Application of spontaneously immortalized odontoblast cells in tooth regeneration.

Here, we report on the first attempt to bioengineer tooth using a spontaneously immortalized mesenchymal cell line. To assess the odontogenic potential of this cell line, odontoblast-lineage cells (OLC) were re-associated with competent dental epithelium isolated from E14.5 mice. A novel three-dimensional organ germ culture method was applied to nurture the constructs in vitro. Additionally, recombinants were transplanted under the kidney capsule in host animals for 2 weeks. Transplants developed into tooth tissues in one-third of the cases. OLC-derived GFP-positive cells could be identified in mineralizing tooth germs by immunohistochemistry. OLCs were capable of intercellular and cell-matrix communication, thus they eventually differentiated into functional odontoblasts. In summary, we managed to utilize OLCs for dental mesenchyme substitution in tooth regeneration experiments. Therefore, our spontaneously transformed cell line proved its potential for future complex, tooth developmental and bioengineering studies.

Expression and localization of regenerating gene I in a rat liver regeneration model.

Regenerating gene (Reg) I has been identified as a regenerative/proliferative factor for pancreatic islet cells. We examined Reg I expression in the regenerating liver of a rat model that had been administered 2-acetylaminofluorene and treated with 70% partial hepatectomy (2-AAF/PH model), where hepatocyte and cholangiocyte proliferation was suppressed and the hepatic stem cells and/or hepatic progenitor cells were activated. In a detailed time course study of activation of hepatic stem cells in the 2-AAF/PH model, utilizing immunofluorescence staining with antibodies of Reg I and other cell-type-specific markers, we found that Reg I-expressing cells are present in the bile ductules and increased during regeneration. Reg I-expressing cells were colocalized with CK19, OV6, and AFP. These results demonstrate that Reg I is significantly upregulated in the liver of the 2-AAF/PH rat model, accompanied by the formation of bile ductules during liver regeneration.

Acute effects on the lung and the liver of oral administration of cerium chloride on adult, neonatal and fetal mice.

We evaluated tissue changes associated with cerium chloride administration via gavage to adult mice, via milk to neonatal mice and transplacentally to fetal mice. Change in adults consisted of extensive pulmonary hemorrhage, pulmonary venous congestion, thickened alveolar septae, hepatic necrosis and neutrophil infiltrations. Those in fetal mice consisted of pulmonary and hepatic congestion. These results indicate that gavage cerium administration elicited subtle tissue changes, though oral toxicity is rather low. These changes were less severe in neonatal and fetal mice. When cerium was injected into adult mice through the tail vein, cerium was distributed mainly to the liver, spleen and lung dose-dependently with the cerium concentration gradually decreasing after 3 days. A study of cerium anticoagulation in mouse plasma showed that clotting time was significantly prolonged when cerium was added to plasma. These results suggest that cerium may disturb blood coagulation and cause pulmonary and hepatic vascular congestion.

Mutation and expression of the p53 gene during chemical hepatocarcinogenesis in F344 rats.

Inactivation of the p53 gene is one of the most frequent genetic alterations in carcinogenesis. We studied gene mutations, the mRNA expression of p53, and the accumulation of p53 protein in chemical hepatocarcinogenesis in rats. Samples consisting of 44 precancerous foci and 18 cancerous foci were collected by laser capture microdissection (LCM), and analyzed for mutations in rat p53 gene exons 5-8 by PCR-single-strand conformational polymorphism (PCR-SSCP). We found that 25 PCR-SSCP bands of exons 6/7 and 8 were altered in 22/62 (35.4%) LCM samples. Direct p53 gene sequencing showed that 20/62 (9 precancer, 11 cancer) (32.3%) LCM samples exhibited 34 point mutations. Ten LCM samples exhibited double or triple mutations in exons 6/7 and 8 simultaneously. A quantitative analysis of p53 mRNA showed that p53 mRNA peaked at an early stage (week 6) in the precancerous lesion, 20 times that of adjacent normal tissue, and returned to normal by week 23. Similar to precancer, p53 mRNA in cancer was five times as high as that of adjacent normal tissue at week 12, and was closer to normal at week 23. When p53 mRNA declined from a high to low, positive immunostaining for the p53 protein began to be seen in precancerous and cancerous foci, suggesting that the p53 protein had accumulated in these foci. Results show that p53 gene mutation is present in initial chemical hepatocarcinogenesis and p53 mRNA concentration is clearly elevated before gene mutation. Once the p53 gene has mutated, mRNA concentration progressively declines, suggesting that mutation leads to inactivation of the p53 gene.

