavelength opsin (M-opsin) null-mutation in the retinal cone dysfunction rat.

The disease-causing gene which underlies a naturally occurring X-linked mutant cone dysfunction Sprague-Dawley rat model was investigated. Full-field electroretinogram (ERG) and simple sequence length polymorphism analyses were applied to 441-second filial generation rats that were derived from crossing a mutant rat and a Brown-Norway rat. After identifying a mutation mapping within the telomeric region of chromosome X, a candidate gene related to retinal cone function in this region was further screened using real-time PCR, immunohistochemistry and histological methods. The results showed that a G-to-T substitution at the splice acceptor site of intron 4 was present in the opsin 1, medium-wave sensitive (Opn1mw) gene, thereby causing down-regulated transcription and translation. These changes were consistent with abnormities seen in the ERG response. However, there was no significant histological change in the mutant rat retina. Therefore, we infer from this that the causative gene for the mutation is Opn1mw and consequently term this a middle-wavelength opsin cone dysfunction (MCD) rat model. The deficiency in vision of the MCD rat is similar to the color vision defects that occur in humans with a color vision defect but without recessive retinal degeneration. This rat model may be useful for understanding the mechanism that is responsible for color vision and for developing clinical therapies for several retinal dystrophies caused by cone opsin deficiencies.

National BioResource Project-Rat and related activities.

In order to establish a system to facilitate the systematic collection, preservation, and provision of laboratory rats (Rattus norvegicus) and their derivates, the National BioResource Project-Rat (NBRP-Rat) was launched in July 2002. By the end of 2008, more than 500 rat strains had been collected and preserved as live animals, embryos, or sperm. These rat resources are supplied to biomedical scientists in Japan as well as in other countries. This review article introduces NBRP-Rat and highlights the phenome project, recombinant inbred strains, BAC clone libraries, and the ENU-mutant archive, named the Kyoto University Rat Mutant Archive (KURMA). The future direction of rat resources are also discussed.

An informative set of SSLP markers and genomic profiles in the rat MHC, the RT1 complex.

Almost 10,000 single nucleotide polymorphisms (SNPs) had been identified in the RT1 complex, the major histocompatibility complex of the rat, but less than approximately 0.5% have been characterized. In the context of the incomplete characterization of most SNPs, simple sequence length polymorphism (SSLP) marker development is still valuable for understanding the involvement of genes in the RT1 in controlling disease susceptibility, since SSLPs are user-friendly and cost-effective genetic markers in rat genome analysis. In this study, we developed a set of 67 SSLP markers, including 57 novel markers, to cover the entire RT1 complex and then created genetic profiles across 67 rat strains. These markers are located almost every 50 kb in the RT1 complex and show comparable polymorphism; the average number of alleles was 8.04 +/- 3.44 and the average polymorphic rate was 71 +/- 23%. Interestingly, markers failing to amplify polymerase chain reaction products were highly observed in all strains except for BN/SsNHsd, which suggests the existence of highly variable genomic sequences or genomic rearrangements in the RT1 region across rat strains. Based on the phylogenic tree and individual genotyping data, we identified 28 SSLP marker haplotypes in the RT1 region that roughly consisted of three genomic regions. These findings provided new insight into the genomic organization of the RT1 complex and we recognized the need of additional RT1 genome sequences in different strains. Owing to the accuracy and ease of determination, PCR-based SSLP genotyping could replace serological typing in genetic analyses and characterization of rat major histocompatibility.

Three-dimensional stereotactic ROI template for measuring regional cerebral blood flow in 99mTc-ECD SPECT: comparison with the manual tracing method.

