Quantitative analysis of development of mitochondrial ultrastructure in differentiating mouse hepatocytes during postnatal period

Between birth and 10 days of age, the volume density (volume/unit cytoplasmic volume) of the matrix, and the surface density (area/unit cytoplasmic volume) of the inner membrane and cristae increased in both periportal and perihepatic hepatocytes, and did not differ significantly between the cells of the two zones. After 10 days of age, however, the volume density of the matrix decreased in perihepatic cells and remained unchanged in periportal cells, and, therefore, it became greater in periportal cells than in perihepatic cells in 20-day-old and adult animals. The surface density of the inner membrane and cristae decreased in the cells of both zones. Further, the hepatocyte volume increased markedly, especially in perihepatic zones between 20 days of age and the adult. The results show that, in postnatally differentiating hepatocytes, mitochondria are likely to develop during early postnatal period, then the structural heterogeneity of mitochondria arises, and hepatocyte volume increases markedly during late postnatal period after weaning. Thus, the process of postnatal hepatocyte differentiation includes such several phases of development.     

Glucagon receptors in endothelial and Kupffer cells of mouse liver

To determine whether hepatic sinusoidal cells contain glucagon receptors and, if so, to study the significance of the receptors in the cells, binding of [125I]-glucagon to nonparenchymal cells (mainly endothelial cells and Kupffer cells) isolated from mouse liver was examined by quantitative autoradiography and biochemical methods. Furthermore, the pathway of intracellular transport of colloidal gold-labeled glucagon (AuG) was examined in vivo. Autoradiographic and biochemical results demonstrated many glucagon receptors in both endothelial cells and Kupffer cells, and more receptors being present in endothelial cells than in Kupffer cells. In vivo, endothelial cells internalized AuG particles into coated vesicles via coated pits and transported the particles to endosomes, lysosomes, and abluminal plasma membrane. Therefore, receptor-mediated transcytosis of AuG occurs in endothelial cells. The number of particles present on the abluminal plasma membrane was constant if the amount of injected AuG increased. Therefore, the magnitude of receptor-mediated transcytosis of AuG appears to be regulated by endothelial cells. Kupffer cells internalized the ligand into cytoplasmic tubular structures via plasma membrane invaginations and transported the ligand exclusively to endosomes and lysosomes, suggesting that the ligand is degraded by Kupffer cells.     

Intracellular localization of alkaline phosphatase in freshly isolated foetal rat hepatocytes

Summary The cytochemical localization of alkaline phosphatase activity in foetal rat hepatocytes was examined in relation to the pattern of cell to cell attachment during cell isolation and culture. In foetal hepatocytesin vivo, alkaline phosphatase was exclusively localized on the bile canalicular membrane. In freshly isolated foetal hepatocytes, however, the activity was present in the endoplasmic reticulum, nuclear envelope, Golgi apparatus, tubulo-vesicular organelles, and over the entire plasma membrane. In monolayer cells cultured for one or two days, the activity was localized on the reconstituted bile canalicular membrane, plasma membrane sites adjacent to neighbouring cells and on the bottom surface of the monolayer, but was detected in none of the intracellular organelles. Biochemical alkaline phosphatase activity did not change during isolation of the cells. These results suggest that, in foetal hepatocytes, loss of cell—cell contact may induce a temporal disturbance, or dedifferentiation, in their membrane system.     

High and testosterone-dependent glucose 6-phosphatase activity in epithelium of mouse seminal vesicle

For study of the mechanism of seminal fructogenesis, glucose 6-phosphatase activity was examined cytochemically (a method modified from that of Wachstein and Meisel) and biochemically (the method of Leskes et al.) in seminal vesicles from normal, castrated, and castrated and testosterone-treated mice. The reaction product for the activity was localized in the endoplasmic reticulum and nuclear envelope of all cell types composing the seminal vesicle. In normal seminal vesicle, the reaction product was apparently more abundant in columnar and basal cells than in other cell types. Ten, 20, and 30 days after castration, the abundant amount of reaction product in columnar and basal cells decreased to the level in other cell types. In animals treated with testosterone after castration, however, the reaction product in columnar and basal cells remained abundant. If fructose 6-phosphate was added to the reaction medium in place of glucose 6-phosphate, the amount and pattern of deposition of the reaction product did not change. Changes in biochemical activity in castrated or castrated and testosterone-treated animals paralleled the cytochemical results. The results show that the high activity in columnar and basal cells is under the control of testosterone, and the role of this enzyme is probably to release fructose into the seminal fluid.     