Repeated immunization induces the increase in fucose content on antigen-specific IgG N-linked oligosaccharides.

OBJECTIVE: In order to investigate whether repeated immunization induces changes in IgG glycosylation, we analyzed the composition of oligosaccharides on antigen-specific IgGs obtained from mice that received different amounts of immunization boosts. METHODS: Three groups of mice were immunized with ovalbumin (OVA) and boosted once, twice, or three times, respectively, with an interval of 1 week. The patterns of oligosaccharides present in anti-OVA specific IgGs were analyzed using lectin-enzyme-linked immunosorbent assay (ELISA) and lectin-blot. RESULTS: The repeated injection of OVA induced both the production of specific IgGs and an increase of fucose content in N-linked oligosaccharides of the IgGs. The lowest IgG fucosylation was observed in mice boosted once, whereas the highest fucosylation rate was observed in mice boosted three times. ELISA assay demonstrated that there was a positive relationship between the fucose content and amount of immunization boosts. CONCLUSIONS: IgG fucosylation increases during repeated immunization with ovalbumin. The alteration of IgG fucosylation may have important biological significance.

Orally administrated rare earth element cerium induces metallothionein synthesis and increases glutathione in the mouse liver.

The influence of oral administration of rare earth element cerium (Ce) was studied in relation to metallothionein (MT) and glutathione (GSH) content in the organs of ICR mice, which were administered heavy metal cadmium (Cd) for comparison. Male ICR mice were divided into 9 groups: 1 control group, 4 cerium groups and 4 cadmium groups, each with 4 mice, for a total of 36 mice. Ce groups included a 20 ppm CeCl3 diet (Ce-low) group and a 200 ppm CeCl3 diet (Ce-high) group, as did Cd groups, i.e., a 20 ppm CdCl2 diet (Cd-low) group and a 200 ppm CdCl2 diet (Cd-high) group. Each group was subdivided in 2 groups except a control group: 6-week administration group and 12-week administration group. The level of plasma aspartate aminotransferase(AST) activity, plasma alanine aminotransferase(ALT) activity, plasma cholesterol and plasma triglyceride in the Ce-low, Cd-low, Ce-high, and Cd-high group were higher than that of control group, although there were no significant differences (p > 0.05). By contrast, both Ce and Cd groups had higher levels of MT and GSH in hepatic cells compared to the control group (p < 0.05) and decreased liver tissue level of lipoperoxide (p < 0.05). These groups also had decreased plasma superoxide dismutase (SOD) activity (p < 0.05), and increased plasma level of lipoperoxide (p > 0.05). In conclusion, it is suggested that orally administered Ce increases MT and GSH as an antioxidant in the mouse liver, and these reaction are probably caused by increases in the oxidative stress with Ce.

Protection of hepatocytes from apoptosis by a novel substance from actinomycetes culture medium.

A novel substance, #675, found from an Streptomyces sp. SM675 culture medium, dose-dependently stimulates the proliferation of human functional liver cell 4 (FLC4). When FLC4 cells were incubated under conditions without fetal bovine serum (FBS), typical features of apoptotic cell death such as shrinkage and nuclear condensation appeared; high molecular weight (HMW) DNA fragments were found; and caspase-3 and poly (ADP-ribose) polymerase (PARP) proteins were cleaved. When FLC4 cells were incubated with #675 and without FBS, the cells grew healthy, no HMW DNA fragments were found, and caspase-3 and PARP cleavage weakened, suggesting that #675 protects FLC4 cells from apoptosis induced by FBS-deprivation. The quantitative reverse-transcribed polymerase chain reaction did not show differences in PARP or Bcl-2 mRNA expression in FLC4 cells incubated with or without #675, indicating other genes may be involved in this anti-apoptosis effect. These results show that #675 enhances FLC4 proliferation via an apoptosis-inhibition pathway, implying potential pharmacological and clinical applications.

