Dystrophin-deficient muscular dystrophy in a Toy Poodle with a single base pair insertion in exon 45 of the Duchenne muscular dystrophy gene.

A 10-month-old, intact male Toy Poodle was referred for a postural abnormality. Blood biochemical tests revealed a marked increase in plasma creatine phosphokinase (CPK) concentration. The isoenzyme test showed that 99% of serum CPK consisted of CPK-MM. Histopathological evaluation of muscle biopsy samples confirmed scattered degeneration and necrosis of myofibers. Immunohistochemistry for dystrophin showed an absence of staining in muscle cells. Based on these findings, the dog was diagnosed with dystrophin-deficient muscular dystrophy. Whole genome sequencing using genomic DNA extracted from blood revealed a single base pair insertion in exon 45 of the Duchenne muscular dystrophy (DMD) gene. This is the first report on muscular dystrophy in Toy Poodles and identified a novel mutation in the DMD gene.

Retrospective evaluation of nimustine use in the treatment of feline lymphoma.

BACKGROUND: Nimustine, similar to lomustine, is an alkylating agent from the nitrosourea family. There have been some reports regarding lomustine treatment for tumour-bearing cats. However, information regarding nimustine treatment for tumour-bearing cats is limited. OBJECTIVES: To retrospectively evaluate adverse events and clinical outcomes in tumour-bearing cats receiving nimustine. METHODS: Information regarding diagnosis, treatment condition, adverse events, and clinical outcomes was collected in tumour-bearing cats receiving nimustine through reviews of medical records. RESULTS: Nine cats with lymphoma were treated with nimustine in the primary therapy (n = 2) and in the rescue therapy (n = 7). Median starting dose of nimustine was 25 mg/m2 (range: 20-30 mg/m2 ) with dosing interval of three weeks and 1-11 administrations. Adverse events were mild gastrointestinal toxicity (grade 1) including diarrhoea (n = 2) and vomiting (n = 2) and mild myelosuppression (grade 1 or 2) including thrombocytopenia (n = 3) and neutropenia (n = 1). No severe adverse events were observed. Progression-free survival durations among cats receiving nimustine in the primary therapy and in the rescue therapy were 274-688 days (median: 481 days) and 9-671 days (median: 102 days), respectively. Overall survival durations among cats receiving nimustine in the primary therapy and in the rescue therapy were 275-745 days (median: 510 days) and 14-671 days (median: 109 days), respectively. CONCLUSIONS: Nimustine was well tolerated and showed clinical outcomes similar to lomustine in cats with lymphoma. These findings suggest that nimustine might be an alternative to lomustine in the treatment of feline lymphoma.

The Establishment of an Optimal Protocol for Contrast-Enhanced Micro-Computed Tomography in the Cloudy Catshark Scyliorhinus torazame.

The purpose of this study was to determine the optimal imaging protocol for contrast-enhanced computed tomography (CECT) using micro-CT (µ-CT) for the posterior cardinal vein (PCV), dorsal aorta (DA), hepatic portal vein (HPV), kidney, liver, cephalic arteries (CAs), and gills of Cloudy Catsharks Scyliorhinus torazame. Additionally, we examined the availability of CECT screening for the coelomic organs. Different doses of iopamidol (100, 300, 500, and 700 mg iodine [mgI]/kg) were administered intravenously for 20 s in six sharks. The CT scans from the pectoral girdle to the pelvic girdle were performed at 0-600 s after administration. Contrast-enhanced CT imaging of the CAs, gills, and coelomic organs was examined. Assessment of the signal enhancement value revealed that the PCV was easily visualized with all contrast doses at 25 s. The CAs, gills, and DA were visible at a slightly higher dose (CAs and gills: 200 mgI/kg at 40 s; DA: 300 mgI/kg at 50 s). The HPV was obvious at a dose of at least 500 mgI/kg after a 150-s delay. The parenchyma of the kidney had a contrast effect at 300 mgI/kg, 150 s after the contrast effect of the renal portal system disappeared. The liver, which stores a lot of lipids, had poor overall contrast enhancement that was optimized at the highest dose of 700 mgI/kg. Contrast-enhanced CT screening at 700 mgI/kg and 150 s is likely to obtain the optimal imaging of the reproductive organs, such as the ovary, oviducal gland, uterus, and testis. The present findings can be applied not only to clinical practice but also to academic research and education on elasmobranchs in aquariums.

