Dong Jae Kim

Developmental toxicity and brain aromatase induction by high genistein concentrations in zebrafish embryos

Genistein is a phytoestrogen found at a high level in soybeans. In vitro and in vivo studies showed that high concentrations of genistein caused toxic effects. This study was designed to test the feasibility of zebrafish embryos for evaluating developmental toxicity and estrogenic potential of high genistein concentrations. The zebrafish embryos at 24 h post-fertilization were exposed to genistein (1 3 10−4 M, 0.5 3 10−4 M, 0.25 3 10−4 M) or vehicle (ethanol, 0.1%) for 60 h. Genistein-treated embryos showed decreased heart rates, retarded hatching times, decreased body length, and increased mortality in a dose-dependent manner. After 0.25 3 10−4 M genistein treatment, malformations of survived embryos such as pericardial edema, yolk sac edema, and spinal kyphosis were also observed. TUNEL assay results showed apoptotic DNA fragments in brain. This study also confirmed the estrogenic potential of genistein by EGFP expression in the brain of the mosaic reporter zebrafish embryos. This study first demonstrated that high concentrations of genistein caused a teratogenic effect on zebrafish embryos and confirmed the estrogenic potential of genistein in mosaic reporter zebrafish embryos.

Isolation and characterization of spheroid cells from human malignant melanoma cell line WM-266-4.

Background/Aims: Spheroid cells which can grow as nonattached spheroids in vitro culture condition are considered as tumor-initiating cells that have properties similar to those of stem cells. However, the existence of spheroid cells in WM-266-4, a human malignant metastatic melanoma cell line, has not yet been reported. Methods: Accordingly, we investigated whether WM-266-4 can form spheroids, and characterized these spheroids using qRT-PCR, histology, immunohistochemistry and xenograft. Results: WM-266-4 contains a small subpopulation of cells that grow as spheroids and express genes strongly related to tumor malignancy and stem-like factors. Second, histological analysis of the spheres revealed that they consist of 300–400 round cells per sphere with a high karyoplasmic ratio. They have a basophilic cytoplasm and are highly pleomorphic in size, and sometimes multinucleated and giant. Third, although there were differences between the spheroid and bulk cells, they both have high tumorigenic potential, as both cell types formed a tumor mass upon injection of only 100 cells in nude mice. Conclusion: We characterized the spheroid cells in an established melanoma cell line. We suggest that enriched spheroid cells might contain more dedifferentiated progenitor cells, but we could not conclude spheroid cells are cancer stem cells.

Benomyl induction of brain aromatase and toxic effects in the zebrafish embryo

Benomyl is a benzimidazole fungicide that has been widely used on a variety of food crops and ornamental plants. It is known to cause adverse effects on reproductive systems, including decreased testicular and epididymal weights and reduced epididymal sperm counts and fertility. The brain aromatase gene is up‐regulated by estrogens and estrogen mimics and considered a target gene to screen estrogen mimics. This study was designed to test the estrogenic potential and toxic effects of benomyl in the zebrafish system, and validated this system as a model that may correspond to the effect of benomyl in rodents. Concentrations of 20 × 10−6, 40 × 10−6 and 80 × 10−6 m of benomyl‐treated embryos showed decreased survival, hatching and heart rates, and increased incidence of malformations, such as pericardial edema, spinal lordosis, elongated heart, head edema, eye lens protrusion and caudal fin disappearance. Benomyl induced enhanced green fluorescent protein (EGFP) expression in the mediobasal hypothalamus (MBH) in transient zebrafish embryos with a brain aromatase‐based reporter gene. In this study, we determined that benomyl has estrogenic potential based on zebrafish brain aromatase gene induction, and that benomyl is toxic at 20 × 10−6 m concentration and higher. These results demonstrate the usefulness of zebrafish embryos as an in vivo system to examine the estrogenic and developmental toxic potential of unknown compounds. Copyright

Gastric Lesions and Immune Responses caused by Long-term Infection with Helicobacter heilmannii in C57BL/6 Mice

Helicobacter heilmannii is a gastric micro-organism that can induce gastritis and B-cell MALT (mucosa-associated lymphoid tissue) lymphoma in mice, in a host-dependent manner. The present study was designed to examine gastric lesions and immune responses caused by intragastric H. heilmannii infection of an inbred mouse strain, C57BL/6. Long-term infection led to the formation of gastric nodules and increased mucosal thickness of the stomach, due to gastric epithelial proliferation. Infection also induced the formation of lymphoid follicles in the corpus mucosa and submucosa. The follicular cells were mainly CD45R+ cells that did not produce immunoglobulin. However, scattered in the lamina propria and corpus submucosa, numerous IgA+ cells were found in infected mice, but not in control mice. RT-PCR results showed that H. heilmannii infection led to increased mRNA expression for IFN-gamma (a Th1 cytokine) and IL-10 (a Th2 cytokine) in the mouse stomach, suggesting that both Th1 and Th2 responses are associated with H. heilmannii infection. The mRNA of other cytokines and chemokines (IL-1beta, IL-12p40, TNF-alpha, MCP-1, KC and MIP-2) was also increased by infection.

