Abstract
Transferrin is an essential ingredient used in cell culture media due to its crucial role in regulating cellular iron uptake, transport, and utilization. It is also a promising drug carrier used to increase a drug’s therapeutic index via the unique transferrin receptor-mediated endocytosis pathway. Due to the high risk of contamination with blood–borne pathogens from the use of human or animal plasma-derived transferrin, recombinant transferrin is preferred for use as a replacement for native transferrin.
We expressed recombinant human transferrin in rice (Oryza sativa L.) at a high level of 1% seed dry weight (10 g/kg). The recombinant human transferrin was able to be extracted with saline buffers and then purified by a one step anion exchange chromatographic process to greater than 95% purity. The rice-derived recombinant human transferrin was shown to be not only structurally similar to the native human transferrin, but also functionally the same as native transferrin in terms of reversible iron binding and promoting cell growth and productivity.
These results indicate that rice-derived recombinant human transferrin should be a safe and low cost alternative to human or animal plasma-derived transferrin for use in cell culture-based biopharmaceutical production of protein therapeutics and vaccines
Deshui Zhanga, , , Somen Nandia, 1, Paula Bryana, 2, Steve Pettitb, Diane Nguyena, Mary Ann Santosb and Ning Huanga, b,
Protein Expression and Purification
Volume 74, Issue 1, November 2010, Pages 69-79
Monday
Wednesday
Transferrin as a Luminal Target for Negatively Charged Liposomes in the Inflamed Colonic Mucosa
The need for improved specificity in the local treatment of inflammatory bowel diseases (IBD) led us to use negatively charged liposomes to target the inflamed colonic epithelium. The purpose of the present study was to elucidate the cause for our previous observations that such liposomes accumulate, preferentially, in the inflamed mucosa of rats that were induced with experimental colitis, following luminal administration. Protein analysis (tandem mass spectrometry, verified by Western blot) of inflamed mucosal specimens, extracted at pH 3, 5 and 7, revealed an increased expression of transferrin (TF) at pH 3. Histological examination indicated that the TF was located at the luminal side of the inflamed epithelium. Negatively charged (but not neutral) liposomes adhered to both commercial and mucosal TF at low pH, but not at neutral pH. Moreover, preincubation of negatively charged liposomes with TF profoundly attenuated their adherence to the inflamed mucosa of the rat colon. It is concluded that, at a low pH, typical of the colon lumen in ulcerative colitis, TF mediates specific mucoadhesion of negatively charged liposomes to the inflamed mucosa. This observation could be useful in the rational design of specific drug vehicles aimed at IBD therapy after luminal administration.
Tirosh B, Khatib N, Barenholz Y, Nissan A, Rubinstein A.
Department of Pharmacology and Experimental Therapeutics, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, P.O. Box 12065, Jerusalem 91120, Israel, Department of Pharmaceutics, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, P.O. Box 12065, Jerusalem 91120, Israel, Laboratory of Membrane and Liposome Research, The Hebrew University-Hadassah Medical School, Jerusalem, Israel, and Department of Surgery, Hadassah-Hebrew University Medical Center, Mount Scopus, P.O. Box 24035, Jerusalem 91240, Israel
Tirosh B, Khatib N, Barenholz Y, Nissan A, Rubinstein A.
Department of Pharmacology and Experimental Therapeutics, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, P.O. Box 12065, Jerusalem 91120, Israel, Department of Pharmaceutics, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, P.O. Box 12065, Jerusalem 91120, Israel, Laboratory of Membrane and Liposome Research, The Hebrew University-Hadassah Medical School, Jerusalem, Israel, and Department of Surgery, Hadassah-Hebrew University Medical Center, Mount Scopus, P.O. Box 24035, Jerusalem 91240, Israel
Tuesday
Transferrin and ferritin response to bacterial infection: the role of the liver and brain in fish
Iron is essential for growth and survival, but it is also toxic when in excess. Thus, there is a tight regulation of iron that is accomplished by the interaction of several genes including the iron transporter transferrin and iron storage protein ferritin. These genes are also known to be involved in response to infection. The aim of this study was to understand the role of transferrin and ferritin in infection and iron metabolism in fish. Thus, sea bass transferrin and ferritin H cDNAs were isolated from liver, cloned and characterized. Transferrin constitutive expression was found to be highest in the liver, but also with significant expression in the brain, particularly in the highly vascularized region connecting the inferior lobe of the hypothalamus and the saccus vasculosus. Ferritin, on the other hand, was expressed in all tested organs, but also significantly higher in the liver. Fish were subjected to either experimental bacterial infection or iron modulation and transferrin and ferritin mRNA expression levels were analyzed, along with several iron regulatory parameters. Transferrin expression was found to decrease in the liver and increase in the brain in response to infection and to increase in the liver in iron deficiency. Ferritin expression was found to inversely reflect transferrin in the liver, increasing in infection and iron overload and decreasing in iron deficiency, whereas in the brain, ferritin expression was also increased in infection. These findings demonstrate the evolutionary conservation of transferrin and ferritin dual functions in vertebrates, being involved in both the immune response and iron metabolism.
