Requirement of integrin ß3 for iron transportation during enamel formation.

Rodent incisors exhibit pigmentation on their labial surfaces. Although previous studies have shown that this pigment is composed of iron, the existence of other elements has not been investigated. This study found that the lower incisors of CD61, also known as integrin ß3, null mice (CD61(-/-)) lacked pigmentation. Although ameloblasts differentiated and formed enamel normally, no ferric ion accumulation was observed in maturation-stage ameloblasts in CD61(-/-) mice. Surface elements of control and CD61-/- lower incisors were compared by x-ray photoelectron spectroscopy (XPS). XPS analysis detected C, Ca, N, O, and P on the labial surfaces of lower incisors of both mice, whereas Fe was detected only in control samples. No peak of non-ferrous metal or other element was detected in either group. Quantitative RT-PCR analysis of 18 iron-transportation-related genes with mRNA from maturation-stage ameloblasts and ALC, a pre-ameloblastic cell line, was performed. The results suggested that CD61 regulates the expressions of Slc11a2 and Slc40a1, both of which are involved in iron transportation in epithelial tissues. These results suggested that the pigment on the labial surface of mouse incisors is composed of Fe and that both anemia and reduction of iron-transporting proteins may cause the loss of pigmentation in CD61(-/-) mice.

Flat adenoma-carcinoma sequence with high-malignancy potential as demonstrated by CD10 and beta-catenin expression: a different pathway from the polypoid adenoma-carcinoma sequence.

AIMS: CD10+ colorectal carcinomas have a high risk of giving rise to liver metastasis. The aim was to examine phenotypic expression in colorectal neoplasia and to elucidate changes in such expression through the adenoma-carcinoma sequence. METHODS AND RESULTS: We examined the expression of various proteins immunohistochemically in 111 flat [non-polypoid growth (NPG)] colorectal neoplasms, categorized into 28 low-grade (NPG-LGN), 44 high-grades (NPG-HGN) and 39 cases of invasive neoplasia (NPG-IN), as well as in 96 polypoid [polypoid growth (PG)] neoplasms, categorized into 26 PG-LGN, 39 PG-HGN and 31 PG-IN according to the Vienna classification. CD10 was more frequently expressed in NPG than in PG neoplasia. MUC2 and MUC5AC were more frequently expressed in PG than in NPG neoplasias. Nuclear beta-catenin was more frequently expressed in NPG-LGN than in PG-LGN. No difference in p53 expression was found between NPG and PG neoplasia. CONCLUSIONS: From the viewpoint of the expression of CD10 and beta-catenin, it would appear that NPG-LGN differs significantly from PG-LGN, thereby indicating that NPG-LGN is a precursor of CD10+ carcinoma. It is important to ensure that NPG neoplasia is not overlooked if cases of CD10+ carcinoma are to be detected at an early stage.

Teratoma formation and hepatocyte differentiation in mouse liver transplanted with mouse embryonic stem cell-derived embryoid bodies.

We previously reported that mouse embryonic stem (ES) cells are capable of differentiating into hepatocytes in cultured embryoid bodies (EBs) and that hepatocytes generate in the recipient liver injected with cultured day-9 EB cells via spleen without the formation of a teratoma. Because ES cells frequently form teratomas in recipient mice, we investigated incidence of teratoma formation when day-9 EBs derived from ES cells were transplanted directly into the subcapsule of mouse liver. In contrast to injection of day-9 EB cells through the portal vein via the spleen, direct subcapsular injection of cultured day-9 EB cells into liver, and even of cultured day-15 EBs, resulted in an high incidence of teratoma in the liver. In teratomas of livers injected directly with day-15 EBs, hepatocytes were detected singly and in clusters. These results imply that undifferentiated cells capable of developing into teratomas exist in cultured EBs, and even in cultured day-15 EBs containing differentiated hepatocytes.

Isolation of hepatocyte-like cells from mouse embryoid body cells.

We previously reported that embryoid body (EB) cells derived from embryonic stem (ES) cells are capable of differentiating into functional hepatocyte-like cells both in vitro and in vivo. Because transplantation of EB-derived cells into the liver via the spleen resulted in a low incidence of teratoma formation, purification of hepatocyte-like cells is required to prevent teratoma formation. The aim of this study was to purify hepatocyte-like cells from cultured EBs. For the isolation of hepatocyte-like cells, EBs cultured for 15 days were treated with trypsin-EDTA. The disaggregated cells were plated on a gelatin-coated dish as a monolayer. These cells were separated by Percoll gradient centrifugation, enriched by magnetic cell sorting, and purified by FACS. The purified hepatocyte-like cells in monolayer cultures were positive for immunostaining for albumin and expressed albumin mRNA, but not Oct3/4 mRNA. Transplantation of the purified hepatocyte-like cells derived from mouse ES cells might be an effective treatment for liver failure.

