A HhaI/BstUI polymorphism in a novel gene at human chromosome 11p15.5

We found a HhaI/BstUI polymorphism in the 3′ untranslated region of a novel gene which was localized to 11p15.5. This region is one of prominent imprinting domains and contains multiple imprinted genes, such as H19, IGF2 , KVLQT1, and p57 ^KIP2 , which suggests that regional factors might contribute to the imprinting. This polymorphism will be useful in the allelic analysis of expression and methylation of the novel gene. Received: July 24, 1998 / Accepted: July 29, 1998     

Modification of an <Emphasis Type="Italic">in vivo</Emphasis> Lung Metastasis Model of Hepatocellular Carcinoma by Low Dose N-nitrosomorpholine and Diethylnitrosamine

Previously, we established the in vivo lung metastasis model of rat HCC induced by two hepatocarcinogens, diethylnitrosamine (DEN) and N-nitrosomorpholine (NMOR) at a dose of 120 ppm. This model allows us to investigate modifying factors leading to the inhibition of metastasis formation. However, low survival rates made the evaluation of metastasis formation difficult. The current experiments were conducted to modify the experimental protocol to improve survival and to establish a better animal metastasis model. Lower doses of NMOR (80 or 40 ppm in drinking water) were given to F344 rats for 14 weeks after DEN treatment. Survival rates in the 80 ppm group and in the 40 ppm group were 57% and 81%, respectively and these values were significantly higher than that in 120 ppm. Incidences of lung metastasis in the 40 ppm group steadily increased up to 67% by week 36 while that in the 80 ppm increased sharply up to 86% by week 24. Severity of lung metastases in the 40 ppm group at week 36 was mild compared with the 80 ppm group at week 24. In the second experiment, in order to characterize HCC development and lung metastasis in the 40 ppm group, rats given DEN and then followed with 40 ppm NMOR were killed sequentially. Development of HCC was observed at week 14 and reached 100% incidence at week 20. First lung metastatic lesions were evident at week 22, and incidence of lung metastasis reached 100%. Tumor cells were identified in the blood at week 20 by RT-PCR. The current study revealed that 40 ppm NMOR for 14 weeks after DEN treatment developed HCC without lung metastases at week 22, then HCC with a frequent lung metastasis at week 40. Thus, it can be said that this system is a more appropriate model for elucidation of mechanisms of metastasis and also for analysis of factors to inhibit natural metastasis.     

No association of GSK3beta gene (GSK3B) with Japanese schizophrenia.

Several lines of evidence indicate that glycogen synthase kinase-3beta (GSK3beta) is one of the candidates for schizophrenia-susceptibility factor. However, it has not been reported the association analysis between GSK3beta gene (GSK3B) and Japanese schizophrenia based on linkage disequilibrium (LD). We provide an association analysis using relatively large samples (381 schizophrenia, and 352 controls) after determination of "tag single nucleotide polymorphisms (SNPs)." In this LD mapping, we selected and genotyped for eight polymorphisms (seven SNPs and one diallelic (CAA)(n) repeat), which covered the entire region of GSK3B, and determined two "tag SNPs." In the following association analysis using these two "tag SNPs," we could not find association with Japanese schizophrenia. Furthermore, we also include subgroup analysis considering age-at-onset and subtypes, neither could we find associations. Because our samples provided quite high power, these results indicate that GSK3B may not play a major role in Japanese schizophrenia.     

A histological evaluation for guided bone regeneration induced by a collagenous membrane