PURPOSE: The three-dimensional stereotactic region of interest template (3DSRT) is computer software, which enables the automatic measurement of regional cerebral blood flow (rCBF). This study was undertaken to compare the rCBF values obtained using the 3DSRT method and the conventional manual tracing method. MATERIALS AND METHODS: Twelve patients with normal brains who underwent technetium-99m L,L-ethyl cysteinate dimer single-photon emission computed tomography studies were enrolled in this study. The brains were divided into 12 segments in each hemisphere, and rCBF was measured in each segment. The regions of interest were automatically placed in the segments in the 3DSRT method and were manually traced by five nuclear medicine technicians in the manual tracing method. The rCBF values obtained were compared between the two methods. Interoperator reliability was evaluated in the manual tracing method. RESULTS: The rCBF values were significantly higher in the manual tracing method than in the 3DSRT method in all segments except for the angular segment. A good correlation was seen between the two methods in the rCBF values in 10 (83.3%) of the 12 brain segments (range of coefficient of determinations: 0.73-0.94). A poor correlation, however, was seen in the pericallosal (0.50) and hippocampal (0.53) segments. Interoperator reliability was lower in these two segments than in the other segments in the manual tracing method. CONCLUSION: Although the rCBF values obtained using the manual tracing method and the 3DSRT method show good correlation in most segments, care is required when comparing the results for the pericallosal and hippocampal segments.

Functional polymorphisms in inbred rat strains and their allele frequencies in commercially available outbred stocks.

Polymorphisms that have been proven to influence gene functions are called functional polymorphisms. It is significant to know the distribution of functional polymorphisms in the rat, widely used in animal models for human diseases. In this study, we assessed 16 functional polymorphisms consisting of 3 coat color and 13 disease-associated genes in 136 rat strains, as a part of the genetic profiling program of the National Bio Resource Project for the Rat (NBRP-Rat). Polymorphisms of Cdkn1a, Fcgr3, Grp10, Lss, and Fdft1, which were proven to function in prostate tumorigenesis, glomerulonephritis, hyperphagia, and cholesterol biosynthesis, were shared among various inbred strains. These findings indicated that most rat strains harbored the disease-associated alleles and suggested that many unidentified functional polymorphisms might exist in inbred rat strains. The functional polymorphisms shared in inbred strains were also observed within outbred stocks available commercially. Therefore, this implies that experimental plans based on either rat inbred strains or outbred stocks need to be carefully designed with a full understanding of the genetic characteristics of the animals. To select the most suitable strains for experiments, the NBRP-Rat will periodically improve and update the genetic profiles of rat strains.

Positron emission tomography analysis of the analgesic effects of acupuncture in rhesus monkeys.

The purpose of this study was to examine whether pain-induced brain activation was suppressed by acupuncture analgesia. We investigated the suppression of the pain-induced neuronal activation in specific brain areas of three male rhesus monkeys (aged four years old) using positron emission tomography (PET), in which changes in the regional cerebral blood flow (rCBF) were examined as an index of the neuronal activation. The brain areas such as the thalamus, insula and anterior cingulate cortex were activated by heating the tail of monkeys in 47 degrees C water compared to the heating at 37 degrees C. Those activations were suppressed by electroacupuncture (EA) with a 2 sec alteration of the frequency of 4 Hz/60 Hz at the right ST36 (the upper anterior tibial muscle) and the right LI4 (the back palm between the first and second metacarpal) acupoints. Meanwhile, this EA analgesic effect was confirmed by prolonging the tail withdrawal latencies from hot water in the temperature range from 45 to 50 degrees C.These brain areas were corresponded to the pain-related areas as reported in previous studies. In conclusion, we were able to visualize the acupuncture analgesia in the CNS. We also detected the brain areas activated or inactivated by acupuncture. The areas that responded to acupuncture stimulation at 47 degrees C water were different from the regions at 37 degrees C. We consider that this difference in the response to acupuncture may support the variation of the clinical efficacy of acupuncture in patients bearing pain or other disorders.

A set of highly informative rat simple sequence length polymorphism (SSLP) markers and genetically defined rat strains.

