Enhanced expression of high-affinity iron transporters via H-ferritin production in yeast.

Our heterologous expression system of the human ferritin H-chain gene (hfH) allowed us to characterize the cellular effects of ferritin in yeasts. The recombinant Saccharomyces cerevisiae (YGH2) evidenced impaired growth as compared to the control, which was correlated with ferritin expression and with the formation of core minerals. Growth was recovered via the administration of iron supplements. The modification of cellular iron metabolism, which involved the increased expression of high-affinity iron transport genes (FET3 and FTR1), was detected via Northern blot analysis. The findings may provide some evidence of cytosolic iron deficiency, as the genes were expressed transcriptionally under iron-deficient conditions. According to our results examining reactive oxygen species (ROS) generation via the fluorescence method, the ROS levels in YGH2 were decreased compared to the control. It suggests that the expression of active H-ferritins reduced the content of free iron in yeast. Therefore, present results may provide new insights into the regulatory network and pathways inherent to iron depletion conditions.

Induction of autoimmunity by immunization with hapten-modified hen egg lysozyme in hen egg lysozyme-transgenic mice.

To understand the mechanism of autoimmunity induction, hen egg lysozyme (HEL)-transgenic (Tg) C57BL/6 (B6) mice were immunized with HEL or phosphorylcholine-conjugated HEL (PC-HEL). Repeated immunization of HEL-Tg mice with native HEL failed to induce the antibody response against HEL. However, immunization with PC-HEL generated a significant anti-HEL antibody response. Immunization of the Tg mice with dominant (HEL(74-88)) or cryptic (HEL(47-61)) T-cell epitope peptide stimulated the corresponding T-cell response and similarly yielded the anti-HEL antibody response. Predominance of immunoglobulin G1 (IgG1) anti-HEL antibody response in the HEL-Tg mice and preferential IL-4 production by HEL-specific T cells suggested the dependency of the antibody response to the presence of T helper 2. HEL-Tg mice received HEL-primed B6 T cells, but not HEL-primed Tg T cells, were able to generate anti-HEL antibody response following PC-HEL immunization. The pattern and the level of epitope peptides generated by splenic antigen-presenting cells indicated that PC-HEL results in much more efficient processing as compared to HEL. These results strongly suggest that the enhancement of antigen processing by hapten (PC) conjugation to the antigen facilitates more efficient stimulation of T cells reactive to self antigen, HEL in HEL-Tg mice resulting in the production of anti-self HEL antibody.

Recovery from iron deficiency in rats by the intake of recombinant yeast producing human H-ferritin.

OBJECTIVE: This study examined whether iron accumulated in ferritin-producing recombinant microbes is bioavailable to rats with iron deficiency. METHODS: Rats induced with iron deficiency were treated with iron preparations of ferrous ammonium sulfate, horse spleen ferritin, control yeast, and ferritin-producing recombinant yeast for 14 d. The bioavailability of iron was examined by measuring hemoglobin concentration, hematocrit value, and tissue iron stores. Differences between dietary groups were determined by one-way analysis of variance, and P < 0.05 was considered statistically significant. RESULTS: Based on hemoglobin concentration and hematocrit value, iron in ferrous ammonium sulfate, horse spleen ferritin, and ferritin-producing yeast were bioavailable to rats and cured iron deficiency. The efficacy of ferritin and ferritin-producing yeast was also confirmed in establishing tissue iron stores after induction of iron deficiency. CONCLUSIONS: The iron sources of ferritin and ferritin-producing yeast were as effective in recovery from iron deficiency as the iron compounds of ferric citrate and ferrous ammonium sulfate. The results suggest that the iron stored in the ferritin of recombinant yeast is bioavailable, and that recombinant yeast may contribute widely as a source of iron to resolve the global problem of iron deficiency.

Suppression of distant pulmonary metastasis of MDA-MB 435 human breast carcinoma established in mammary fat pads of nude mice by retroviral-mediated TIMP-2 gene transfer.

BACKGROUND: Previous studies have shown that TIMP-2 overexpression is a useful therapeutic tool for inhibiting tumor growth and invasion in animals. However, it has not been reported whether genetic manipulation for TIMP-2 overexpression can induce an inhibitory effect on spontaneous metastasis from the primary tumor site to other organs such as lungs or lymph nodes in an animal model. METHODS: The present studies describe the effects of retrovirus-mediated TIMP-2 gene transfer into human breast cancer cell lines on the in vitro invasion of the tumor cells or the in vivo growth in nude mouse. Here we also used retroviral-mediated TIMP-2 overexpression by intratumoral injection for suppression of metastasis in human breast carcinoma established in the mammary fat pad of nude mice. RESULTS: As expected, overexpression of TIMP-2 inhibited matrix metalloprotenase (MMP) activity and invasion of the tumor cells. Also, the growth rate of tumors grafted with the breast cancer cells transduced with the retrovirus vector encoding TIMP-2 cDNA was significantly slower than the growth rate of tumors grafted with the breast cancer cells transduced with a control retrovirus vector. Furthermore, single intratumoral injection of the TIMP-2 retrovirus-producing cells into human breast tumor tissue established in mammary fat pads of nude mice showed a dramatic decrease in size and number of lung metastatic tumors. CONCLUSIONS: Retrovirus-mediated TIMP-2 gene transfer into human breast cancer cells is able to down-regulate invasion and show that tumor-derived angiogenesis is reduced. In this model, retroviral-mediated transduction of TIMP-2 cDNA into a limited population of human tumor cells inhibits tumor growth and prevents distant pulmonary metastasis. These results indicate that it may not be necessary to deliver and express these genes in every single tumor cell as long as the level of expression in a limited number of transduced cells is sufficient to prevent the excessive breakdown of the extracellular matrix.