Cytochemical glucose-6-phosphatase activity in the cells of mouse pancreas and submandibular gland

Ultrastructural localization of glucose-6-phosphatase activity was studied in the cells of the pancreas and submandibular gland of the mouse using a incubation medium modified from that of Wachstein & Meisel (1956). In pancreatic acinar cells, the reaction product for the enzyme activity was not found even after 90 min of incubation with three changes of the medium. However, the reaction product was localized in the endoplasmic reticulum and nuclear envelope of all other cell types composing the pancreas and submandibular gland. The reaction product appeared in moderate to abundant amounts in acinar cells and striated duct cells of the submandibular gland, and in the B cells, A and D cells of the pancreatic islet, but it was scarce in other cell types.     

Multilocus loss of DNA methylation in individuals with mutations in the histone H3 Lysine 4 Demethylase KDM5C

Background

A number of neurodevelopmental syndromes are caused by mutations in genes encoding proteins that normally function in epigenetic regulation. Identification of epigenetic alterations occurring in these disorders could shed light on molecular pathways relevant to neurodevelopment.

Results

Using a genome-wide approach, we identified genes with significant loss of DNA methylation in blood of males with intellectual disability and mutations in the X-linked KDM5C gene, encoding a histone H3 lysine 4 demethylase, in comparison to age/sex matched controls. Loss of DNA methylation in such individuals is consistent with known interactions between DNA methylation and H3 lysine 4 methylation. Further, loss of DNA methylation at the promoters of the three top candidate genes FBXL5, SCMH1, CACYBP was not observed in more than 900 population controls. We also found that DNA methylation at these three genes in blood correlated with dosage of KDM5C and its Y-linked homologue KDM5D. In addition, parallel sex-specific DNA methylation profiles in brain samples from control males and females were observed at FBXL5 and CACYBP.

Conclusions

We have, for the first time, identified epigenetic alterations in patient samples carrying a mutation in a gene involved in the regulation of histone modifications. These data support the concept that DNA methylation and H3 lysine 4 methylation are functionally interdependent. The data provide new insights into the molecular pathogenesis of intellectual disability. Further, our data suggest that some DNA methylation marks identified in blood can serve as biomarkers of epigenetic status in the brain.     

Autologous Transplantation of Oral Mucosal Epithelial Cell Sheets Cultured on an Amniotic Membrane Substrate for Intraoral Mucosal Defects

The human amniotic membrane (AM) is a thin intrauterine placental membrane that is highly biocompatible and possesses anti-inflammatory and anti-scarring properties. Using AM, we developed a novel method for cultivating oral mucosal epithelial cell sheets. We investigated the autologous transplantation of oral mucosal epithelial cells cultured on AM in patients undergoing oral surgeries. We obtained specimens of AM from women undergoing cesarean sections. This study included five patients without any history of a medical disorder who underwent autologous cultured oral epithelial transplantation following oral surgical procedures. Using oral mucosal biopsy specimens obtained from these patients, we cultured oral epithelial cells on an AM carrier. We transplanted the resultant cell sheets onto the oral mucosal defects. Patients were followed-up for at least 12 months after transplantation. After 2–3 weeks of being cultured on AM, epithelial cells were well differentiated and had stratified into five to seven layers. Immunohistochemistry revealed that the cultured cells expressed highly specific mucosal epithelial cell markers and basement membrane proteins. After the surgical procedures, no infection, bleeding, rejection, or sheet detachment occurred at the reconstructed sites, at which new oral mucous membranes were evident. No recurrence was observed in the long-term follow-up, and the postoperative course was excellent. Our results suggest that AM-cultured oral mucosal epithelial cell sheets represent a useful biomaterial and feasible method for oral mucosal reconstruction. However, our primary clinical study only evaluated their effects on a limited number of small oral mucosal defects.     