Neves JV, Wilson JM, Rodrigues PN.
IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Portugal
Neves JV, Wilson JM, Rodrigues PN.
IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Portugal
Wednesday
Ceruloplasmin/Transferrin System Is Related to Clinical Status in Acute Stroke
Background and Purpose— In acute stroke, Iron (Fe) may amplify reperfusion injury by catalyzing the conversion of superoxide and hydrogen peroxide into highly reactive radicals. Transferrin (Tf) is the main protein regulating Fe homeostasis, whereas Ceruplasmin (CP) is a circulating ferroxidase enzyme able to oxidize ferrous ions to less toxic ferric forms. This study aims at investigating whether CP, Copper (Cu), Tf, and Fe play a role in the pathophysiology of acute stroke.
Methods— We enrolled 35 acute stroke patients and 44 controls. All patients underwent: neurological examination assessed by National Institutes of Health Stroke Scale (NIHSS), ultrasound evaluation of carotid atherosclerosis, brain MRI to quantify ischemic lesion volume and measurement of serum levels of CP, Cu, Tf, Fe, hydro-peroxides, and Total plasmatic antioxidant capacity.
Results— In patients, NIHSS scores were associated with Tf (r=–0.48, P=0.004), hydro-peroxides (r=0.34, P=0.046), CP (r=0.43, P=0.012), and lesion volume (r=0.50, P=0.004). Lesion volume was inversely associated with Tf (r=–0.44, P=0.012). CP and hydro-peroxides were also largely related (r=0.81, P<0.001). The model multiple R was 0.57, resulting in a 32.5% of explained NIHSS variance with Tf accounting for 23.4% and CP for 9.1%.
Conclusions— CP and Tf levels are representative of clinical status in acute stroke patients. Our findings suggest a protective role of Tf in acute stroke and a possible ambivalent role of CP.
Methods— We enrolled 35 acute stroke patients and 44 controls. All patients underwent: neurological examination assessed by National Institutes of Health Stroke Scale (NIHSS), ultrasound evaluation of carotid atherosclerosis, brain MRI to quantify ischemic lesion volume and measurement of serum levels of CP, Cu, Tf, Fe, hydro-peroxides, and Total plasmatic antioxidant capacity.
Results— In patients, NIHSS scores were associated with Tf (r=–0.48, P=0.004), hydro-peroxides (r=0.34, P=0.046), CP (r=0.43, P=0.012), and lesion volume (r=0.50, P=0.004). Lesion volume was inversely associated with Tf (r=–0.44, P=0.012). CP and hydro-peroxides were also largely related (r=0.81, P<0.001). The model multiple R was 0.57, resulting in a 32.5% of explained NIHSS variance with Tf accounting for 23.4% and CP for 9.1%.
Conclusions— CP and Tf levels are representative of clinical status in acute stroke patients. Our findings suggest a protective role of Tf in acute stroke and a possible ambivalent role of CP.
Friday
The Usefulness of the Serum Transferrin Receptor to Serum Ferritin Ratio for Discriminating between Iron Deficiency Anemia and Anemia of Inflammation
BACKGROUND: The incidence of iron deficiency anaemia in infants, which is caused by the increased iron demand for rapid growth during this period, is reported to range from 10 to 40%. This age group also suffers from a number of acute illnesses (urinary tract infection, pneumonia and other viral illness). The aim of this study was to evaluate the usefulness of soluble transferrin receptor (sTfR) values and the different methods of calculating the sTfR and serum ferritin (SF) ratio for differentiating anemia of inflammation (AI) from iron deficiency anemia (IDA) or a mixture of these two types of anemia.