Canine idiopathic chylothorax: Anatomic characterization of the pre- and postoperative thoracic duct using computed tomography lymphography.

Surgical treatment has improved the prognosis of canine idiopathic chylothorax, although a recurrence of the disease occurs occasionally after the procedure. An improved understanding of possible causes for this recurrence would be helpful for prognosis and treatment planning in affected patients. In this retrospective case series study, we described the detailed pre- and postoperative computed tomographic lymphography (CTLG) imaging characteristics for a group of dogs with surgically confirmed idiopathic chylothorax. Preoperative CTLG was performed in 12 of 14 dogs diagnosed with idiopathic chylothorax. Thoracic ducts were present on the right side in 10 dogs, left side in one dog, and bilaterally in one dog. All the 14 dogs received a combination therapy of pericardiectomy and thoracic duct ligation (TDL) by video-assisted thoracoscopic surgery. One week after surgery, a postoperative CTLG was performed, and the thoracic ducts were apparent in seven of 14 dogs. Three dogs had an unchanged course of the thoracic duct, which could have resulted from a missed duct. Four dogs were identified as having a bypass formation: the oblique duct originated at the ligation site and connected to the duct on the other side. Our findings indicated that one of the possible causes for postoperative recurrence of chylothorax in dogs could be "invisible or sleeping" fine ducts that are collapsed and not visible in preoperative CTLG scans. After TDL causes a change in the pressure of lymphatic flow, these fine thoracic ducts may become apparent using postoperative CTLG.

HSP110 expression in canine mammary gland tumor and its correlation with histopathological classification and grade.

Heat shock proteins (HSPs) play critical roles as molecular chaperones, thereby promoting cellular homeostasis. HSPs are overexpressed in many types of human tumors and their serum concentration is elevated in cancer patients. Recent studies have suggested that HSPs may promote tumorigenesis via interactions with tumor-related proteins. There are only a few studies that address the expression of HSPs in canine tumors. In our previous study, we identified elevated levels of HSP110 expression in canine mammary gland tumors (cMGTs). In this study, we examined both serum concentrations and tissue expression of HSP110 in dogs with cMGT. We found that serum HSP110 concentrations were not significantly different in a comparison between dogs with cMGT (3.44 ± 1.27 µg/mL) and healthy controls (3.23 ± 1.18 µg/mL). By contrast, significant differences in levels of HSP110 expression were identified in comparisons between simple carcinoma and benign mixed tumor (p = 0.001), simple carcinoma and non-neoplastic lesions (p < 0.001), complex carcinoma and benign mixed tumor (p = 0.015), complex carcinoma and non-neoplastic lesions (p < 0.001), simple adenoma and benign mixed tumor (p = 0.041), and simple adenoma and non-neoplastic lesions (p = 0.007). Similarly, significantly different levels of HSP110 expression were identified when comparing grade Ⅲ with non-neoplastic lesion (p = 0.026), grade Ⅱ with benign tumor (p = 0.015), grade Ⅱ with non-neoplastic lesion (p < 0.001), and grade Ⅰ with non-neoplastic lesion (p < 0.001). Taken together, our results indicate that expression of HSP110 correlates with the malignancy in this cohort of dogs diagnosed with cMGT. These findings also suggest that HSP110 is associated with tumorigenesis and the relative malignancy of cMGT.

Efficacy of en bloc thoracic duct ligation in combination with pericardiectomy by video-assisted thoracoscopic surgery for canine idiopathic chylothorax.

