Identification of independent APP locus duplication in Japanese patients with early-onset Alzheimer disease.

BACKGROUND: The occurrence of duplications of the amyloid precursor protein gene (APP) has been described in European families with early-onset familial Alzheimer disease (EO-FAD) and cerebral amyloid angiopathy. However, the contribution of APP duplication to the development of AD in other ethnic populations remains undetermined. METHODS: The occurrence of APP duplication in probands from 25 families with FAD and 11 sporadic EO-AD cases in the Japanese population was examined by quantitative PCR and microarray-based comparative genomic hybridisation analyses. APP expression level was determined by real-time quantitative reverse-transcription (RT) PCR analysis using mRNA extracted from the peripheral blood of the patients. RESULTS: We identified APP locus duplications in two unrelated EO-FAD families. The duplicated genomic regions in two patients of these families differed from each other. No APP duplication was found in the late-onset FAD families or sporadic EO-AD patients. The patients with APP duplication developed insidious memory disturbance in their fifties without intracerebral haemorrhage and epilepsy. Quantitative RT-PCR analysis showed the increased APP mRNA expression levels in these patients compared with those in age- and sex-matched controls. CONCLUSIONS: Our results suggest that APP duplication should be considered in patients with EO-FAD in various ethnic groups, and that increased APP mRNA expression level owing to APP duplication contributes to AD development.

Clinical, molecular and histopathological features of short stature syndrome with novel CUL7 mutation in Yakuts: new population isolate in Asia.

BACKGROUND: In total, 43 patients having short stature syndrome in 37 Yakut families with autosomal recessive prenatal and postnatal nonprogressive growth failure and facial dysmorphism but with normal intelligence have been identified. METHODS: Because Yakuts are considered as a population isolate and the disease is rare in other populations, genomewide homozygosity mapping was performed using 763 microsatellite markers and candidate gene approach in the critical region to identify the causative gene for the short stature syndrome in Yakut. RESULTS: All families shared an identical haplotype in the same region as the identical loci responsible for 3-M and gloomy face syndromes and a novel homozygous 4582insT mutation in Cullin 7 (CUL7) was found, which resulted in a frameshift mutation and the formation of a subsequent premature stop codon at 1553 (Q1553X). Yakut patients with short stature syndrome have unique features such as a high frequency of neonatal respiratory distress and few bone abnormalities, whereas the clinical features of the other Yakut patients were similar to those of 3-M syndrome. Furthermore, abnormal vascularisation was present in the fetal placenta and an abnormal development of cartilage tissue in the bronchus of a fetus with CUL7 mutation. CONCLUSION: These findings may provide a new understanding of the clinical diversity and pathogenesis of short stature syndrome with CUL7 mutation.

Stimulative effect of epinephrine on glucose production and utilization rates in sheep using a stable isotope.

The rates of the production and utilization of blood glucose were measured during intravenous epinephrine infusion (2.0 nmol kg-1 min-1 for 1 hr) in sheep. An isotope dilution method with an infusion of [U-13C]glucose and nonsteady-state equations were used for the determination of blood glucose kinetics. Heart rate and concentrations of blood glucose, plasma free fatty acids, and lactate increased (P < 0.01), whereas plasma insulin concentrations tended to decrease (P < 0.06) during epinephrine infusion. The blood glucose turnover rate was 1.9 +/- 0.1 mg kg-1 min-1 before epinephrine infusion. The rate of blood glucose production increased (P < 0.01) to 6.4 +/- 0.5 mg kg-1 min-1 at 20 min after the initiation of epinephrine infusion. The blood glucose utilization rate increased (P < 0.05) gradually and reached 4.1 +/- 1.2 mg kg-1 min-1 at 60 min after the initiation of epinephrine infusion. These results suggest that in sheep, epinephrine stimulates the rates of both the production and the utilization of blood glucose and that hyperglycemia induced by epinephrine infusion is mainly due to a rapid enhancement in the rate of the production of blood glucose.

Chromium supplementation does not influence glucose metabolism or insulin action in response to cold exposure in mature sheep.