METHODS: 173 infants among all the infants who visited Gyeongsang National University Hospital from 2000 to 2006 were enrolled in this study. The hemoglobin (Hb), SF and sTfR values were checked and the infants were divided into the Al subgroup (Hb <11g/dL and SF > 50microgram/L), the IDA subgroup (Hb <11g/dL and SF < 12microgram/L), the normal group (Hb > or =11g/dL and SF > or =12microgram/L), and the unclassified anemia (UCA) group (Hb <11g/dL and SF 12~50microgram/L).
RESULTS: The mean sTfR and sTfR/Log SF values in the AI group were 3.89 and 10.6microgram/mL, respectively (P<0.01). These values in the IDA group were 1.9 and 36.11, respectively (P<0.01). The mean Log (sTfR/SF) was statistically significant between all the subgroups (1.35 in AI, 3.29 in IDA, 1.76 in Nor and 2.35 in UCA). All the infants in the IDA group had a Log (sTfR/SF) value >2.55 whereas all the infants classified in AI group had a Log (sTfR/SF) value <2.55.
CONCLUSION: The Log (sTfR/SF) value is a useful criterion for discriminating between AI and IDA.
METHODS: 173 infants among all the infants who visited Gyeongsang National University Hospital from 2000 to 2006 were enrolled in this study. The hemoglobin (Hb), SF and sTfR values were checked and the infants were divided into the Al subgroup (Hb <11g/dL and SF > 50microgram/L), the IDA subgroup (Hb <11g/dL and SF < 12microgram/L), the normal group (Hb > or =11g/dL and SF > or =12microgram/L), and the unclassified anemia (UCA) group (Hb <11g/dL and SF 12~50microgram/L).
RESULTS: The mean sTfR and sTfR/Log SF values in the AI group were 3.89 and 10.6microgram/mL, respectively (P<0.01). These values in the IDA group were 1.9 and 36.11, respectively (P<0.01). The mean Log (sTfR/SF) was statistically significant between all the subgroups (1.35 in AI, 3.29 in IDA, 1.76 in Nor and 2.35 in UCA). All the infants in the IDA group had a Log (sTfR/SF) value >2.55 whereas all the infants classified in AI group had a Log (sTfR/SF) value <2.55.
CONCLUSION: The Log (sTfR/SF) value is a useful criterion for discriminating between AI and IDA.
Thursday
The Usefulness of the Serum Transferrin Receptor to Serum Ferritin Ratio for Discriminating between Iron Deficiency Anemia and Anemin of Inflamation
BACKGROUND:
The incidence of iron deficiency anaemia in infants, which is caused by the increased iron demand for rapid growth during this period, is reported to range from 10 to 40%. This age group also suffers from a number of acute illnesses (urinary tract infection, pneumonia and other viral illness). The aim of this study was to evaluate the usefulness of soluble transferrin receptor (sTfR) values and the different methods of calculating the sTfR and serum ferritin (SF) ratio for differentiating anemia of inflammation (AI) from iron deficiency anemia (IDA) or a mixture of these two types of anemia.
METHODS:
173 infants among all the infants who visited Gyeongsang National University Hospital from 2000 to 2006 were enrolled in this study. The hemoglobin (Hb), SF and sTfR values were checked and the infants were divided into the Al subgroup (Hb <11g/dL and SF > 50microgram/L), the IDA subgroup (Hb <11g/dL and SF < 12microgram/L), the normal group (Hb > or =11g/dL and SF > or =12microgram/L), and the unclassified anemia (UCA) group (Hb <11g/dL and SF 12~50microgram/L).
RESULTS:
The mean sTfR and sTfR/Log SF values in the AI group were 3.89 and 10.6 microgram/mL, respectively (P<0.01). These values in the IDA group were 1.9 and 36.11, respectively (P<0.01). The mean Log (sTfR/SF) was statistically significant between all the subgroups (1.35 in AI, 3.29 in IDA, 1.76 in Nor and 2.35 in UCA). All the infants in the IDA group had a Log (sTfR/SF) value >2.55 whereas all the infants classified in AI group had a Log (sTfR/SF) value <2.55.