This study was designed to evaluate the histological changes during ossification and cellular events including osteogenic differentiation responding to collagenous bioresorbable membranes utilized for GBR. Standardized artificial bony defects were prepared at rat maxillae, and covered with a collagenous bioresorbable membrane. These animals were sacrificed at 1, 2, 3 and 4 weeks after the GBR-operation. The paraffin sections were subject to tartrate resistant acid phosphatase (TRAP) enzyme histochemistry and immunohistochemistry for alkaline phosphatase (ALP), osteopontin (OP) and osteocalcin (OC). In the first week of the experimental group, woven bone with ALP-positive osteoblasts occupied the lower half of the cavity. The collagenous membrane included numerous ALP-negative cells and OP-immunoreactive extracellular matrices. At 2 weeks, the ALP-, OP- and OC-immunoreactivity came to be recognizable in the region of collagenous membrane. Since ALP-negative soft tissue separated the collagenous membrane and the new bone originating from the cavity bottom, the collagenous membrane appeared to induce osteogenesis in situ. At 3 weeks, numerous collagen fibers of the membrane were embedded in the adjacent bone matrix. At 4 weeks, the membrane-associated and the cavity-derived bones had completely integrated, showing the same height of the periosteal ridge as the surrounding alveolar bones. The collagen fibers of a GBR-membrane appear to participate in osteogenic differentiation.     

Pseudotype hepatitis C virus enters immature myeloid dendritic cells through the interaction with lectin

Dendritic cells (DC) are the most potent antigen-presenting cells that regulate immune responses. One of the mechanisms for hepatitis C virus (HCV) persistence is the ability of HCV to suppress DC function. Direct HCV infection to blood DC has been implicated for DC dysfunction. To clarify the susceptibility of each DC subset to HCV, we used pseudotype vesicular stomatitis virus (VSV) coated with chimeric HCV envelope glycoproteins (E1 and E2). We demonstrate that pseudotype VSV enters myeloid DC (MDC) but not plasmacytoid DC (PDC). The highest efficiency of pseudotype VSV entry to MDC was observed when MDC were cultured with GM-CSF. Such efficiency decreased when MDC are matured with the treatment of IL-4, CpG oligodeoxynucleotide, or CD40 ligand. Mannan inhibited pseudotype VSV entry to MDC, but Ca(2+) chelators failed to do so. These results show that pseudotype VSV possessing HCV-E1 and E2 enters immature MDC through the interaction with lectins in a Ca(2+)-independent manner.     

Expression and intracellular localization of FKHRL1 in mammary gland neoplasms

FKHRL1 (FOXO3a), a member of the Forkhead family of genes, has been considered to be involved in the development of breast tumors; however, the in vivo expression and activation status of FKHRL1 in breast tumors still remains unclear. We immunohistochemically demonstrated the expression and intracellular localization of FKHRL1 in human breast tumors by the novel anti-FKHRL1 antibody which is available for formalin-fixed paraffin-embedded specimens. In a total of 51 cases of benign tumors, FKHRL1 was diffusely expressed in all cases, and its intracellular localization was revealed to be cytoplasmic (inactive form) in 94% of cases of intraductal papillomas (16/17) and 91% cases of fibroadenomas (31/34), with a similar pattern to normal glandular epithelium. In invasive ductal carcinomas, 83% of the cases (93/112) diffusely expressed FKHRL1; however, unlike benign tumors, 71% of the cases (66/93) showed the nuclear-targeted, active form of FKHRL1. Moreover, activated FKHRL1 was predominantly observed in scirrhous (29/36, 81% of the cases) and papillotubular (30/38, 79% of the cases) subtypes, compared to the solid-tubular subtype (7/19, 37% of the cases). Furthermore, the cases with nuclear-targeted FKHRL1 showed a tendency to have lymph nodal metastasis with statistical significance (P < 0.0001). Thus, the activation of FKHRL1 seems to be recognized as one of the specific features of invasive ductal carcinoma of the breast.     

Real-time imaging of individual fluorescent-protein color-coded metastatic colonies in vivo