OBJECTIVE: To compare the outcomes of pericardiectomy performed with conventional clipping thoracic duct ligation (C-TDL) to those with en bloc thoracic duct ligation (EB-TDL) using video-assisted thoracoscopic surgery (VATS) for canine idiopathic chylothorax. STUDY DESIGN: Retrospective consecutive case series. ANIMALS: Thirteen client-owned dogs with idiopathic chylothorax. METHODS: Medical records of dogs treated with pericardiectomy in combination with TDL by VATS without intraoperative contrast were reviewed. Five and seven dogs underwent C-TDL and EB-TDL, respectively, and 11 dogs were evaluated by preoperative and 7- to 10-days-postoperative computed tomography-lymphography (CTLG). No clinical symptoms with absent or minimal pleural effusion was defined as clinical improvement. Long-term remission (LTR) was defined as rapid resolution of pleural effusion and no recurrence for more than 1 year. Anesthesia time, operation time, the duration of hospitalization, and time until pleural effusion resolution were compared. RESULTS: Clinical improvement was achieved in 91.7% of the cases (C-TDL, 4/5; EB-TDL, 7/7), excluding one case of intraoperative death. The LTR rate was significantly higher with EB-TDL (6/7 [85.7%]) than with C-TDL (1/5 [20%]). Anesthesia time, operation time, and time until pleural effusion resolution were significantly better with EB-TDL than with C-TDL. The rates of thoracic ducts visualization by postoperative CTLG were 100% (5/5) with C-TDL and 42.9% (3/7) with EB-TDL. CONCLUSION: En bloc TDL was an effective treatment for canine idiopathic chylothorax in this patient population. It compared favorably to C-TDL, although missed branches at the time of surgery may explain the difference between C-TDL and EB-TDL in this small population of cases. CLINICAL SIGNIFICANCE: En bloc TDL by VATS was an effective minimally invasive treatment for canine idiopathic chylothorax. Computed tomography-lymphography can be used for surgical planning and postoperative evaluation.

Correlation between toll-like receptor 4 and nucleotide-binding oligomerization domain 2 (NOD2) and pathological severity in dogs with chronic gastrointestinal diseases.

Toll-like receptor 4 (TLR4), nucleotide-binding oligomerization domain 2 (NOD2), and TNF-α play important roles in human inflammatory bowel diseases. The aim of this study was to elucidate the relationship between Toll-like receptor 4, NOD2, and TNF-α and the severity of chronic gastrointestinal diseases in dogs. We examined the expression levels of TLR4, NOD2, and TNF-α in the stomach, duodenum, ileum, colon, and rectum obtained from 21 dogs with chronic gastrointestinal disease, including inflammatory bowel disease, high-grade lymphoma, food responsive enteropathy, chronic pancreatitis, low-grade lymphoma, inflammatory colorectal polyp, and chronic colitis. Next, we demonstrated whether there is good correlation between the expression levels of TLR4, NOD2, and TNF-α and the histopathological analysis of each sample. We found that the level of TLR4 expression in the ileum of dogs with chronic gastrointestinal disease was positively associated with the histopathological severity. We also found that the level of NOD2 expression in the duodenum, stomach, and rectum was positively associated with the histopathological severity. However, there was no correlation between TNF-α expression in the 5 regions tested in this study and the histopathological severity. These findings indicate that TLR4 and NOD2 are remarkably associated with the severity of chronic gastrointestinal disease in dogs.

Prevalence of microorganisms associated with feline gingivostomatitis.

OBJECTIVES: Feline gingivostomatitis (FGS) is a painful chronic inflammatory disease of the oral cavity. The purpose of this study was to examine the frequency of detection of certain common feline bacteria and viruses to determine any potential associations with FGS. METHODS: A multicentre case-control study design was conducted. In total, 72 control cats and 32 cats with FGS were included in the study. Oral swabs were cultured for bacterial identification and a PCR assay was carried out to examine the infection of feline calicivirus (FCV), feline herpesvirus-1 (FHV-1), Chlamydia felis, Mycoplasma felis and Bordetella bronchiseptica. RESULTS: There was a significant difference in age distribution between the control and the FGS group. Based on a PCR assay, the positive rate of FCV was significantly higher in FGS cats than control animals. For other infectious pathogens, including FHV-1, C felis and M felis, there was no significant difference. Bacterial culture of oral swabs revealed that Pasteurella multocida was most frequently detected, but the detection rate was significantly lower in FGS cats. In FGS cats, the incidence of Enterococcus faecalis and anaerobic bacteria were more frequently isolated than in control cats. CONCLUSIONS AND RELEVANCE: This study indicates that the positive rate of FCV was significantly higher in cats with FGS, and the microflora of the oral cavity of cats with FGS might be disrupted, although additional studies are required to compare the oral microbiome in cats of a variety of ages.