BACKGROUND: The National Bio Resource Project for the Rat in Japan (NBRP-Rat) is focusing on collecting, preserving and distributing various rat strains, including spontaneous mutant, transgenic, congenic, and recombinant inbred (RI) strains. To evaluate their value as models of human diseases, we are characterizing them using 109 phenotypic parameters, such as clinical measurements, internal anatomy, metabolic parameters, and behavioral tests, as part of the Rat Phenome Project. Here, we report on a set of 357 simple sequence length polymorphism (SSLP) markers and 122 rat strains, which were genotyped by the marker set. RESULTS: The SSLP markers were selected according to their distribution patterns throughout the whole rat genome with an average spacing of 7.59 Mb. The average number of informative markers between all possible pairs of strains was 259 (72.5% of 357 markers), showing their high degree of polymorphism. From the genetic profile of these rat inbred strains, we constructed a rat family tree to clarify their genetic background. CONCLUSION: These highly informative SSLP markers as well as genetically and phenotypically defined rat strains are useful for designing experiments for quantitative trait loci (QTL) analysis and to choose strategies for developing new genetic resources. The data and resources are freely available at the NBRP-Rat web site 1.

WTC deafness Kyoto (dfk): a rat model for extensive investigations of Kcnq1 functions.

KCNQ1 forms K+ channels by assembly with regulatory subunit KCNE proteins and plays a key role in the K+ homeostasis in a variety of tissues. In the heart, KCNQ1 is coassembled with KCNE1 to produce a cardiac delayed rectifier K+ current. In the inner ear, the KCNQ1/KCNE1 complex maintains the high concentration of K+ in the endolymph. In the stomach, KCNQ1 is coassembled with KCNE2 to form the K+ exflux channel that is essential for gastric acid secretion. In the colon and small intestine, KCNQ1 is coassembled with KCNE3 to play an important role in transepithelial cAMP-stimulated Cl- secretion. For further understanding of Kcnq1 function in vivo, an animal model has been required. Here we reported the identification of a coisogenic Kcnq1 mutant rat, named deafness Kyoto (dfk), and the characterization of its phenotypes. WTC-dfk rats carried intragenic deletion at the Kcnq1 gene and showed impaired gain of weight, deafness, and imbalance resulting from the marked reduction of endolymph, prolonged QT interval in the electrocardiogram (ECG), and gastric achlorhydria associated with hypertrophic gastric mucosa. Surprisingly, WTC-dfk rats showed hypertension, which suggested that Kcnq1 might be involved in the regulation of blood pressure. These findings suggest that WTC-dfk rats could represent a powerful tool for studying the physiological functions of KCNQ1 and for the establishment of new therapeutic procedures for Kcnq1-related diseases.

Glucose concentration-dependent potentiation of insulin secretion by a new chemical entity, KCP256.

The insulinotropic activity of KCP256 [(R)-8-benzyl-2-cyclopentyl-7, 8-dihydro-4-propyl-1H-imidazo[2,1-i]purin-5(4H)-one hydrochloride] was examined using MIN6 cells (a pancreatic beta-cell line) and pancreatic islets isolated from rats. Unlike sulfonylurea anti-diabetic drugs, KCP256 dose-dependently (0.1-10 microM) enhanced insulin secretion from MIN6 cells and its insulinotropic effect was exerted only at high concentrations of glucose (8.3-22 mM) but not at low concentrations of glucose (3.3-5.5 mM). Furthermore, the action mechanism of KCP256 was different because, unlike sulfonylurea drugs, KCP256 did not displace the binding of [3H]glibenclamide, and did not inhibit the 86Rb+ efflux nor K(ATP) channel activity. In isolated islets, KCP256 also enhanced insulin secretion in a dose- and a glucose-concentration-dependent manner. Plasma levels of insulin after glucose challenge in KCP256-administrated rats were higher than those in vehicle-administrated animals, indicating that KCP256 can enhance insulin secretion in vivo. Since the insulinotropic activity of KCP256 only occurs at high concentrations of glucose, this novel drug may exhibit a decreased risk of drug-induced hypoglycemia compared with sulfonylurea drugs when treating patients with diabetes.