We have established stable, bright green fluorescent protein (GFP)- or red fluorescent protein (RFP)-expressing HT-1080 human fibrosarcoma clones. These cell lines showed similar cell proliferation rates and high-frequency experimental lung metastasis. The HT-1080-GFP and -RFP clones enable simultaneous real-time dual-color imaging in the live animal. HT-1080 cells were transduced with retroviral vectors containing GFP or RFP and the neomycin resistance gene. Stable transformants were selected stepwise with G418 up to 800 μl/ml. Subsequently, high GFP- or RFP-expressing clones, HT-1080-GFP or HT-1080-RFP, respectively, were selected. 3×10⁶ cells from each clone were mixed and injected into the tail vein of SCID mice. The cells seeded the lung at high frequency with subsequent formation of pure green and pure red colonies as well as mixed yellow colonies with different patterns visualized directly on excised lungs. The lung metastases were also visualized by external fluorescence imaging in live animals through skin-flap windows over the chest wall. Lung metastases were observed on the lung surface of all mice. SCID mice well tolerated multiple surgical procedures for direct-view imaging via skin-flap windows. Real-time metastatic growth of the two different colored clones in the same lung was externally imaged with resolution and quantification of green, red, or yellow colonies in live animals. The color coding enabled determination of whether the colonies grew clonally or were seeded as a mixture with one cell type eventually dominating, or whether the colonies grew as a mixture. The simultaneous real-time dual-color imaging of metastatic colonies described in this report gives rise to the possibility of color-coded imaging of clones of cancer cells carrying various forms of gene of interest. This revised version was published online in July 2006 with corrections to the Cover Date.     

Preparation of ceramic microspheres for in situ radiotherapy of deep-seated cancer

Radiotherapy is one of the most effective treatments for cancers. However, external irradiation provides only small doses to deep-seated cancers, and often causes damage to healthy tissues. It has been reported that 20-30 microm diameter 17Y(2)O(3)-19Al(2)O(3)-64SiO(2) (mol%) glass microspheres are useful for the in situ irradiation of cancers. Yttrium-89 (89Y) in this glass can be neutron bombarded to form the beta-emitter 90Y (half-life=64.1h). When injected in the vicinity of the cancer, such activated glass microspheres can provide a large localized dose of beta-radiation. The Y(2)O(3) content of the glass in the microspheres is limited to only 17 mol%. Chemically durable microspheres with a higher Y(2)O(3) content need to be developed. Phosphorus-31 (31P) with 100% natural abundance can also be activated by neutron bombardment to form the beta-emitter 32P (half-life=14.3d). Chemically durable microspheres containing a high phosphorus content are expected to be more effective for cancer treatment. We prepared pure Y(2)O(3) and YPO(4) microspheres using a high-frequency induction thermal plasma melting technique, and investigated the resulting structure and chemical durability. We successfully prepared smooth, highly spherical polycrystalline Y(2)O(3) and YPO(4) microspheres with diameters in the range 20-30 microm. Both the Y(2)O(3) and YPO(4) microspheres showed high chemical durability in saline solutions buffered at pH=6 and 7. These microspheres are expected to be more effective than the conventional glass microspheres for the in situ radiotherapy of cancer.     

Prostaglandin E1-initiated immune regulation during human mixed lymphocyte reaction

Prostaglandin E1 (PGE1) has therapeutic value for transplantations due to its microvascular activity. Interleukin (IL)-18, which is elevated in plasma during the acute rejection after organ transplantation, elicits the expression of intercellular adhesion molecule (ICAM)-1, B7.1, B7.2, CD40, and CD40 ligand (CD40L) on monocytes as well as the production of interferon (IFN)-gamma and IL-12 and proliferation of T-cells during the human mixed lymphocyte reaction (MLR) in an in vitro model of acute rejection. In contrast, PGE1 inhibits all the adhesion molecule expression, cytokine production and T-cell proliferation in the presence of IL-18. The effects of PGE1 depend on stimulation of the IP/EP2/EP4-receptor, and thus, PGE1 might have therapeutic potential for treating acute rejection due to its immune regulatory effect.     

Axonal trajectory of single group Ia and Ib fibers in the cat spinal cord

Intracellular staining with HRP of physiologically identified group Ia and Ib afferent fibers in the adult cat lumbosacral cord revealed that group Ia and Ib fibers take a similar course in the dorsal funiculus, but the collaterals emerging from them show a different topographical distribution and a different mode of branching in the gray matter. Ia collaterals terminate in laminae VI, VII, IX, and sometimes VIII, whereas Ib collaterals terminate only in lamina VI, or both VI and IX. In lamina IX, two large motor-type neurons received terminations of both Ia and Ib fibers at the same time.