Report of fatal mixed infection with Cryptosporidium parvum and Giardia intestinalis in neonatal calves.

In the production and management of beef and dairy cattle, controlling diarrhea is one of the important concerns. Pathogenic agents of the disease, protozoan parasites including Cryptosporidium spp., are difficult to control, making prevention, diagnoses, and treatment of diarrhea. In the present study, we investigated a farm with a history of calf deaths over a period of 10 years in order to determine the cause of disease and to clarify the detailed distribution of the pathogens. In four examined calves that were reared in calf pens, all were positive with Cryptosporidium and/or Giardia, while the other breeding stock and adult cattle were negative. Molecular analyses revealed that the isolates from calves were C. parvum subtype IIaA15G2R1 as a zoonotic and G. intestinalis assemblage E. Other pathogenic bacteria and diarrhea-causing viruses were not detected. After treating the calf pens with boiling water and milk of lime (Ca[OH]2), oocysts of C. parvum and cysts of G. intestinalis were not found and no additional calves died. This is the first report to describe the mixed infection of both parasites in Japan.

Localization of heat shock protein 110 in canine mammary gland tumors.

Heat shock proteins (HSPs) function as molecular chaperones in the regulation of protein folding, conformation, and assembly; in addition, they also protect cells from protein-protein aggregation resulting from cellular stress. Recently, HSPs were shown to be overexpressed in several human cancer cells compared with normal cells. HSPs are considered to be related to apoptosis-associated proteins, and inhibition of apoptosis promotes tumor growth. Canine mammary gland tumors have received a great deal of attention from researchers due to the many common biological and histological characteristics that they share with human tumors. We previously confirmed that HSP110 is a canine mammary gland tumor antigen and reported that HSP110 mRNA expression significantly increased in tumor tissue. We have now created a functional recombinant canine HSP110 protein and a rabbit anti-HSP110 polyclonal antibody. This recombinant protein can refold heat-denatured firefly luciferase at 42°C. Immunohistochemical analysis showed that HSP110 was mainly localized in the cytoplasm of epithelial and interstitial cells in canine mammary gland tumors. Extensive genomic research has revealed genetic similarities between humans and dogs; comparative oncological studies between these species have made remarkable progress. The results reported here contribute valuable oncological knowledge for the development of novel therapeutic methods in both veterinary science and human medicine.

Characteristics of bone fractures and usefulness of micro-computed tomography for fracture detection in rabbits: 210 cases (2007-2013).

OBJECTIVE: To characterize bone fractures and the usefulness of micro-CT for imaging fractures in pet rabbits. DESIGN: Retrospective case series. ANIMALS: 210 client-owned rabbits with bone fractures. PROCEDURES: Medical records of rabbits evaluated for bone fractures from 2007 through 2013 were examined. Information was collected on signalment and nature of fractures, and radiographic and micro-CT images of fractures were reviewed. RESULTS: Almost half (n = 95 [47.7%]) of fractures were in rabbits < 3 years old. Accidental fall was the most common cause. Vertebral fracture was the most common type of fracture with a nonneoplastic cause (n = 46 [23.2%]) and was most common in the L4-L7 region. The tibia was the most common site for limb fracture among all fractures with a nonneoplastic cause (45 [22.7%]). Twelve (5.7%) fractures had a neoplastic cause, and 7 of these were associated with metastatic uterine adenocarcinoma. Females were significantly more likely to have a fracture caused by neoplasia than were males. Compared with radiography, micro-CT provided more detailed fracture information, particularly for complicated fractures or structures (eg, skull, pelvic, vertebral, and comminuted limb fractures). CONCLUSIONS AND CLINICAL RELEVANCE: Findings were useful for understanding the nature of fractures in pet rabbits and supported the use of micro-CT versus radiography for fracture detection and evaluation.

