Miiuy croaker transferrin gene and evidence for positive selection events reveal different evolutionary patterns.

Transferrin (TF) is a protein that plays a central role in iron metabolism. This protein is associated with the innate immune system, which is responsible for disease defense responses after bacterial infection. The clear link between TF and the immune defense mechanism has led researchers to consider TF as a candidate gene for disease resistance. In this study, the Miichthys miiuy (miiuy croaker) TF gene (MIMI-TF) was cloned and characterized. The gene structure consisted of a coding region of 2070 nucleotides divided into 17 exons, as well as a non-coding region that included 16 introns and spans 6757 nucleotides. The deduced MIMI-TF protein consisted of 689 amino acids that comprised a signal peptide and two lobes (N- and C-lobes). MIMI-TF expression was significantly up-regulated after infection with Vibrio anguillarum. A series of model tests implemented in the CODEML program showed that TF underwent a complex evolutionary process. Branch-site models revealed that vertebrate TF was vastly different from that of invertebrates, and that the TF of the ancestors of aquatic and terrestrial organisms underwent different selection pressures. The site models detected 10 positively selected sites in extant TF genes. One site was located in the cleft between the N1 and N2 domains and was expected to affect the capability of TF to bind to or release iron indirectly. In addition, eight sites were found near the TF exterior. Two of these sites, which could have evolved from the competition for iron between pathogenic bacteria and TF, were located in potential pathogen-binding domains. Our results could be used to further investigate the function of TF and the selective mechanisms involved.

Effect of selenium supplementation with sodium selenite and selenium nanoparticles on iron homeostasis and transferrin gene expression in sheep: a preliminary study.

The present research aimed at evaluating the effects of sodium selenite and selenium nanoparticles (Se NPs) on iron homeostasis and the expression of transferrin and its receptor-binding protein genes. Twenty one Lori-Bakhtiary sheep were randomly allocated into 3 groups. Groups 1 and 2 orally received Se NPs and sodium selenite (1 mg kg(-1)) for 10 consecutive days, respectively. Group 3 served as the control. Blood and sternal bone marrow samples were collected at different supplementation intervals. Various factors such as serum iron concentration, total iron binding capacity (TIBC), and transferrin saturation percent were determined. The expression of transferrin and transferrin binding receptor genes was also studied. Results showed a decreasing trend in serum iron concentration particularly during the early and middle stages of supplementation (0-20 days) with Se NPs or selenium ions. Conversely, the TIBC level increased in sera especially during these periods (0-20 days) in animals that received selenium NPs or selenium ions. Our results also showed that expression of transferrin and its receptor genes was considerably increased during supplementation of the animals by both selenium compounds for 10 or 20 days. After this period, the expression of the mentioned genes significantly decreased, especially in animals that received selenium ions.

Effects of dietary cadmium exposure on tissue-specific cadmium accumulation, iron status and expression of iron-handling and stress-inducible genes in rainbow trout: influence of elevated dietary iron.

Recent evidences suggest that dietary cadmium (Cd) uptake likely occurs via the dietary iron (Fe) uptake pathway in freshwater fish, at least in part. The present study investigated the interactive effects of dietary Cd and Fe in juvenile rainbow trout (Oncorhynchus mykiss). Fish were treated for four weeks with four different diets: normal Fe, high Fe, normal Fe plus Cd, and high Fe plus Cd. Physiological parameters, tissue-specific Fe and Cd level, plasma Fe status, and tissue-specific mRNA expression of transferrin, metallothioneins (MT-A and MT-B) and heat shock proteins 70 (HSP70a and HSP70b) were analyzed. Exposure to dietary Cd increased Cd burden in the following order: intestine>kidney>stomach>liver>gill>carcass. Interestingly, high dietary Fe reduced Cd accumulation in the stomach and intestine as well as in the wholebody of fish. Dietary Cd increased hepatic transferrin mRNA expression and total Fe binding capacity in the plasma, indicating the effect of Cd on Fe handling in fish. The mRNA expression of MTs and HSP70s was also increased in various tissues following dietary Cd exposure, however the response profile of different MT and HSP70 genes was not consistent among different tissues. In general, MT-A was more responsive to Cd exposure in the intestine and liver, whereas MT-B was more responsive in the kidney. Similarly, HSP70a expression was more sensitive to Cd exposure than HSP70b, particularly in the intestine. Interestingly, high Fe diet suppressed Cd-induced induction of transferrin, MT and HSP70 genes in various tissues. Overall, our study suggests that elevated dietary Fe can reduce Cd accumulation and ameliorate Cd-induced stress responses in freshwater fish.