An isotope dilution method using [U-(13)C] glucose infusion and a glucose clamp approach were applied to determine the effects of supplemental Cr and cold exposure on blood glucose turnover rate and tissue responsiveness and sensitivity to insulin in eight sheep. The daily profiles of blood metabolites and hormones were also determined. The sheep consumed diets containing either 0 or 1 mg of Cr/kg from a high-Cr yeast and were exposed from a thermoneutral environment (20 degrees C) to a cold environment (0 to 4 degrees C) for 9 d. The experiment used a crossover design. Body weight was lost (P = 0.02) during cold exposure, regardless of Cr supplementation. Blood glucose turnover rate and the maximal glucose infusion rate did not differ between diets, but both were higher (P = 0.0004 and P = 0.0001, respectively) during cold exposure than in the thermoneutral environment. The plasma insulin concentration at half-maximal glucose infusion rate changed with neither diet nor environment. Plasma concentrations of glucose and NEFA increased (P < 0.05) during cold exposure for both diets. In sheep, Cr supplementation, 1 mg/kg of diet as high-Cr yeast, has little influence on blood glucose metabolism and insulin action, whereas cold exposure enhances both without further modification by Cr supplementation.

Vimentin/cytokeratin coexpression foci in a well-differentiated canine hepatocellular carcinoma.

A number of pale-stained cell foci were observed within a well-differentiated hepatocellular carcinoma which developed in a 10-year-old male mongrel dog. The foci were composed of hepatocyte-like cells, but did not contain glycogen granules in their cytoplasm. Immunohistochemically, the focus cells coexpressed both bile duct type cytokeratin and vimentin. Electronmicroscopically, they were abundant in cytoplasmic organelles and contained bile pigments. Bile canaliculi were noted between the focus cells. The focus cells in the present case were considered to be neoplastic hepatocytes expressed bipotential features of hepatic stem cells.

Immunohistochemical and ultrastructural studies on the sinusoidal lining cells of canine hepatocellular carcinoma.

Immunohistochemical and ultrastructural studies were performed on the sinusoidal lining cells of eight canine hepatocellular carcinomas. The sinusoidal endothelial cells of the tumors had a positive reaction for both Factor VIII-related antigen and peanut agglutinin, but did not bind with Ulex europaeus agglutinin-1. Desmin- and lysozyme-positive cells were present along the sinusoids and perisnusoidal spaces of the tumor tissues, respectively, but were fewer in number compared with those of normal canine liver. Alpha-Smooth muscle actin-positive cells outlining the sinusoids were frequently observed. Electron microscopy revealed that basement membranes were often formed beneath the sinusoidal endothelial cells, with rare fenestration. Macrophages were present around or within the sinusoids and tended to increase in number relative to the degree of tumor differentiation. Myofibroblast-like cells with various morphological features, consistent with alpha-smooth muscle actin-positive cells, were frequently found in the perisinusoidal space. The present study indicates that the sinusoidal lining cells of canine hepatocellular carcinoma have some phenotypic characteristics.

Combined hepatocellular and cholangiocellular carcinoma in a dog.

A transitional type of combined hepatocellular and cholangiocellular carcinoma developed in a 12-year-old male Yorkshire terrier dog. The tumor was histologically composed of both hepatocellular carcinoma and cholangiocellular carcinoma components, and both elements were closely intermingled. Intraluminal mucin accumulation in cytokeratin-positive tubular/glandular structures was observed within the cholangiocellular carcinoma components and this feature was useful histological marker for a differential diagnosis between combined hepatocellular and cholangiocellular carcinoma and a pseudoglandular type of hepatocellular carcinoma. This primary hepatic tumor is extremely rare in dogs.

Morphological and immunohistochemical studies on porcine serum-induced rat liver fibrosis.

In order to clarify the pathogenesis of porcine serum (PS)-induced rat liver fibrosis, three experiments differing in dose of PS or duration of treatment were performed on male Fischer 344 rats. The rats were given an intraperitoneal injection of PS twice a week for 3 to 16 weeks and euthanized 7 days after the last injection for each treatment group. Liver tissues from these animals were subjected to detailed morphological and immunohistochemical examinations. Biochemical tests on treated rat serum revealed an increase in globulin concentration but no elevation in AST, ALT and ALP activities. There were no relationships among the dose of PS, the extent of fibrosis, and the anti-PS antibody titer. A number of alpha-smooth muscle actin-positive non-myofibroblastic cells, desmin-positive cells, and lipofuscin-laden Kupffer cells were found around the central veins and in the fibrous septa. In advanced stages of fibrosis, a proliferation of elastic fibers were observed in the septa. These findings were considered to indicate gradually occurred hepatocellular necrosis. The vascular endothelial cells in the fibrous septa expressed factor VIII-related antigen, exhibited fenestration accompanied by basement membrane formation, and were surrounded by Ito cells. Most of the portal vein branches showed hypertrophic thickening of the smooth muscle layer, resulting in narrowing of the lumen. These vascular changes suggested that hemodynamic alterations of the intrahepatic circulation induced hepatocellular necrosis/apoptosis and played an important role in the pathogenesis of porcine serum-induced liver fibrosis in rats.