The fossil record of Glypturus Stimpson, 1866 (Crustacea,Decapoda, Axiidea,Callianassidae) revisited,with notes on palaeoecology and palaeobiogeography

Abstract: The fossil record of the callianassid genus Glypturus (Decapoda, Axiidea) is re‐evaluated. Our systematic revision, both of extant and fossil taxa, is based on major cheliped morphology only, thus providing an important impetus for palaeontological studies. Both spination and tuberculation of chelipeds are herein considered of great taxonomic importance. Presence of spines on the upper margins of the merus and propodus and the lower margin of the carpus are significant for generic assignment, whereas the extent of tuberculation on lateral surfaces of the propodus is important for assignment at the species level. Altogether, four extant and six exclusively fossil species of Glypturus are recognized. Several extinct callianassid taxa are now transferred to the genus, namely Callianassa berryi, Callianassa fraasi, Callianassa munieri, Callianassa pugnax and Callianassaspinosa; Callianassa pseudofraasi is considered a junior synonym of C. fraasi. Based on a comparison of ecological preferences of extant representatives, the presence of Glypturus in the fossil record is considered to be linked with tropical to subtropical, nearshore carbonate environments of normal salinity. We argue that Glypturus is of Tethyan origin, with a stratigraphical range going as far back as the Eocene. Since then, the genus migrated both westwards and eastwards, establishing present‐day communities in the western Atlantic and Indo‐West Pacific which both comprise several distinct species. In the presumed area of origin, the genus does no longer occur today. The exlusively fossil (middle Eocene) genus Eoglypturus from Italy is considered closely related to Glypturus and is thus assigned to the subfamily Callichirinae as well.     

Postnatal changes in sublobular distribution of NADPH-cytochrome P-450 reductase in rat liver

Summary Immunohistochemical distribution of NADPH-cytochrome P-450 reductase (NADPH-ferrihaemoprotein reductase; EC 1.6.2.4.) in the liver lobule was examined during development of the rat. From the 19th day of gestation to 4 days after birth, the enzyme was distributed uniformly throughout the lobule. The immunostaining for the enzyme was weak before birth, and became slightly stronger after birth. A slightly uneven distribution of immunoreactivity, stronger in perivenular zones, appeared at 5 days after birth. Then, the staining intensity in perivenular zones became progressively stronger with age, except for a slight increase between 10 and 20 days of age. The intensity in periportal zones also increased gradually, although it remained weaker than that in perivenular zones. Around 30 days of age, the distribution of the immunostaining, stronger in perivenular than in periportal zones, was similar to that seen in the lobules of adult animals. thus, heterogeneity among hepatocytes with respect to the enzyme content is not present in fetal and newborn rats but develops gradually during postnatal development; the postnatal growth of the liver is accompanied by a change in the pattern of the distribution of this enzyme within the lobule.     

Immunohistochemical localization of type II and type I collagens in articular cartilage of the femoral head of dexamethasone-treated rats

The immunohistochemical localization of type II and type I collagens was examined in the articular cartilage of the femoral head of growing rats injected systemically with 5 mg kg−1 dexamethasone for 2 weeks every other day. The intensities of immunostaining for type II collagen, measured by microphotometry, was highest in the flattened cell layer and high in the hypertrophic cell layer, moderate in the proliferative cell and transitional cell layers and low in the superficial layer. After dexamethasone administration, the intensities decreased markedly in the flattened cell layer and slightly in the hypertrophic cell layer, although the decreases in other layers were negligible. The staining intensities for type I collagen were highest in the flattened cell layer, low in the superficial and transitional cell layers and very low in the proliferative and hypertrophic cell layers. After dexamethasone administration, the intensities increased markedly in the flattened cell layer and slightly in the superficial and proliferative cell layers, but did not change in the transitional and hypertrophic cell layers. Thus, dexamethasone administration caused a decrease in type II collagen and an increase in type I collagen in the matrix of the surface portion of articular cartilage. The composition of isoforms of collagen in the matrix changed after the steroid administration. The results strongly suggest that the shift in collagen composition from type II to type I predominance is a cause of the degeneration of the articular cartilage after glucocorticoid administration. This revised version was published online in November 2006 with corrections to the Cover Date.