Profiling of serum metabolites in canine lymphoma using gas chromatography mass spectrometry.

Canine lymphoma is a common cancer that has high rates of complete remission with combination chemotherapy. However, the duration of remission varies based on multiple factors, and there is a need to develop a method for early detection of recurrence. In this study, we compared the metabolites profiles in serum from 21 dogs with lymphoma and 13 healthy dogs using gas chromatography mass spectrometry (GC-MS). The lymphoma group was separated from the control group in an orthogonal projection to latent structure with discriminant analysis (OPLS-DA) plot using ions of m/z 100-600, indicating that the metabolites profiles in lymphoma cases differed from those in healthy dogs. The lymphoma group was also separated from the control group on OPLS-DA plot using 29 metabolites identified in all serum samples. Significant differences were found for 16 of these metabolites with higher levels in the lymphoma group for 15 of the metabolites and lower levels for inositol. An OPLS-DA plot showed separation of the lymphoma and healthy groups using these 16 metabolites only. These results indicate that metabolites profile with GC-MS may be a useful tool for detection of potential biomarker and diagnosis of canine lymphoma.

Elongation factor-1α is a novel protein associated with host cell invasion and a potential protective antigen of Cryptosporidium parvum.

The phylum Apicomplexa comprises obligate intracellular parasites that infect vertebrates. All invasive forms of Apicomplexa possess an apical complex, a unique assembly of organelles localized to the anterior end of the cell and involved in host cell invasion. Previously, we generated a chicken monoclonal antibody (mAb), 6D-12-G10, with specificity for an antigen located in the apical cytoskeleton of Eimeria acervulina sporozoites. This antigen was highly conserved among Apicomplexan parasites, including other Eimeria spp., Toxoplasma, Neospora, and Cryptosporidium. In the present study, we identified the apical cytoskeletal antigen of Cryptosporidium parvum (C. parvum) and further characterized this antigen in C. parvum to assess its potential as a target molecule against cryptosporidiosis. Indirect immunofluorescence demonstrated that the reactivity of 6D-12-G10 with C. parvum sporozoites was similar to those of anti-ß- and anti-γ-tubulins antibodies. Immunoelectron microscopy with the 6D-12-G10 mAb detected the antigen both on the sporozoite surface and underneath the inner membrane at the apical region of zoites. The 6D-12-G10 mAb significantly inhibited in vitro host cell invasion by C. parvum. MALDI-TOF/MS and LC-MS/MS analysis of tryptic peptides revealed that the mAb 6D-12-G10 target antigen was elongation factor-1α (EF-1α). These results indicate that C. parvum EF-1α plays an essential role in mediating host cell entry by the parasite and, as such, could be a candidate vaccine antigen against cryptosporidiosis.

Phylogenetic identification of Cystoisospora spp. from dogs, cats, and raccoon dogs in Japan.

Cystoisospora spp. from feces in dogs, cats, and raccoon dogs were isolated, sequenced at the small subunit ribosomal RNA gene locus and compared to other Cystoisospora spp. Cystoisospora oocysts from dogs and raccoon dogs were morphologically similar with those of C. ohioensis, and cat isolates were similar with those of C. felis. The sequences from dogs and raccoon dogs, and cats have a homology with C. ohioensis and C. felis, respectively. Phylogenetic analysis of the DNA sequences showed that the dog and raccoon dog isolates were nested in a clade with other Cystoisospora spp. including C. ohioensis, C. belli, and C. orlovi. The cat isolate formed a sister group with C. felis that was a separate clade from the dog and raccoon dog group. We report sequence variation in these Cystoisospora sequences and have identified raccoon dogs as another carnivore host for Cystoisospora spp. infecting dogs.

