Identification of a lactoferrin-derived peptide possessing binding activity to hepatitis C virus E2 envelope protein

Bovine and human lactoferrins (LF) prevent hepatitis C virus (HCV) infection in cultured human hepatocytes; the preventive mechanism is thought to be the direct interaction between LF and HCV. To clarify this hypothesis, we have characterized the binding activity of LF to HCV E2 envelope protein and have endeavored to determine which region(s) of LF are important for this binding activity. Several regions of human LF have been expressed and purified as thioredoxin-fused proteins in Escherichia coli. Far-Western blot analysis using these LF fragments and the E2 protein, expressed in Chinese hamster ovary cells, revealed that the 93 carboxyl amino acids of LF specifically bound to the E2 protein. The 93 carboxyl amino acids of LFs derived from bovine and horse cells also possessed similar binding activity to the E2 protein. In addition, the amino acid sequences of these carboxyl regions appeared to show partial homology to CD81, a candidate receptor for HCV, and the binding activity of these carboxyl regions was also comparable with that of CD81. Further deletion analysis identified 33 amino acid residues as the minimum binding site in the carboxyl region of LF, and the binding specificity of these 33 amino acids was also confirmed by using 33 maltose-binding protein-fused amino acids. Furthermore, we demonstrated that the 33 maltose-binding protein-fused amino acids prevented HCV infection in cultured human hepatocytes. In addition, the site-directed mutagenesis to an Ala residue in both terminal residues of the 33 amino acids revealed that Cys at amino acid 628 was determined to be critical for binding to the E2 protein. These results led us to consider the development of an effective anti-HCV peptide. This is the first identification of a natural protein-derived peptide that specifically binds to HCV E2 protein and prevents HCV infection.     

Antirotaviral potential of lactoferrin from different origin: effect of thermal and high pressure treatments

Rotaviral gastroenteritis causes a high rate of infant mortality and severe healthcare implications worldwide. Several studies have pointed out that human milk and dairy fractions, such as whey and buttermilk, possess antirotaviral activity. This activity has been mainly associated with glycoproteins, among them lactoferrin (LF). Thermal treatments are necessary to provide microbiological safety and extend the shelf life of milk products, though they may diminish their biological value. High hydrostatic pressure (HHP) treatment is a non-thermal method that causes lower degradation of food components than other treatments. Thus, the main objective of this study was to prove the antirotaviral activity of LFs from different origin and to evaluate the effect of several thermal and HHP treatments on that activity. LF exerted a high antirotaviral activity, regardless of its origin. Native LFs from bovine, ovine, swine and camel milk, and the human recombinant forms, at 1 mg/mL, showed neutralizing values in the range 87.5–98.6%, while human LF neutralized 58.2%. Iron saturation of bovine LF did not modify its antirotaviral activity. Results revealed interspecies differences in LFs heat susceptibility. Thus, pasteurization at 63 °C for 30 min led to a decrease of 60.1, 44.5, 87.1, 3.8 and 8% of neutralizing activity for human, bovine, swine, ovine and camel LFs, respectively. Pasteurization at 75 °C for 20 s was less harmful to the activity of LFs, with losses ranging from 0 to 13.8%. HHP treatment at 600 MPa for 15 min did not cause any significant decrease in the neutralizing activity of LFs.     

Detection and isolation of a hepatic membrane receptor for ferritin

A ferritin receptor has been detected on isolated rat hepatocytes and has been partially purified from rat liver using affinity chromatography. Isolated hepatocytes exhibit approximately 30,000 ferritin binding sites/cell with a binding association constant (Ka) of 1 x 10(8) mol-1 liter. A binding assay has been developed which utilizes a hepatic ferritin receptor coupled to a microparticulate support to facilitate separation of bound and free ligand. This method yielded a Ka of 3 x 10(8) mol-1 liter for the purified hepatic ferritin receptor. Binding of ferritin to the insolubilized receptor was partially inhibited by human lactoferrin but unaffected by 200-fold molar excess of bovine albumin, rat transferrin, or human asialoorosomucoid.     