NaCl plus chitosan as a dietary salt to prevent the development of hypertension in spontaneously hypertensive rats.

The effect of NaCl plus 3% chitosan on the systolic blood pressure of spontaneously hypertensive rats (SHR) were evaluated and compared with NaCl plus KCl (NaCl, 49.36% + KCl 49.36%) and chitosan or NaCl treatment alone. In SHR, administration of NaCl plus chitosan (44 mM Na/day) for two months significantly decreased the systolic blood pressure greater than of NaCl plus KCl and NaCl alone. NaCl plus chitosan resulted, though not statistically significant, in decreased urinary Na+ excretion and decreased blood urea nitrogen levels. Urinary creatinine of NaCl plus chitosan was slightly decreased compared to 3 treated groups. Serum electrolytes levels, however, remained unchanged. The combination of NaCl and chitosan may be superior to the conventional use of NaCl plus KCl or NaCl alone in the prevention of hypertension. Even though these supplementary diets have demonstrated potential anti-hypertensive effects in the experimental animal model, further research is needed before any recommendations can be made.

Search for potential target site of nucleocapsid gene for the design of an epitope-based SARS DNA vaccine

Abstract It is believed today that nucleocapsid protein (N) of severe acute respiratory syndrome (SARS)-CoV is one of the most promising antigen candidates for vaccine design. In this study, three fragments [N1 (residues: 1–422); N2 (residues: 1–109); N3 (residues: 110–422)] of N protein of SARS-CoV were expressed in Escherichia coli and analyzed by pooled sera of convalescence phase of SARS patients. Three gene fragments [N1 (1–1269 nt), N2 (1–327 nt) and N3 (328–1269 nt)—expressing the same proteins of N1, N2 and N3, respectively] of SARS-N were cloned into pVAX-1 and used to immunize BALB/c mice by electroporation. Humoral (by enzyme-linked immunosorbent assay, ELISA) and cellular (by cell proliferation and CD4+:CD8+ assay) immunity was detected by using recombinant N1 and N3 specific antigen. Results showed that N1 and N3 fragments of N protein expressed by E. coli were able to react with sera of SARS patients but N2 could not. Specific humoral and cellular immunity in mice could be induced significantly by inoculating SARS-CoV N1 and N3 DNA vaccine. In addition, the immune response levels in N3 were significantly higher for antibody responses (IgG and IgG1 but not IgG2a) and cell proliferation but not in CD4+:CD8+ assay compared to N1 vaccine. The identification of antigenic N protein fragments has implications to provide basic information for the design of DNA vaccine against SARS-CoV. The present results not only suggest that DNA immunization with pVax-N3 could be used as potential DNA vaccination approaches to induce antibody in BALB/c mice, but also illustrates that gene immunization with these SARS DNA vaccines can generate different immune responses.

Protective and therapeutic effects of an extract mixture of alder tree, labiate herb, milk thistle green bean-rice bran fermentation, and turnip against ethanol-induced toxicity in the rat.

An herbal extract mixture and yogurt added to the herbal extract mixture were tested for their protective and therapeutic effects on ethanol-induced liver injury. The herbal extract mixture, yogurt and commercial drugs were used for treatment for two weeks prior to administering a single oral dose of ethanol (3 g/kg body weight). The herbal extract mixture and yogurt added to the herbal extract mixture were found to provide protection against ethanol-induced toxicity comparable to the commercial drug treatment, according to the serum and histopathological analysis. It was also shown that co-treatment with herbal extract mixture and yogurt against a triple oral dose of ethanol (2 g/kg body weight, over one week) provided protection against ethanol toxicity. After the initial set of experiments, the herbal extract mixture and yogurt treatments were extended for three more weeks. When compared to the positive control, further treatment with both the herbal extract and yogurt significantly reduced liver injury and resulted in a lower grade of lipid deposition.

Production of specific antibodies against SARS-coronavirus nucleocapsid protein without cross reactivity with human coronaviruses 229E and OC43.

Severe acute respiratory syndrome (SARS) is a life-threatening disease for which accurate diagnosis is essential. Although many tools have been developed for the diagnosis of SARS, false-positive reactions in negative sera may occur because of cross-reactivity with other coronaviruses. We have raised polyclonal and monoclonal antibodies (Abs) using a recombinant form of the SARS virus nucleocapsid protein. Cross-reactivity of these anti-SARS Abs against human coronavirus (HCoV) 229E and HCoV OC43 were determined by Western blotting. The Abs produced reacted with recombinant SARS virus nucleocapsid protein, but not with HCoV 229E or HCoV OC43.