In order to study the role of the protein shell in both iron uptake and iron core formation of ferritin, we constructed a deletion mutant of the ferritin gene and expressed the mutant gene in Escherichia coli, This mutant was obtained by introducing an amber mutation at position Pro-157 and a deletion of the 19 amino acid residues at the carboxy-terminus of the recombinant tadpole H-chain ferritin. The deleted amino acids correspond to E-helix forming the hydrophobic channel in the protein. E. coli harboring the plasmid pTHP157, which contains the deleted gene, was grown at $23^{\circ}C$ in the presence of 0.1 mM IPTG, and the induced protein appeared to be partly soluble. Nondenaturing polyacrylamide gel electrophoresis showed that the expressed mutant H-chains coassemble into holoprotein, suggesting that E-helix is not necessary for assembly of the subunits as reported for human H-chain ferritin. Its ability in iron core formation was proven in an Fe staining gel, the result disagreeing with the observation that the hydrophobic channel is necessary for iron core formation in human H-chain ferritin.
These studies were carried out for the elucidation of liming effect on the growth of rice seedlings and the chemical characteristics of an acid sulphate paddy that shows not only extremely high acidity of soil but also poor growth of rice plants, consequently low yield. Thus the liming effect on the changes of acidity, oxidation-reduction potential, and the contents of iron, aluminium, sulphate, and phosphorus fractions in the soil was investigated under the waterlogging and drying condition. The reclaimable or inhibitory effect of phosphorus, iron and aluminium on the growth of rice seedlings was also investigated under liming. The results are summarized as follows: 1. After liming, the pH of the acid sulphate subsoil decreased again on drying. 2. The oxidation-reduction potential reached a minimum after 5 days of flooding and greatly decreased on liming but increased after drying. 3. The contents of ferrous iron soluble in water-and Morgan's solution reached a maximum after 15 days of flooding and only the content of water soluble ferrous iron was greatly decreased. 4. The content of aluminium soluble in water-and Morgan's solution decreased by flooding and liming, and showed a tendency to increase on drying. 5. In the limed acid sulphate soil, the content of water soluble calcium showed a highly significant negative correlation with the content of sulphate and liming decreased sulphate content in the soil. 6. The contents of total phosphorus was 496.3 ppm in the acid sulphate topsoil and 387.5 ppm in the subsoil. The content of each phosphorus fraction was in the order of Fe-P>Occ. Fe-P>Ca-P>Occ. Al-P>Al-P and Fe-P content in the soil was the highest fraction among them. 7. Lime application increased greatly Ca-P and Al-P, and Occ. Fe-P and Occ. Al-P only slightly, but decreased Fe-P differently in each soil. 8. Effect of phosphorus on the dry matter yield of rice seedlings was great. The optimum amount of phosphorus to produce maximum dry matter yield of rice seedlings appeared to be 6.8% of maximum absorption (absorption coefficient) without liming and 10.0% with liming. 9. In rice seedlings liming increased the content and uptake of calcium and silica but decreased those of iron and aluminium. Phosphorus application increased the content and uptake of phosphorus and decreased iron while the application of iron and aluminium increased their contents and uptake but decreased those of phosphorus. 10. Liming greatly alleviated such toxicity of iron and aluminium. 11. When phosphorus was applied, the dry matter yield of rice seedlings showed highly significant positive correlations with uptake of phosphorus, calcium and silica each. When iron and aluminium were applied, dry matter yields indicated significant positive correlations with the contents or uptake of calcium and silica each, but significant negative correlations with the content or uptake of iron and aluminium. 12. Under the application of phosphorus and lime, dry matter yields showed significant positive correlations with pH and Morgan's extractable calcium each of the soil samples after harvest. Under the application of lime, iron and aluminium, dry matter yields showed significant positive correlations with pH, calcium and silica each, but negative correlations with iron and aluminium contents each of the soil samples after harvest.