Demonstration of the specific binding of bovine transferrin to the human transferrin receptor in K562 cells: evidence for interspecies transferrin internalization

Specific binding of ferric bovine transferrin to the human transferrin receptor was investigated using K562 cells propagated in serum-free medium without transferrin supplemented with 10(-5) elemental iron. Affinity chromatography of solubilized extracts of K562 cells surface-labeled with 125I was performed using bovine transferrin- and human transferrin-Sepharose 4B resins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of resin eluates reveal that bovine transferrin specifically binds a Mr = 188,000 protein which dissociates into a Mr = 94,000 protein under reducing conditions, a finding identical to what is seen with human transferrin. The Mr = 94,000 reduced protein isolated by bovine transferrin resin shows an identical one-dimensional partial proteolytic digestion map with that of the human transferrin receptor. Unlabeled bovine transferrin was shown to specifically compete 125I-labeled human transferrin from the human transferrin receptor on the surface of K562 cells at 4 degrees C in a similar manner as unlabeled human transferrin; however, approximately a 2,000-fold higher concentration of bovine ligand was required to achieve comparable competition (50% inhibition of binding). Indirect immunofluorescence cytolocalization of bovine transferrin in K562 cells grown in serum-free medium supplemented with ferric bovine transferrin reveal patterns similar to those seen for human transferrin (both focal perinuclear and diffuse cytoplasmic fluorescence). Monensin treatment results in a dramatic accumulation of bovine ligand in perinuclear aggregates, suggesting that it is recycled through the Golgi, as is human transferrin. K562 cells grown in serum-free medium supplemented with either 300 micrograms/ml of ferric human or ferric bovine transferrin were found to demonstrate superimposable growth curves.     

Species specificity of iron delivery in hybridomas

Summary Studies with Human x Human (HxH), Human x Mouse (HxM), and Mouse x Mouse (MxM) hybridomas have enabled us to define specific factors that affect hybridoma growth in a species-specific manner. Three transferrins and three lipophilic iron chelates have been tested for their ability to support hybridoma proliferation and antibody production. The results of these studies demonstrate that HxH hybridomas do not respond to bovine transferrin a⁺ concentrations up to 100 μg/ml and are approximately 100-fold less responsive to mouse transferrin than to human transferrin. HxM and MxM hybridomas respond equally to human or mouse transferrin but are 100-fold less sensitive to bovine transferrin. An antibody to the human transferrin receptor inhibited the growth-promoting activity of human or mouse transferrin on HxH hybridomas but was ineffective on HxM hybridomas. This semonstrated the functionality of the human transferrin receptor in HxH hybridomas and that human, mouse, and bovine transferrin were interacting through the mouse transferrin receptor in HxM hybridomas. HxH and HxM hybridomas respond similarly to three different iron chelates exhibiting 80 to 110% of the growth response to human transferrin. MxM hybridomas fail to respond to the iron chelates at similar concentrations, suggesting that the human genome present in the other hybridoma species confers a unique ability for utilizing iron when delivered in this form.     

Albumin prevents nonspecific transferrin binding and iron uptake by isolated hepatocytes

Bovine serum albumin inhibits binding of transferrin by hepatocytes in suspension by 60-70%. Iron uptake is inhibited by less than 20%. A Scatchard analysis of the transferrin-binding data reveals a biphasic plot in the absence of bovine serum albumin, but a monophasic plot in the presence of bovine serum albumin. Bovine serum albumin inhibits low-affinity binding of transferrin (125000 molecules/cell), but has no effect on high-affinity binding (38000 molecules/cell). In pronase-treated cells, transferrin binding is reduced by 40%, and when bovine serum albumin is added, the binding is reduced by a further 40%. Corresponding figures for iron uptake are 70 and 10%, respectively. The results are strong evidence that the major part of iron uptake by hepatocytes occurs from transferrin bound to the plasma membrane transferrin receptor.     

Delivery of iron to human cells by bovine transferrin. Implications for the growth of human cells in vitro.

Following suggestions that transferrin present in fetal-bovine serum, a common supplement used in tissue-culture media, may not bind well to human cells, we have isolated the protein and investigated its interaction with both human and bovine cells. Bovine transferrin bound to a human cell line, K562, at 4 degrees C with a kd of 590 nM, whereas human transferrin bound with a kd of 3.57 nM, a 165-fold difference. With a bovine cell line, NBL4, bovine transferrin bound with the higher affinity, kd 9.09 nM, whereas human transferrin bound with a kd of 41.7 nM, only a 5-fold difference. These values were reflected in an 8.6-fold difference in the rate of iron delivery by the two proteins to human cells, whereas delivery to bovine cells was the same. Nevertheless, the bovine transferrin was taken up by the human cells by a specific receptor-mediated process. Human cells cultured in bovine diferric transferrin at 40 micrograms/ml, the concentration expected in the presence of 10% fetal-bovine serum, failed to thrive, whereas cells cultured in the presence of human transferrin proliferated normally. These results suggest that growth of human cells in bovine serum could give rise to a cellular iron deficiency, which may in turn lead to the selection of clones of cells adapted for survival with less iron. This has important consequences for the use of such cells as models, since they may have aberrant iron-dependent pathways and perhaps other unknown alterations in cell function.     