Effect of low pH on the morphology and viability of Cryptosporidium andersoni sporozoites and histopathology in the stomachs of infected mice.

The genus Cryptosporidium includes many common parasites infecting animals and humans, and is a major cause of diarrheal illness worldwide. The biology of gastric Cryptosporidium spp., including replication in the stomach, has not been well documented. This study evaluated the viability of Cryptosporidium andersoni sporozoites in gastric environments after excystation and examined the endogenous development and histopathological changes in the stomachs of infected mice, using a novel type of C. andersoni. Sporozoites were affected by low pH (61.6% viability after 3h at pH2.0). Electron microscopy revealed developmental parasites on the gastric foveolae but not on the surface of the gastric mucosa. Histopathological examinations at 1, 2, 4 and 12 weeks p.i. uncovered three different lesions. The gastric mucosa of foveolae filled with parasites was extended and the amount of neutral mucopolysaccharide at the mucosal surface was decreased with the first type of lesion. The gastric mucosa was atrophied, some gastric glands were disrupted and the amount of acid mucopolysaccharide at the mucosal surface was increased with the second type. Finally, the gastric mucosa was slightly extended and goblet cells were present in the gastric mucosa, indicating intestinal metaplasia, in the third type. No parasites were detected in these areas with increased acidic mucin and indications of metaplasia. The results suggest that C. andersoni parasites could not survive in acidic environments for a long period before invading host cells and preferentially develop in neutral sites of the gastric mucosa, resulting in histopathological changes and chronic shedding of oocysts.

Survey and molecular characterization of Cryptosporidium and Giardia spp. in owned companion animal, dogs and cats, in Japan.

Compared with other countries, surveys of these parasites have been rarely performed in companion animals of Japan in spite of their significance for public health. Here, we investigated pet dogs and cats in Japan for the first time, and genetically analyzed the isolates to evaluate the risk of zoonotic infections. Seventy-seven fecal samples were collected from privately owned dogs and 55 samples from owned cats in Osaka city, Japan. Cryptosporidium oocysts were identified in 3/77 dogs (3.9%) and 7/55 cats (12.7%), and Giardia infection in 2/77 dogs (2.6%) and 1/55 cats (1.8%). Amplification of the target regions for genotyping was successful, Cryptosporidium isolates in dogs and cats were identified as C. canis and C. felis, respectively, and those of Giardia in dogs and cats were G. intestinalis Assemblages D and F. The discharge period of the oocysts varied within 3-16 weeks and that of the cysts was 12 weeks. To date, zoonotic types of both parasites have been identified in other animals in Japan, and further large-scale studies are needed to determine the distribution of zoonotic genotypes in these animals, especially those closely associated with humans.

Expression of calbindin-D28K in sporadic nephroblastomas of the chicken.

Calbindin-D28K (Ca-D28K) is a calcium-binding protein. In the kidney, Ca-D28K is present in the distal nephron, but not in the proximal nephron. This site-specific distribution in the kidney indicates that Ca-D28K is a potential marker for the differentiation of the distal nephron. In this study, we have examined the expression of Ca-D28K in 25 sporadic cases of chicken nephroblastomas. All cases of nephroblastomas were composed of atypical tubular structures, blastemal cells, and fibrous stroma in varying degrees of differentiation. Immunohistochemically in all nephroblastoma specimens, Ca-D28K was expressed in the epithelial cells of the subsets of tubular structures, but not in the blastema or the stroma. These results suggested that the tubuli in the nephroblastomas are able to differentiate into the phenotype of distal nephrons. Furthermore, Ca-D28K might develop as a novel diagnostic marker for nephroblastomas because this molecule is reported to be completely negative in other renal tumors, including renal cell carcinoma, chromophobe carcinomas, and oncocytoma.

Inhibition of male chick phenotypes and spermatogenesis by Bisphenol-A.