Expression analyses of zebrafish transferrin, ifabp, and elastaseB mRNAs as differentiation markers for the three major endodermal organs: liver, intestine, and exocrine pancreas.

In the present work, three zebrafish cDNA clones encoding transferrin, intestinal fatty acid binding protein (IFABP), and elastaseB were cloned and their expression patterns in early zebrafish development were characterized as differentiation markers for the three major endoderm organs: liver, intestine, and exocrine pancreas. transferrin and ifabp mRNAs exhibit a biphasic expression pattern during early development. transferrin mRNAs were first expressed at approximately 7 hours postfertilization (hpf) in the yolk syncytial layer (YSL) and later in the liver rudiment (from approximately 48 hpf) and in the esophagus transiently (72-96 hpf). Ifabp mRNAs were initially expressed in the YSL at the ventral side during late epiboly (8-9 hpf), spread throughout the YSL of later stage embryos, and appeared in the intestine rudiment at approximately 36 hpf. In contrast to the transferrin and ifabp mRNAs, elastaseB mRNAs were not expressed in the yolk sac or YSL, and these transcripts were detected exclusively in the exocrine pancreas after approximately 56 hpf.

Honey bee (Apis mellifera) transferrin-gene structure and the role of ecdysteroids in the developmental regulation of its expression.

Social life is prone to invasion by microorganisms, and binding of ferric ions by transferrin is an efficient strategy to restrict their access to iron. In this study, we isolated cDNA and genomic clones encoding an Apis mellifera transferrin (AmTRF) gene. It has an open reading frame (ORF) of 2136 bp spread over nine exons. The deduced protein sequence comprises 686 amino acid residues plus a 26 residues signal sequence, giving a predicted molecular mass of 76 kDa. Comparison of the deduced AmTRF amino acid sequence with known insect transferrins revealed significant similarity extending over the entire sequence. It clusters with monoferric transferrins, with which it shares putative iron-binding residues in the N-terminal lobe. In a functional analysis of AmTRF expression in honey bee development, we monitored its expression profile in the larval and pupal stages. The negative regulation of AmTRF by ecdysteroids deduced from the developmental expression profile was confirmed by experimental treatment of spinning-stage honey bee larvae with 20-hydroxyecdysone, and of fourth instar-larvae with juvenile hormone. A juvenile hormone application to spinning-stage larvae, in contrast, had only a minor effect on AmTRF transcript levels. This is the first study implicating ecdysteroids in the developmental regulation of transferrin expression in an insect species.

Cloning and expression of a putative transferrin cDNA of the spruce budworm, Choristoneura fumiferana.

A spruce budworm (Choristoneura fumiferana) transferrin cDNA (CfTf) was isolated and cloned from a cDNA library that was constructed using mRNA from fifth to sixth instar larvae. CfTf cDNA encoded a predicted protein of 681 amino acids with a molecular mass of approximately 76 kDa. CfTf shared 72% and 74% identities at the amino acid level with transferrins of Manduca sexta and Bombyx mori, respectively. Like other transferrins, CfTf retains most of the N-terminal, iron-binding amino acid residues. Northern blot analyses indicated that CfTf mRNA was present at high levels after ecdysis, but that the expression level was low prior to ecdysis at the fourth-sixth instar stages. The highest level of CfTf expression was detected in the fat body. Relatively low levels of expression were detected in the epidermis and no expression was found in the midgut. Expression of CfTf mRNA could be induced by bacteria but not fungi. Expression of CfTf mRNA was suppressed by iron load.

Plasma protein regulation by thyroid hormone.

Thyroid hormones (THs) regulate growth, development, differentiation and metabolic processes by interacting and activating thyroid hormone receptors (TRs). Although much progress has been made in our understanding of the transcriptional regulation of many TR target genes, little is known of the regulation of plasma protein gene expression by TRs. To investigate the role of TRs in plasma protein expression we used human hepatocellular carcinoma cell lines and carried out cDNA microarray analysis. Our results indicate that several plasma proteins including transferrin, prothrombin, angiotensinogen, haptoglobin, alpha-2-HS-glycoprotein alpha and beta chain, complement, lipoproteins and fibrinogen are up-regulated by THs. Furthermore, clusterin, alpha-2-macroglobulin precursor, prothymosin alpha and alpha-fetoprotein were found to be down-regulated by THs.Transferrin, an iron-binding protein expressed in all mammals, and mainly synthesized in the liver, was investigated further. Immunoblot and Northern blot analyses revealed that exposure of HepG2-TRalpha1 sub-lines and HepG2-Neo cells to tri-iodothyronine (T(3)) induced time- and dose-dependent increases in the abundance of transferrin mRNA and protein, with the extent of these effects correlating with the level of expression of TRalpha1. Nuclear run-on experiments indicate that this induction is functioning at the transcriptional level. Moreover, cyclohexamide treatment did not eliminate the induction of transferrin by TH. Thus, our results suggest that the induction of transferrin by TH is direct and may in fact be mediated by an as yet unidentified response element in the promoter region.