Pathways both mediated by and independent of transferrin(Tf) and the TfR have been described for the accumulation of iron. Although it is not clear whether the same systems take up iron and gallium, these pathways may suggest the contention that uptake of Ga-67 can, in fact, occur by both Tf-independent and Tf-dependent systems and may share with Fe-59 in part the same mechanism for uptake. The predominant system by which uptake of both radiometals occurs may be different in the degree of the transformation of tumor. Transformed(MMSV/3T3) and untransformed(BALB/3T3) cells were incubated with luM of Ga-67-citrate of Fe-59-chloride for 15 min. at $37^{\circ}C$ in either the presence or absence of Tf. After then, the monolayers were washed with HBSS or PBS, and the cells were solubilized in 1% SDS for gamma well counting and protein determinations. There were similarities, as well as differences, in the pattern of uptake of Fe-59 and Ga-67 presented both in ionic from and as bound to Tf. Both radiometals appeared gain to cells in either ionic or Tf-bound forms. Transformed cells appeared to accumulate more radiometal, either Ga-67 or Fe-59 in the presence of Tf than do the their untransforemd counterparts. Conversly the presentation of either radiometal in ionic form resulted in significantly greater accumulation of metal by the untransformed cells than those transformed. The efficiency for uptake of Ga-67 or Fe-59 in the absence of Tf was greater than for uptake of the Ga-Tf or Fe-Tf. However, the magnitude of difference in efficiency of uptake was greater for Fe-59(10-fold) than for Ga-67 (3-fold). Our results Supports the theory that both Tf-independent and Tf-dependent systems for the uptake of Ga-67 both systems operate oppositely between transformed cells and those untransformed, with uptake by the predominating in transformed cells by the Tf-mediated system and in untransformed cells by the Tf-independent. The uptake of Ga-67 by tumor may share with Fe-59 in part the same mechanism.
Choe, Yunjeong;Yoo, Ah Young;Kim, Sam Woong;Hwang, Jihwan;Kang, Ho Young
Journal of Life Science
/
v.27
no.1
/
pp.72-77
/
2017
Inorganic iron is essential for various metabolic processes, including RNA synthesis, electron transport, and oxygen detoxification in microorganisms. Many bacterial pathogens compete for iron acquisition in diverse environmental condition such as host. Salmonella Typhimurium SL1344 also requires inorganic iron as a cofactor for growth. When a M9 minimal liquid medium was supplemented with ethylenediamine di-o-hydroxyphenylactic acid (EDDA) which acts as an iron-chelating agent, growth of Salmonella Typhimurium SL1344 in the supplemented medium was completely arrested by deficient of useful iron under iron-depleted condition. However, a number of siderophores, which are small, high-affinity iron chelating compounds secreted by microorganisms such as bacteria and fungi, were produced for utilization of restricted iron under iron-depleted condition. A M9 minimal liquid medium complemented with human transferrin (hTf)-iron complex turned completely off production of siderophores, but growth of Salmonella Typhimurium SL1344 maintained level similar to compare one complemented with iron (III) chloride (FeCl3). This means that human transferrin (hTf)-bound iron can utilize via directly interaction with Salmonella Typhimurium SL1344 without productions of siderophores. Through construction and analysis of negative mutant for utilization of human transferrin (hTf)-bound iron, we confirm that the bacterium can directly use human transferrin (hTf)-bound iron without extracellularly intermediated carriers such as siderophores.
Iron (Fe) is an essential metal in biological processes, which maintains a homeostasis in the human body. Divalent metal transporter 1 (DMT1) has been known as an iron transporting membrane protein, which is involved in the uptake Fe at the apical portion of intestinal epithelium, and may transport Fe across the membrane of acidified endosome in peripheral tissues. In this study, we studied the tissue distribution of DMT1 in the Fe supplemented (FeS) diet fed rats, and the regulation of DMT1 expression by depleting body Fe. Sprague-Dawley rats were divided into two groups, and fed FeS (120 mg Fe/kg) diet or Fe deficient (FeD, 2∼6 mg Fe/kg) diet for 4 weeks. The evaluation of body Fe status was monitored by measuring sFe, UIBC and tissue Fe concentration. Additionally, DMT1 mRNA levels were analyzed in the peripheral tissues by using the quantitative real time RT-PCR method. In the FeS diet fed rats, the tissue Fe was maintained at a relatively high level, and DMT1 was eventually expressed in all tissues studied. DMT1 was highly expressed in the testis, kidney and spleen, while a moderate levels of DMT1 expression was detected in the brain, liver and heart. In the digestive system, the highest level of DMT1 was found in the duodenum. Feeding the FeD diet caused a reduced body weight gain and depletion of body Fe with finding of decreased sFe, increased UIBC and decreased tissue Fe concentration. The depletion of body Fe upregulated DMT1 expression in the peripheral tissue. The expression of DMT1 was very sensitive to the body Fe depletion in the small intestine, especially in the duodenum, showing dramatically higher levels in the FeD rats than those of the FeS group. In the FeD diet fed animals, the expression of DMT1 was low significantly in other tissues compared with the duodenum. The expression of DMT1, however, was 60∼120% higher in the testis, kidney and spleen, and 30∼50% higher in the lung, liver and heart, compared to the FeS diet fed rats. In summary, DMT1 expression was ubiquitous in mammalian tissue, and the level of expression was the organ-dependent. The expression of DMT1 in peripheral tissues was upregulated by depletion of body Fe. Duodenum was the most sensitive tissue among organs studied during Fe depletion, and expressed the greatest level of DMT1, while other tissues were less higher than in duodenum. This study supports that DMT1 plays a role in maintaining the body Fe level through intestinal uptake as well as homeostasis of Fe in the peripheral tissue.