Bisphenol-A (BPA) has been reported to bind to the estrogen receptor (ER) and also to act as a xenoestrogen on the reproductive system of many species. In our previous study, a high dose of BPA disturbed the growth of the comb and testes of male chickens. In this study, the exposure of relatively low doses of BPA on the growth of the male chicken phenotypes was investigated. White Leghorn male chicks were orally administered various doses of BPA (2 microg to 200 mg/kg) from 2 weeks of age, and thereafter the comb, wattle and testes were examined at 5, 10, 15, 20 and 25 weeks of age. Although the body weight showed no significant difference among the birds of all ages, the growth of above organs was significantly affected in the chicks even with a minimal dose of 2-microg BPA. These inhibitory effects appeared in a dose-dependent manner. Histologically, the growth of the testes was negatively affected by exposure to over 20-microg/kg BPA: namely, the development of seminiferous tubuli and spermatogenesis were severely inhibited. The mRNA expressions of ERalpha and the aromatase gene (p450arom) increased in the testes in a dose-dependent manner after BPA administration. Accordingly, even low doses of BPA delayed the growth of the male chicken phenotype either by a direct effect or by an indirect response resulting in an increase in both of the endogenous estrogen levels and hyper-sensitivity to estrogen.

Hypothalamo-pituitary-adrenal gland axis in mice inhaling toluene prior to low-level long-term exposure to formaldehyde.

We studied the change in the hypothalamo-pituitary-adrenal gland (HPA) axis upon adding prior toluene inhalation to our previous formaldehyde inhalation experiments to determine whether short term exposure to relatively high levels of toluene triggers multiple chemical sensitivity (MCS). Data come from immunocytochemical, morphometrical and RT-PCR measurements. Four groups of adult female mice were exposed to differing concentrations (0, 80, 400, and 2,000 ppb) of formaldehyde for 16 hr/day, 5 days/week for twelve weeks, after the mice were exposed intranasally to 500 ppm toluene per mouse for 6 hr/day, for 3 days. We found that the number of corticotropin releasing hormone (CRH)-immunoreactive (ir) neurons was up-regulated according to the amount of formaldehyde as well as inhalation of formaldehyde alone in our previous experiment. The proportion of adrenocorticotropin hormone (ACTH)-ir cells increased according to the formaldehyde concentration, though there was no significant difference between the 400 and 2,000 groups. The number of ACTH-ir cells was higher in the 400 group than in the other groups (0, 80, and 2,000). Expression of ACTH-mRNA was also up-regulated according to the quantity of formaldehyde. The sinusoid in the anterior pituitary showed more dilatation in the 400 and 2,000 groups than in the control group, especially in the 2,000 group. We propose that exposure to toluene prior to inhalation of formaldehyde has no effect on the HPA axis and as a trigger of MCS, although greater sinusoid dilatation was found in the anterior pituitary gland at higher concentrations of formaldehyde.

Molecular cloning and analysis of the mouse gicerin gene.

Gicerin is a cell adhesion molecule, which has five immunoglobulin-like loop structures in an extracellular domain followed by a single transmembrane domain and a short cytoplasmic tail. We have reported that gicerin participates in neurite extension and structural organization of the nervous system, and its expression in the nervous system is high during the development and dramatically decreased after birth. To elucidate the mechanism how the expression of gicerin is regulated, we performed a genomic cloning of a mouse gicerin. A fragment of 16 kbp genomic clone contained 8 kbp gicerin gene composed of 16 exons with 6 kbp upstream region. Genomic cloning revealed that two isoforms of gicerin were generated by an alternative splicing of exon 15 results in cytoplasmic domains composed of either 63 or 21 amino acids. As for an expressional regulation of gicerin, we found that the mRNA content of gicerin in PC12 cells was regulated by cAMP. Quantitative-PCR analysis revealed that forskolin induced four-fold increase of gicerin mRNA. To characterize the involvement of its promoter region, we examined the promoter activity in PC12 cells by a luciferase-reporter assay. We found that a CRE site located at 60 bp upstream of gicerin gene was responsible for the increase of its mRNA induced by forskolin.