Beneficial effect of tetrahydrobiopterin on the survival of rats exposed to hepatic ischemia-reperfusion injury.

INTRODUCTION: The protective role of nitric oxide (NO) against hepatic ischemia-reperfusion (I/R) injury remains controversial. In this study we investigated the effect of tetrahydrobiopterin (BH4) on the survival of rats exposed to an hepatic I/R injury. METHODS: The rats were subjected to 100 minutes of 70% hepatic ischemia 30 minutes after administration of BH4 or saline. A specific inducible NO synthase (iNOS) blocker, 1400W, was used to evaluate endogenous iNOS. NOS protein measured the histological appearance of the liver by Western blotting, and survival was evaluated after reperfusion. RESULTS: The 1-week survival rate was 60% among the BH4 group and 10% for the saline group. The serum ALT and bilirubin values in the BH4 group were significantly lower than the saline group. Histological examination of the liver revealed only a small necrotic area in the BH4 group as opposed to massive necrosis and cell infiltration in the saline group. Injection of 1400W significantly decreased the prolongation of survival produced by BH4. CONCLUSIONS: BH4 significantly improved the survival rate, the histological findings, and the liver function, thereby reducing liver failure. Western blotting showed a higher level of iNOS protein in the BH4 group than the saline group, 1400W suppressed this effect of BH4. Taken together, these observations suggest that NO derived from reactions driven by BH4-induced iNOS exerts a protective effect against reperfusion injury.

Raman scattering study of water clusters around polyrotaxane and pseudopolyrotaxane supramolecular assemblies.

We have measured Raman spectra of collective O-H stretching vibration of water clusters in polyrotaxane and pseudopolyrotaxane aqueous solutions and the aqueous solutions of their constituent molecules. The intensities of the collective bands of water clusters in the polyrotaxane and pseudopolyrotaxane solutions were approximately equal to that of their solvents. On the other hand, those in the solutions of linear polymeric chains and cyclic molecules were smaller. These results indicate that the water molecules in the solvents cannot approach to interact with the hydrophobic parts of the constituent molecules sterically when the constituent molecules form the inclusion complexes. Thus, the polyrotaxane and pseudopolyrotaxane molecules are observed as inert in terms of molecular interaction with water, although the constituent molecules have hydrophobic parts in their structure.

Hand-assisted laparoscopic hepatic resection.

Thanks to recent advances, performance of liver resection is now possible using laparoscopic procedures. However, still there are some difficulties to overcome. The hand-assisted method lends safety and reliability to the laparoscopic procedure. A 54-year-old man diagnosed with hepatocellular carcinoma (HCC) was referred for hepatectomy. Angiography with computed tomography (CT) scans revealed a 2-cm hepatocellular carcinoma (HCC) at segment V, close to the gallbladder. A hand-assisted laparoscopic hepatic resection was performed. Four 10-mm trocars, one for wall lifting and three for working, were placed in the upper abdomen. A small incision was added at the right side of umbilicus, and the operator's left hand was inserted through it. A microwave tissue coagulator and laparoscopic ultrasonic dissector were used for liver resection. Total operation time was 162 min; blood loss was 20 g. The postoperative course was uneventful, and the postoperative hospital stay was 7 days. We thus demonstrated that laparoscopic liver resection is safer and easier when the hand of the operator can be inserted into the abdomen. The small incision does not greatly diminish the benefits that accrue from minimally invasive laparoscopic surgery. The hand-assisted procedure allows better access to the tumor. In addition, hand assistance restores the sense of touch to the operator and is an effective means of controlling sudden and unexpected bleeding.

Modulatory factors on temperature-synchronized degradation of dextran grafted with thermoresponsive polymers and their hydrogels.

Several types of dextran grafted with poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide [dextran-g-poly(NIPAAm-co-DMAAm)] with different graft lengths and numbers were synthesized in a preciously controlled fashion, and their enzymatic degradation was examined by viscosity measurement and gel permeation chromatography as a function of temperature. Degradation of dextran-g-poly(NIPAAm-co-DMAAm)s decreased with increasing the graft length below their lower critical solution temperatures (LCSTs). Above the LCST, enzymatic degradation was independent of the graft length. A larger amount of the graft chain with increasing the graft length rather than the graft number was effective to modulate the temperature-synchronized degradation. Hydrogels were prepared by cross-linking the graft copolymers using 1,6-hexamethylenediamine. While all the hydrogels have water content of about 93-96% in a wide range of temperatures, their degradation behaviors show a significant dependence on a temperature change. Such a unique property is closely related to the structure of graft copolymers such as graft lengths. Consequently, introducing thermoresponsive grafts with longer length to dextran and its hydrogels is suggested to be an important factor for modulating enzymatic degradation of dextran in synchronization with temperature.