Lymphangiosarcoma in a dog.

Lymphangiosarcoma was seen in the subcutis of right chest in a 11-year-old female Poodle. No metastasis was observed clinically. Tumor cells were vimentin positive and formed irregular space or slit without erythrocytes in the tumor tissue. Lymphocytic foci and edema were seen in the stroma. Only a few tumor cells had factor VIII-related antigen. Electronmicroscopically, tumor cells did not accompany with basement membrane and intercellular junctional complex.

Study on the pathogenesis of porcine serum-induced liver fibrosis in rats with special reference to the effects of hypertension.

To investigate the pathogenesis of porcine serum (PS)-induced liver fibrosis in rats, two experiments were carried out, taking into consideration of hypertension and vascular changes. In Experiment I, spontaneous hypertensive rats (SHRs), two-kidney, one clip hypertensive F344 rats (2K1C rats), and normotensive F344 rats were given an intraperitoneal injection of PS of 0.5 ml twice a week for 8 weeks. Histopathological, immunohistochemical, and electron microscopical examinations were performed on the liver from each rat. Histological features of liver fibrosis in hypertensive and normotensive rats were essentially identical. However, in the PS-treated SHRs, 2 of 5 animals showed the most severe fibrosis in all PS-treated groups. Electron microscopically, degranulated mast cells, eosinophils, and macrophages engulfing apoptotic cells were rarely observed in the late stage of fibrous septa (FS) in the PS-treated SHR liver. In Experiment II with normotensive F344 rats, histopathological features of early FS in the liver were compared with those of late FS observed in Experiment I using serial sections, and we found that FS developed along the wall of newly formed vessels to connect between neighboring central veins. However, the effect of hypertension on this fibrosis could not be clearly demonstrated in the present study using SHRs and 2K1C rats.

Morphological study on a case of canine hepatic nodular fibrosis.

Hepatic nodular fibrosis occurred in an 8-year-old male Papillon dog. Fibrous nodules, consisting of broad bands of collagen fibers, spindle cells, and lipofuscin-laden foamy macrophages, were well-circumscribed and frequently linked up with the portal areas. Because the spindle cells were positive for desmin and/or alpha-smooth muscle actin, they might be Ito cells or myofibroblasts. These results suggest that both the spindle cells and macrophages play an important role in the pathogenesis of hepatic nodular fibrosis, which might arise from the portal area.

Hepatoblastoma in a dog.

A hepatoblastoma was found in a 13-year-old female Maltese dog. Histologically, the tumor showed a wide trabecular pattern and was frequently accompanied with vascular lake formation. Tumor cells were positive for cytokeratin and neuron specific enolase, but negative for chromogranin. Electronmicroscopically, tumor cells were accompanied with continuous basement membrane and had poorly developed desmosomes. Sinusoidal endothelia had fenestration and were surrounded by myofibroblast-like cells. To the best of our knowledge, this paper is the first report of morphological studies on canine hepatoblastoma.

Lipid peroxides and antioxidants in serum of neonatal calves.

OBJECTIVE: To examine the association between oxidative stress and antioxidants in neonatal calves after birth. SAMPLE POPULATION: Sera from 6 healthy Holstein-Friesian cows and 7 of their newborn calves were obtained at various intervals after birth. PROCEDURE: Lipid peroxides in serum of cows and their newborn calves were estimated by measuring concentrations of thiobarbituric acid-reactive substances (TBARS). The antioxidative activities of neonatal sera were evaluated by measuring their superoxide-scavenging activities, ferroxidase activities, and the concentration of bilirubin-associated albumin. RESULTS: Concentration of TBARS in neonatal sera within 1 day after birth was significantly higher than concentrations > or = 2 days after birth and concentrations in serum of the dams. In contrast, antioxidative activities of neonatal sera, evaluated on the basis of superoxide-scavenging activities, ferroxidase activities, and concentration of bilirubin-associated albumin 3 hours after birth, were significantly lower than antioxidative activities in sera of the dams. CONCLUSIONS: Susceptibility of calves to oxidative stress during the neonatal period may be explained by the immature defense system against superoxide radicals.