Silk fabrics were dyed by Cochineal extracts after mordanted with various mordants and their properties of dyeing, fastness and change of surface color were investigated. Silk fabrics were mordanted with aluminum potassium sulfate, copper acetate, tin chloride, iron sulfate or chromium potassium sulfate at $60^\circ{C}$ for 30 minutes and subsequently dyed at $60^\circ{C}$ for 60 minutes unless mentioned otherwise. The dye-uptake of silk fabrics increased with the concentration of Cochineal extracts and mordants used. Maximum dye-uptake was obtained at pH 3.5-4, regardless of the type of mordants used. Surface color of silk fabrics changed from red purple to purple when mordanted with Al or Cu mordant, from purple to purple blue with Fe mordant and showed red purple with Sn or Cr mordant, respectively. It also changed according to pH of dyeing bath. Dyed silk fabrics showed poor light fastness but good dry-cleaning, perspiration and rubbing fastness.
Seo, Ji-Hyoung;Bae, Jin-Ho;Jeong, Sin-Young;Lee, Jae-Tae;Lee, Kyu-Bo;Ahn, Byeong-Cheol
The Korean Journal of Nuclear Medicine
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v.38
no.1
/
pp.109-110
/
2004
Purpose: Tc-99m MDP bone scan was performed to evaluate a generalized bone pain in a 24-year-old male chronic myelogenous leukemia patient who received bone marrow transplantation at 7 months ago. The patient had received large amounts of blood transfusion for managing symptoms related to anemia. Bone scan revealed substantial splenic tracer uptake. Magnetic resonance image and laboratory evidence of hemochromatosis suggests that the presence of large quantities of iron in the spleen of this patient may have been responsible for the splenic uptake of the bone scanning agent. The authors report a case of splenic hemochromatosis incidentally found on Tc-99m MDP bone scan.
The $P_{entC}$ promoter of the entCERA operon encoding enzymes for enterobactin biosynthesis in Escherichia coli is tightly regulated by the availability of iron in the culture medium. In iron-rich conditions, the $P_{entC}$ promoter activity is strongly repressed by the global transcription regulator Fur (ferric uptake regulator), which complexes with ferrous ions and binds to the Fur box 19-bp inverted repeat. In this study, we have constructed the expression vector pOS2 containing the $P_{entC}$ promoter and characterized its repression, induction, and modulation by quantifying the expression of the lacZ reporter gene encoding $\beta$-galactosidase. $\beta$-Galactosidase activities of E. coli transformants harboring pOS2-lacZ were highly induced in the presence of divalent metal ion chelators such as 2,2'-dipyridyl and EDTA, and were strongly repressed in the presence of excess iron. It was also shown that the basal level $\beta$-galactosidase expression by the $P_{entC}$ promoter was drastically decreased by incorporating the fur gene into the expression vector. Since the newly developed iron chelator-inducible expression system is efficient and cost-effective, it has wide applications in recombinant protein production.
Kim, Hyoung-Won;An, Eun-Kyoung;Shin, Mi-Young;Kim, In-Ho
한국생물공학회:학술대회논문집
/
2003.10a
/
pp.544-547
/
2003
Fusion $ferritin(F_H+F_L),$ an iron-binding protein, was purified from recombinant E. coli by gel filtration chromatography after two-step sonications. Unfolded ferritin was refolded by GFC with various refolding enhancing additives. 50 mM Tris-HCI(pH 7.4) buffers containing 2 M urea and additive was used in GFC. Objective was to characterize the structure change at various conditions. Molecular weight was determined using GF-HPLC and RP-HPLC was used to quantify the unfolded and refolded proteins. Activity was confirmed by iron-uptake reaction.
Bressler Joseph P.;Olivi Luisa;Cheong Jae Hoon;Kim Yongbae;Bannon Desmond
Proceedings of the Korean Society of Applied Pharmacology
/
2004.11a
/
pp.50-61
/
2004
Lead and cadmium are potent environmental toxicants that affect populations living in Europe, Americas, and Asia. Identifying transporters for lead and cadmium could potentially 1 help us better understand possible risk factors. The iron transporter divalent metal transporter 1(DMT1) mediates intestinal transport of cadmium, and lead in yeast and fibroblasts overexpressing DMT1. In human intestinal cells knocking down expression of DMT1 attenuated uptake of cadmium and iron but not lead. A possible explanation is the expression of a second transporter for lead in intestine. In astrocytes, however, DMT1 appears to transport lead in an extracellular buffer at pH value. At neutral pH, transport was not mediated by DMT1 but rather by a transporter that is stimulated by bicarbonate and inhibited by 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid. The identity of this lead transporter is under study.
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