Cloning and expression of the transferrin and ferritin genes in a marsupial, the brushtail possum (Trichosurus vulpecula).

Transferrin and ferritin cDNAs have been isolated and characterised from the common brushtail possum (Trichosurus vulpecula), the first marsupial examples of these genes. The transferrin cDNA encodes a 711 amino acid pre-protein which shows high levels of amino acid identity with eutherian transferrins (58-60%) and lactoferrins (54-56%). Phylogenetic analysis suggests that the possum transferrin has evolved independently along a pathway distinct from that of the eutherian transferrins and lactoferrins. Possum H-ferritin is a 182 residue protein which shares 86-94% amino acid identity with mammalian, avian and amphibian sequences. Ferritin mRNA was detected in all tissues tested, whereas transferrin was highly expressed in possum liver and mammary gland, and at lower levels in heart, testis and lung. In the possum mammary gland, ferritin mRNA was expressed throughout lactation with higher levels during the first 30 days which coincides with the high iron concentration of milk at this time. The transferrin gene was differentially expressed during lactation with peak mRNA levels detected during the first 6 days of lactation and after day 106 throughout late lactation. The pattern of transferrin mRNA expression in the mammary gland was identical to that of another whey protein, the late lactation protein, suggesting that the transcription of these genes may be regulated by a similar mechanism in this tissue.

[Effect of recombinant human growth hormone with total parenteral nutrition on albumin synthesis in patients with peritoneal sepsis].

OBJECTIVE: To investigate the effect of recombinant human growth hormone (rhGH) in combination with total parenteral nutrition (TPN) on albumin synthesis in patients with peritoneal sepsis. METHOD: 17 patients with peritoneal sepsis were divided randomly into two groups. The control group received TPN only for 7 days, and the GH group received both rhGH (12 U/d) and TPN for 7 days. The TPN scheme and other treatment were the same in the two groups. RESULT: Serum albumin, prealbumin, and transferrin concentration were increased in patients in the GH group (P < 0.01), but no apparent effect was observed in the control group (P > 0.05). CONCLUSION: In condition of serious peritoneal sepsis, TPN can not increase albumin, prealbumin, and transferrin synthesis alone, whereas rhGH in combination with TPN significantly increase the synthesis of visceral proteins.

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.

Nucleotide sequence of transferrin cDNAs and tissue-specific of the transferrin gene in Atlantic cod (Gadus morhua).

Atlantic cod (Gadus morhua) transferrin cDNAs were isolated from a liver cDNA library using a cod transferrin-derived polymerase chain reaction product as a hybridization probe. The composite nucleotide sequence of two overlapping clones was 2223 bp in length excluding the poly(A) sequence and was equivalent to 87% of the 3' end of the Atlantic salmon transferrin cDNA sequence. Comparison of the deduced amino acid sequence of cod, salmon, Xenopus and several mammalian transferrins revealed that the two fish sequences are more similar with respect to their amino acid sequence and the position of additions/deletions than to other vertebrate transferrins. Conservation of the iron-binding domains and cysteine residues involved in disulphide bridges indicates that all transferrins share similar tertiary structure and support the hypothesis that extant vertebrate transferrin genes were derived from a gene duplication before the divergence of fish, frogs and mammals. Cod transferrin mRNA was detected in both brain and liver RNA and to a much lesser extent in RNA isolated from kidney and heart in contrast to salmon and several other vertebrates in which the transferrin gene is not expressed in brain.

Transferrin synthesis by mouse lymph node and peritoneal macrophages: iron content and effect on lymphocyte proliferation.

Transferrin is an essential requirement for lymphocyte proliferation, because it supplies activated lymphocytes with iron needed for cell proliferation. However, during inflammation or an immune response, the iron content of circulating transferrin, which is of hepatic origin, decreases. It is hypothesized that activated lymphocytes may therefore obtain transferrin-iron from an alternative source, and we have investigated the possibility that transferrin is synthesized locally in lymphoid tissues. It was found that lymph node cells from mice stimulated in vivo with Freund's complete adjuvant were able to synthesize transferrin, and this was because of the macrophage rather than the lymphocyte population. Transferrin synthesized by mouse lymph node or peritoneal macrophages contained iron and was able to promote mouse lymphocyte proliferation. Peritoneal macrophages activated in vivo synthesized more transferrin, released more transferrin-bound iron, and were more effective than resident macrophages at enhancing lymphocyte proliferation. These results suggest that transferrin synthesized by macrophages acts in a paracrine manner to support lymphocyte proliferation, thus eliminating possible detrimental effect of hypoferremia on the immune system.