Total deletion and a missense mutation of ITPR1 in Japanese SCA15 families.

BACKGROUND: Spinocerebellar ataxia type 15 (SCA15) is a progressive neurodegenerative disorder characterized by pure cerebellar ataxia, very slow progression, and distinct cerebellar atrophy. The locus for SCA15 was first mapped to 3p24.2-3pter in an Australian family. We have subsequently mapped two Japanese families presenting with ataxia and postural tremor of the head, arm, or trunk to the SCA15 locus. Recently, partial deletions involving both the type 1 inositol 1,4,5-triphosphate receptor (ITPR1) and sulfatase modifying factor 1 (SUMF1) genes have been identified in Australian and British families with SCA15. METHODS: We conducted fine haplotype analysis on the region including ITPR1. To identify the deletion, we conducted gene dosage analysis and array-based comparative genomic hybridization (aCGH) analysis. Gene expression analysis was performed using quantitative real-time reverse transcription PCR. Mutational analyses of ITPR1 and SUMF1 were also performed. RESULTS: We have identified a 414-kb deletion including the entire ITPR1 and exon 1 of SUMF1 in patients in family A. The expression levels of ITPR1 and SUMF1 mRNAs of the patient were half those of the normal control. Furthermore, in family B, we have identified a C-to-T substitution at position 8581 of ITPR1, resulting in the amino acid substitution of leucine for proline at codon 1059, which is highly conserved among species. CONCLUSIONS: Our results strongly confirm that ITPR1 is the causative gene for SCA15 and suggest that we need to investigate the point mutation in ITPR1 in the patients with autosomal dominant cerebellar ataxia and tremor.

The bone-titanium interface in vitro.

Commercially pure 5-mm-diameter titanium (cpTi) discs received droplet inoculations of cells derived from rat bone marrow and were maintained in supplemented culture medium for 2-3 weeks. The cells and extracellular matrix (ECM) were processed for observation by light (LM), scanning (SEM), and transmission electron (TEM) microscopy. The latter was achieved by freeze-fracturing the solid metal from the resin-embedded tissue using a method which preserved the interface. Surface staining of whole discs revealed cells separated from the metal substratum by areas of ECM which stained positively using von Kossa's method to identify mineralization. At SEM, the ECM comprised dense interwoven collagen fiber networks which were partially obscured by globular masses (GMs). Individual GMs were associated with collagen fibers, especially at fiber intersections. EDAX line scan analysis confirmed the presence of Ca and P in these areas which were assumed to be spheritic foci of calcification since the Ca and P peaks diminished in areas which demonstrated only collagen fibers or the underlying cpTi. TEM examination confirmed the presence of globular mineralization and also revealed the presence of an interfacial zone between the metal substratum and the mineralized ECM elaborated by osteoblasts during the culture period. The interfacial zone comprised two layers, a bonding zone containing few collagen fragments and a ruthenium red positive layer containing more densely packed collagen fibers. We believe that this is the first report of both the formation of bonelike tissue on solid titanium substrata in vitro and demonstration of an interface which bears close morphological similarities to that known to develop in vivo.

A stereologic analysis of collagen phagocytosis by fibroblasts in three soft connective tissues with differing rates of collagen turnover.

The purpose of this study was to assess the importance of the phagocytic mechanism of collagen resorption in the normal turnover and remodelling of soft connective tissues. Collagen phagocytosis by fibroblasts in rat skin, attached gingiva, and periodontal ligament was quantitated using the methodology of electron microscopic stereology. Periodontal ligament contained five and 15 times as much phagocytosed collagen as attached gingiva and skin respectively. Also, for each tissue examined, a positive correlation was observed between the amount of collagen phagocytosed and the known rate of mature collagen turnover.