Microarray analysis of cytokine and chemokine genes in the brains of macaques with SHIV-encephalitis

Human immunodeficiency virus (HIV)-encephalitis results from a cascade of viral-host interactions that lead to cytokine and chemokine imbalance, which then leads to neuropathologic manifestations of the disease. These include macrophage/microglia activation, astrocytosis and neuronal dysfunction or death. As the molecular mechanisms of this process are poorly understood, we used Atlas human cytokine or cytokine receptor microarray analysis to highlight gene expression profiles that accompanied encephalitis in Simian human immunodeficiency virus (SHIV) 89.6P-infected macaques. Of the 277 genes screened, marked upregulation of monocyte chemoattractant protein-1, interferon-inducible peptide IP-10 and interleukin-4 were observed specifically in the encephalitic brains. These genes are collectively known to promote macrophage infiltration and activation and virus replication. In contrast, genes regulating neurotrophic functions, such as brain-derived neurotrophic factor were downregulated. We also found that some of the apoptosis genes were up- or down-regulated. These data provide a comprehensive spectrum of gene expression that underscores the two major clinical manifestations of this unique syndrome: enhanced virus replication in brain macrophages and dystrophic changes in neurons.     

Gene expression signature of human HepG2 cell line

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and is associated with various clinico-pathological characteristics such as genetic mutations and viral infections. Therefore, numerous laboratories look out for identifying always new putative markers for the improvement of HCC diagnosis/prognosis. Many molecular profiling studies investigated gene expression changes related to HCC. HepG2 represents a pure cell line of human liver carcinoma, often used as HCC model due to the absence of viral infection. In this study we compare gene expression profiles associated with HepG2 (as HCC model) and normal hepatocyte cells by microarray technology. Hierarchical cluster analysis of genes evidenced that 2646 genes significantly down-regulated in HepG2 cells compared to hepatocytes whereas a further 3586 genes significantly up-regulated. By using the Ingenuity Pathway Analysis (IPA) program, we have classified the genes that were differently expressed and studied the functional networks correlating these genes in the complete human interactome. Moreover, to confirm the differentially expressed genes as well as the reliability of our microarray data, we performed a quantitative Real time RT-PCR analysis on 9 up-regulated and 11 down-regulated genes, respectively. In conclusion this work i) provides a gene signature of human hepatoma cells showing genes that change their expression as a consequence of liver cancer in the absence of any genetic mutations or viral infection, ii) evidences new differently expressed genes found in our signature compared to previous published studies and iii) suggests some genes on which to focus future studies to understand if they can be used to improve the HCC prognosis/diagnosis.     

Intrinsic electric fields promote epithelization of wounds in the newt, Notophthalmus viridescens

The lateral electric fields (LFs) in the vicinity of small wounds made in hindlimb digit tip skin of Notophthalmus viridescens have been measured and manipulated. Healing of these wounds was assessed by crystal violet staining and by histology. Paired experiments were conducted on the animals: the healing of one digit's wound was compared with healing of the contralateral digit's wound whose LF was changed from that of its contralateral wound. When currents were passed through the animal (into one wound and out of the contralateral wound) so that the LF of one wound was zero while the contralateral wound had an enhanced LF, the wounds with the enhanced LF healed more rapidly than the wounds with the zero LF. When digits on one side were treated with 30 microM benzamil in an artificial pond water so that their wound LFs were reduced to approximately zero, and the contralateral wounds were kept in artificial pond water without benzamil so that they had normal wound LFs, there was significantly less epithelization of the benzamil-treated wounds than of the control wounds. This effect on wound healing was reversed by adding currents that restored the normal wound LFs, but not by adding currents that reversed the wound LFs to the opposite polarity. When currents were added to reverse the wound LFs on one side of the animal, leaving the contralateral wounds free of added currents, the wounds with the reversed LFs healed more poorly than the contralateral wounds with normal LFs. These results are consistent with the hypothesis that the intrinsic LFs in the vicinity of wounds promote epithelization of these wounds.     