CONCLUSION:
The Log (sTfR/SF) value is a useful criterion for discriminating between AI and IDA.
The incidence of iron deficiency anaemia in infants, which is caused by the increased iron demand for rapid growth during this period, is reported to range from 10 to 40%. This age group also suffers from a number of acute illnesses (urinary tract infection, pneumonia and other viral illness). The aim of this study was to evaluate the usefulness of soluble transferrin receptor (sTfR) values and the different methods of calculating the sTfR and serum ferritin (SF) ratio for differentiating anemia of inflammation (AI) from iron deficiency anemia (IDA) or a mixture of these two types of anemia.
METHODS:
173 infants among all the infants who visited Gyeongsang National University Hospital from 2000 to 2006 were enrolled in this study. The hemoglobin (Hb), SF and sTfR values were checked and the infants were divided into the Al subgroup (Hb <11g/dL and SF > 50microgram/L), the IDA subgroup (Hb <11g/dL and SF < 12microgram/L), the normal group (Hb > or =11g/dL and SF > or =12microgram/L), and the unclassified anemia (UCA) group (Hb <11g/dL and SF 12~50microgram/L).
RESULTS:
The mean sTfR and sTfR/Log SF values in the AI group were 3.89 and 10.6 microgram/mL, respectively (P<0.01). These values in the IDA group were 1.9 and 36.11, respectively (P<0.01). The mean Log (sTfR/SF) was statistically significant between all the subgroups (1.35 in AI, 3.29 in IDA, 1.76 in Nor and 2.35 in UCA). All the infants in the IDA group had a Log (sTfR/SF) value >2.55 whereas all the infants classified in AI group had a Log (sTfR/SF) value <2.55.
CONCLUSION:
The Log (sTfR/SF) value is a useful criterion for discriminating between AI and IDA.
Tuesday
Taking iron measures - Human Transferrin
A more accurate method to measure iron in clinical samples is proving ahead of its time, say researchers in Spain.
The group at the University of Oviedo in Spain, led by Alfredo Sanz-Medel, has developed a technique that allows many variables that can indicate iron-related disease to be measured simultaneously and with great precision.
"Iron is used in numerous enzymes and processes throughout the human body"Any imbalance in the amount of iron in the body can lead to disease, said Sanz-Medel. But many different parameters need to be measured to detect such pathologies - since the metal is used not just as an oxygen transporter in the blood but also in numerous enzymes and processes throughout the human body. Until now these parameters have had to be measured separately, often needing multiple steps.
Sanz-Medel's method avoids this and uses transferrin (Tf), a blood plasma protein that transports iron around the body, to measure iron levels in serum. The group saturate the transferrin with either naturally-occurring iron or a non-radioactive isotope and use high performance liquid chromatography (HPLC) and inductively coupled plasma-mass spectrometry (ICP-MS) techniques to measure the amounts of the metal and protein. By comparing the iron isotope ratios, the data can be used to extrapolate clinically useful parameters including the amount of iron bound to tranferrin (Tf) and unbound in serum.
The group at the University of Oviedo in Spain, led by Alfredo Sanz-Medel, has developed a technique that allows many variables that can indicate iron-related disease to be measured simultaneously and with great precision.
"Iron is used in numerous enzymes and processes throughout the human body"Any imbalance in the amount of iron in the body can lead to disease, said Sanz-Medel. But many different parameters need to be measured to detect such pathologies - since the metal is used not just as an oxygen transporter in the blood but also in numerous enzymes and processes throughout the human body. Until now these parameters have had to be measured separately, often needing multiple steps.
Sanz-Medel's method avoids this and uses transferrin (Tf), a blood plasma protein that transports iron around the body, to measure iron levels in serum. The group saturate the transferrin with either naturally-occurring iron or a non-radioactive isotope and use high performance liquid chromatography (HPLC) and inductively coupled plasma-mass spectrometry (ICP-MS) techniques to measure the amounts of the metal and protein. By comparing the iron isotope ratios, the data can be used to extrapolate clinically useful parameters including the amount of iron bound to tranferrin (Tf) and unbound in serum.
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