High density cultivation of BSK cells on sintered alumina ceramic foam support.

A perfusion system which utilizes a porous ceramic core has been tested for the cultivation of transformed BHK cells which produce human transferrin. A design is presented in which cells are immobilized within the porous ceramic particle and are fed by continuous perfusion of batch liquid medium. It was found that more than 5 x 10(9) BHK cells could be supported within the 40 mL ceramic matrix, a ten-fold increase in cell density per unit surface area over the standard roller bottle cultures or a five-fold increase in volumetric cell density over suspension cultures. The cell specific productivity of human transferrin was similar to that observed in suspension culture. The system offer the advantages of significant reduction in serum requirements and the potential for scale-up.

Peripheral parenteral nutrition is no better than enteral nutrition in acute exacerbation of Crohn's disease: a prospective trial.

The use of parenteral nutrition in patients with exacerbation of regional enteritis is controversial, the clinical dictum being bowel rest and nutritional repletion. In order to address this issue, on the short-term at least, a prospective randomized trial compared peripheral parenteral alimentation and elemental feedings for 2 weeks in patients hospitalized with regional enterities. Both groups had significant objective clinical improvement on their respective nutritional supplementation regimens pre- versus posttherapy as assessed by the Crohn's Disease Activity Index (CDAI) (p less than 0.05). However, there was no significant difference in improvement between parenteral versus enteral groups as assessed by the CDAI. Changes in nutritional assessment parameters, including retinol binding protein, nitrogen balance, total lymphocyte count, and transferrin, were related to the quantity of calories consumed rather than the mode of delivery. A positive nitrogen balance was obtained in all patients despite weight loss in the majority. The route of nutrient delivery in acute exacerbation of regional enteritis does not appear to have an impact on the short-term outcome.

Expression of the amino-terminal half-molecule of human serum transferrin in cultured cells and characterization of the recombinant protein.

A human liver cDNA library was screened with a synthetic oligonucleotide, complementary to the 5' region of human transferrin mRNA, as a hybridization probe. The full-length human cDNA clone isolated from this screen contained part of the 5' untranslated region, the complete coding region for the signal peptide and the two lobes of transferrin, the 3' untranslated region, and a poly(A) tail. By use of oligonucleotide-directed mutagenesis in vitro, two translational stop codons and a HindIII site were introduced after the codon for Asp-337. This fragment was inserted into two different expression vectors that were then introduced into Escherichia coli. As judged by NaDodSO4-polyacrylamide gel electrophoresis and Western blot analysis, however, recombinant hTF/2N was undetectable in bacteria transformed by these plasmids. Concurrently, we developed a plasmid vector for the expression of recombinant hTF/2N in eukaryotic cells. In this case, a DNA fragment coding for the natural signal sequence, the hTF/2N lobe, and the two stop codons was cloned into the expression vector pNUT, such that the expression of hTF/2N was controlled by the mouse metallothionein promoter and the human growth hormone termination sequences. Baby hamster kidney cells containing this hTF/2N-pNUT plasmid secreted up to 20 mg of recombinant hTF/2N per liter of tissue culture medium. Recombinant hTF/2N was purified from the medium by successive chromatography steps on DEAE-Sephacel, Sephadex G-75, and FPLC on Polyanion SI. The purified protein was characterized by NaDodSO4-PAGE, urea-PAGE, amino-terminal sequence analysis, UV-visible spectroscopy, iron-binding titration, and proton NMR.(ABSTRACT TRUNCATED AT 250 WORDS)

[Regulation of ferritin and transferrin synthesis in hepatocytes depending on iron status of rats].

