Sanae Hisayasu

Complete sequence analysis of rat transferrin and expression of transferrin but not lactoferrin in the digestive glands

Rat milk and digestive juices contain transferrin but not lactoferrin, which is a major iron-binding protein in these secretions of human and mouse. To compare the structure of rat transferrin to that of transferrins and lactoferrins in other species, we isolated a cDNA clone containing the entire coding region of transferrin from rat liver and determined its sequence. The amino-acid sequence of rat transferrin had 69.8% identity with that of human transferrin and 48.8% identity with that of human lactoferrin. Rat transferrin, like other transferrins, had the potential N-linked glycosylation site only in the C-terminal domain, although lactoferrins characterized so far contained the glycosylation sites in both the N- and C-terminal domains. Southern and Northern analyses showed that there was the gene specifically hybridized with the mouse lactoferrin cDNA in rat genomic DNA, but only the transferrin mRNA was detected in mammary gland, submaxillary gland and pancreas of rat. These results suggest that the rat lactoferrin gene is silent in the mammary gland, and transferrin can serve as a functional substitute for lactoferrin in rat.

Targeted Gene Delivery to Selected Liver Segments Via Isolated Hepatic Perfusion

BACKGROUND Development of targeted gene transfer technologies is essential for in vivo gene therapy. In this study, we examined the feasibility of physically targeting an adenoviral vector to selected liver segments in rats by isolating the hepatic perfusion (IHSP) and clamping the portal vein between the upper and lower segments. MATERIALS AND METHODS The rats were divided into two groups: IHSP group and the inferior vena cava (IVC) group. The adenoviral vector, which harbored the beta-galactosidase (beta-gal) gene, was administered via the portal vein, after which unbound vector particles were washed out with phosphate-buffered saline (PBS) and removed via the cannulated inferior vena cava (IVC) in IHSP group, while the IVC group received the transgene directly via the IVC without isolation of the hepatic perfusion. RESULTS With this configuration (IHSP group), >99% of the beta-gal activity was limited to the targeted hepatic lobes, findings which were confirmed by histochemical staining with X-gal. We also found there to be significant differences in transgene expression among the hepatic lobes in the IVC group. CONCLUSIONS Taken together, these results indicate that the IHSP technique is useful for local gene delivery to selected liver segments, and that when evaluating the efficacy of IHSP in the treatment of liver disease (e.g., nonresectable tumors), interlobar differences must be given careful consideration to ensure that sufficient drug or vector is delivered to all targeted hepatic lobes.

Milk growth factor (MGF)-induced differentiation of NT2/D1 cells.

The differentiation activity of milk growth factor (MGF, 200 ng/ml), which also has proliferative activity, was investigated in NT2/D1 cells relative to that of retinoic acid (RA, 10(-7) M). MGF suppressed the proliferation of NT2/D1 cells to the same extent as RA after cultivation for 2x4 days. MGF enhanced Fas expression in NT2/D1 cells and prevented the decrease of Fas expression when RA was also added. MGF induced the synthesis of alpha-smooth muscle actin (alpha-SM-actin) in NT2/D1 cells without fibrils, but RA did not have such a potent activity. MGF extended glial fibrillary acidic protein (GFAP) that existed in a local area of NT2/D1 cell cytoplasm. On the other hand, RA enhanced GFAP expression and dispersed it throughout the cells. MGF slightly induced neurofilament-medium size (NF-M) synthesis in NT2/D1 cells that RA induced in the cells. MGF was less effective than RA in stimulating the synthesis of epinephrine in the cells, and the additive effect of MGF and RA enhanced epinephrine synthesis. While dopamine synthesis was less effectively stimulated by MGF than by RA, an additive effect of MGF and RA for dopamine synthesis was not observed in the cells. It was thus found that MGF differentiated NT2/D1 cells through alpha-SM-actin-synthesis.

Decreased mucosal iron induces an increase in intestinal iron absorption in the early stage of iron-deficiency

To investigate the contribution of non-heme iron and ferritin in the intestinal mucosa in regulating intestinal iron absorption during the early stage of iron-deficiency, we performed an in vivo study using rats fed an iron-deficient diet. Intestinal iron absorption was measured using the ligated intestine method. Rats fed the iron-deficient diet for 2 days showed no change in hematological parameter values (blood hemoglobin, plasma iron, the total iron binding capacity) and no change in the content of non-heme iron and ferritin in the liver. However, uptake of iron into the mucosa (mucosal uptake) and transfer of iron from mucosal cells to blood (serosal transfer) was significantly increased compared with the initial levels. At that time, the content of non-heme iron in the mucosa significantly decreased, however, no significant change in mucosal ferritin was observed. Extending the period of feeding the iron-deficient diet resulted in a small additional decrease in the level of mucosal non-heme iron, but led to no significant increase in mucosal uptake and serosal transfer. These findings suggest that a decrease in non-heme iron content in the intestinal mucosa may induce an increase in intestinal iron absorption during the early stage of iron-deficiency. In addition, it was evident that there is an upper limit in intestinal iron absorption. This limit in iron absorption may play a role in preventing iron-overload in the body.

Effects of vitamin A on eicosanoids production in the lung of rat treated by thermal injury.

After the treatment of thermal injury, contents of leukotriene C4 (LTC4) increased significantly at early stage and then kept the high level of LTC4 during the inflammation. Administration of vitamin A (VA) on vitamin A deficient- (AD) rats suppressed LTC4 induction in the lung and also inflammation and then gradually induced the significant decrease of LTC4 contents to the normal level 3 days after treatment of thermal injury. Synthesis of 6KPGF1α in the lung of AD rat was induced gradually until 24 hours after thermal injury. While the administration of VA to AD-rat enabled in the lung to induce the synthesis of 6-KPGF1α enough at early stage of thermal injury. After this induction, the production of 6KPGF1α gradually decreased. These results suggest that VA will suppress specifically the induction of lipoxygenase products but will not interfere the synthesis of cyclooxygenase products.