Effects of slightly acidic electrolysed drinking water on mice.

Slightly acidic electrolysed (SAE) water is a sanitizer with strong bactericidal activity due to hypochlorous acid. We assessed the safety of SAE water as drinking water for mice at a 5 ppm total residual chlorine (TRC) concentration to examine the possibility of SAE water as a labour- and energy-saving alternative to sterile water. We provided SAE water or sterile water to mice for 12 weeks, during which time we recorded changes in body weight and weekly water and food intakes. At the end of the experiment, all of the subject animals were sacrificed to assess serum aspartate aminotransferase, alanine aminotransferase and creatinine levels and to examine the main organs histopathologically under a light microscope. In addition, we investigated the bacteria levels of both types of water. We found no difference in functional and morphological health condition indices between the groups. Compared with sterile water, SAE water had a relatively higher ability to suppress bacterial growth. We suggest that SAE water at 5 ppm TRC is a safe and useful alternative to sterile water for use as drinking water in laboratory animal facilities.

Gadolinium chloride suppresses styrene-induced cytochrome P450s expression in rat liver.

To assess the effect of gadolinium (Gd) on the expression of several forms of cytochrome P450 (P450s) and antioxidant enzymes, we treated rats with gadolinium chloride (25 mg as Gd/kg body weight) 4 h after styrene (a multiple P450 inducer) treatment (600 mg/kg). Gd treatment significantly suppressed styrene-inducible cytochrome P4502B1 (CYP2B1), CYP2B2, CYP2E1, and CYP3A2 mRNA expressions to 48.6%, 69.8%, 61.1%, and 38.5%, accompanying with the reduction of proteins expression to 1.42%, 31.2%, 21.1% and 21.1%, respectively, compared with styrene alone treatment. Gd suppressed styrene-inducible CYP1A2 expression, but only at the protein level. On the other hand, styrene treatment caused a decrease in reduced form of glutathione (GSH), as well as increases in lipid peroxide and serum ALT and AST activities, suggesting the occurrence of hepatic damage probably due to styrene-induced oxidative stress in rat liver. Post-treatment of Gd attenuated this styrene-caused hepatic damage. Moreover, mRNA expressions of cellular antioxidant enzymes such as catalase, CuZn-superoxide dismutase (CuZnSOD) and glutathione peroxidase (GPX) were hardly changed by styrene and/or Gd treatment. In summary, Gd suppressed styrene-inducible expression of not only CYP2B1 but also several forms of P450 at both the mRNA and protein levels, along with attenuation of styrene-caused liver damage. These findings suggested that Gd is a chemo-preventive agent against hepatic damage caused by xenobiotics requiring biotransformation.

Differentiation of liver epithelial (stem-like) cells into hepatocytes induced by coculture with hepatic stellate cells.

The liver is believed to contain stem cells that can differentiate into either hepatocytes or biliary epithelial cells. In the present study, we established a nonhepatocytic epithelial cell line from the normal livers of adult rats. The established cells, designated HSL cells, were immunoreactive against alpha-fetoprotein, but neither albumin nor cytokeratin 19. To demonstrate the differentiation potential of HSL cells in vitro, the cells were cocultured with hepatic stellate cells as a mixture or separately using insert wells. Consequently, although coculture with hepatic stellate cells rendered HSL cells able to produce albumin, the mixed coculture system mimicking the hepatic environment elicited this phenomenon more effectively than the separated coculture system. In conclusion, HSL cells have immature properties and the potential to differentiate into mature cells. Not only the extracellular matrices but also soluble factors, which are produced by hepatic stellate cells, induce this maturation, demonstrating the importance of the hepatic environment for hepatocyte differentiation.