In order to examine the control mechanism of ferritin (Fr) and transferrin (Tf) synthesis depending on intracellular iron levels, the rate of 14C-leucine incorporation into those proteins was investigated by cell culture of isolated hepatocytes obtained from iron deficient, iron injected and control rats. The effects of iron (ferric ammonium citrate: FeAc) and (diferric Tf: 2FeTf) or desferrioxamine (Dfo) in culture media were also examined. Serum iron, TIBC and Hb levels of iron deficient rats, which were fed an iron deficient diet for 2 and 4 weeks were significantly lower than the control. However serum iron and TIBC levels of iron injected rats which had received 30 and 45 mg iron as iron dextran 18 h before sacrifice were approximately ten times higher than the control group. The time course of 14C-leucine incorporation into Fr and Tf was investigated at the 60, 120 and 180 minutes stages of the culture. Fr synthesis was increased by the amounts of iron injected, whereas Tf synthesis showed a negative response to iron. The 14C activities in Fr and Tf detected from culture media were proportional to those in hepatocytes. The percentage of nonglycosylated Fr was 82.0-91.4% for total Fr in the culture media in every experiment, which was measured by the affinity of glycosylated Fr to Con A-Sepharose (Con A). This result suggested the leakage of cytosol Fr through the cell membrane instead of specific secretion of the sialyl protein. The efficiency of Fr and Tf synthesis was positively or negatively proportional to cellular iron contents respectively. And the curves of 14C-leucine incorporation into both proteins, calculated as the sum of those in hepatocytes and culture media intersected at the point between the 30 mg iron injected and control groups. The addition of FeAC or 2FeTf into the culture media had an indistinct effect on Fr and Tf synthesis, whereas there was a significant decrease for Fr and a slight increase for Tf formations in the Dfo supplement. These results showed the influence of cellular iron levels in Fr and Tf synthesis.

Transferrin synthesis by inducer T lymphocytes.

Transferrin (Tf) is a growth factor that transports iron in plasma. It is essential for proliferation of activated T lymphocytes. Previous studies have suggested that peripheral blood cells are capable of synthesizing Tf. Using in situ hybridization techniques and human Tf complementary DNAs as probes, peripheral blood cells have been examined for sites of Tf messenger RNA (mRNA) transcription. The studies described here demonstrate that Tf is synthesized by a specific subset of T lymphocytes, the T4+ inducer subset. T lymphocyte proliferation is dependent upon the presence of both interleukin 2 (IL-2) and Tf, even though resting cells do not possess receptors for either. The present studies indicate that during T cell activation, induction of IL-2 mRNA transcription and IL-2 receptor expression precede the transcription of Tf mRNA and expression of Tf receptors, respectively. These events in turn precede the initiation of DNA synthesis. Transferrin and its receptor appear to be involved in an autocrine pathway which is functionally linked to the IL-2/IL-2 receptor autocrine loop.

The effect of cortisol on the synthesis of rat plasma albumin, fibrinogen and transferrin.

A decrease of absolute synthesis of albumin, no change in that of fibrinogen and an increased fractional synthesis of transferrin were observed 3h after intraperitoneal administration of a pharmacological dose of 5 mg of cortisol to 220g rats in the post-absorptive state and previously kept on a diet with 40% protein. The concentration in liver of total free amino acids was practically unchanged at this time. Intraperitoneal administration of a mixture of amino acids with the cortisol raised this concentration and was accompanied by an almost complete de-repression of the synthesis of albumin, with no real effect on that of fibrinogen. In considerable contrast, in rats studied at 24h after intraperitoneal administration of cortisol, and who had been fed once in the interim (but who had received no amino acids intraperitoneally), there was a marked increase in the absolute synthesis of albumin and fibrinogen, with an increase in fractional synthesis that was less proportionately but still very significant and which included transferrin. The amino acid concentrations had risen above the supplemented values at 3h but not as much proportionately as the fractional synthesis rates, and of course not as much as the absolute synthesis rates, of albumin and fibrinogen. These time-dependent effects of cortisol suggest to us that our studies resolve the apparently conflicting results of the effect of cortisol on the synthesis of albumin reported by others.

The acute effect of ethanol on albumin, fibrinogen and transferrin synthesis in the rat.

Decrease of absolute synthesis of albumin and fractional synthesis of transferrin was observed within 3h of orally administering ethanol (4ml/kg) to rats maintained on a 40%-protein diet. In contrast, absolute synthesis of fibrinogen was unaffected. With this ethanol intake, the changes in protein synthesis occurred without significant ultrastructural change in the liver. When the ethanol intake was greater (8ml/kg) ultrastructural disruption was observed. However, both the decrease of plasma protein synthesis and the ultrastructural alterations could be prevented by the simultaneous administration of a mixture of amino acids with the ethanol. The latter findings, not reported hitherto, suggest that ethanol may interfere with hepatic plasma protein synthesis and ultrastructure more through a disturbance of amino acid metabolism than through direct physical damage to the hepatocyte. An Appendix outlines the deconvolutional method used to correct for losses of labelled protein in the period during which measurements were made. The principle may also be applied to labelled plasma urea. The details of the calculations are given in a supplementary paper that has been deposited as Supplementary Publication 50007 at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1972) 126, 5.