NK recognition of target structures: is the transferrin receptor the NK target structure?

That the transferrin receptor acts as a target antigen for human NK cells has previously been suggested. In this study we used two models to examine the hypothesis that the transferrin receptor is recognized by NK cells. In the first model, we employed mouse cloned NK cells in conjunction with the species-specific monoclonal antibody R17 217, which binds to the murine transferrin receptor. We show that there is no correlation between the amount of transferrin receptor expressed on targets and the susceptibility of these targets to NK lysis or NK binding in cold target competition assays. In the second model, we used human NK cells and transferrin receptor-positive transformants as targets. These transformants were derived from mouse L cells transfected with human DNA and selected for the presence of human transferrin receptor. Results show that, in contrast to the mouse system, there is a correlation between the expression of the human transferrin receptor on targets and the ability of these targets to competitively inhibit the lysis of K562 by NK cells. However, because inhibition is not complete, other cell surface antigens probably play a role in human NK-target interactions.     

Altered profile of gene expression in rat hearts induced by chronic nicotine consumption

Using a cDNA microarray technique, we analyzed the expression profile of 1081 genes in the whole heart tissue of rats. The expressions of three classes of genes encoding cellular energy metabolism enzymes, transmembrane receptors, and intracellular kinase network members were reduced by more than 2.5-fold in cardiac tissues from the rats fed with nicotine (3mg/kg/day) for 3 months. The down-regulated 11 genes included mitochondrial ATP synthase beta subunit, mitochondrial H(+) transporting ATP synthase F1 complex alpha subunit isoform 1, liver mitochondrial aldehyde dehydrogenase 2, glutathione-S-transferase mu type 2, corticotropin-releasing factor receptor 2, metabotropic glutamate receptor 2, N-methyl-D-aspartate receptor subtype 2B, muscarinic acetylcholine receptor M3, transmembrane receptor Unc5H1, glycogen synthase kinase 3alpha, and Ca(2+)/calmodulin-dependent protein kinase II beta subunit. It appears that chronic nicotine treatment affects cardiac function by modulating the expressions of genes involved in energy metabolism and signal transduction.     

Differential gene expression in pancreatic tissues of streptozocin-induced diabetic rats and genetically-diabetic mice in response to hypoglycemic dipeptide cyclo (His-Pro) treatment

Diabetic studies are mostly interested in gene expression in the pancreas, the site of insulin secretion that regulates blood glucose levels. However, a single gene approach has been ruled out for many years in discovering new genes or the molecular networks involved in the induction process of diabetes. To understand the molecular mechanisms by which cyclo (His-Pro) (CHP) affects amelioration of diabetes mellitus, we performed gene expression profiling in the pancreatic tissues of two diabetic animal models, streptozocin (STZ)-induced diabetic rats (T1DM) and genetically-diabetic (C57BL/6J ob/ob) mice (T2DM). To understand the healing process of these diabetic rodents, we examined the effects of CHP on various gene expression in pancreatic tissues of both animal models. Our microarray analysis revealed that a total of 1,175 genes were down-regulated and 629 genes were up-regulated in response to STZ treatment, and the altered expression levels of numerous genes were restored to normal state upon CHP treatment. In particular, 476 genes showed significantly altered gene expression upon CHP treatment. In a functional classification, 7,198 genes were counted as differentially expressed in pancreatic tissues of STZ- and CHP-treated rats compared with control, whereas 1,534 genes were restored to normal states by CHP treatment. Microarray data demonstrated for the first time that overexpression of the genes encoding IL-1 receptor, lipid metabolic enzymes (e.g. Mte1, Ptdss1, and Sult2a1), myo-inositol oxygenase, glucagon, and somatostatin as well as down-regulation of olfactory receptor 984 and mitochondrial ribosomal protein, which are highly linked to T1DM etiology. In genetically-diabetic mice, 4,384 genes were altered in gene expression by more than 2-fold compared to the control mice, when counted differentially expressed. In genetically-diabetic mice, 4,384 genes altered in expression by higher than 2-fold were counted as differentially expressed genes in pancreatic tissues of CHP-treated mice. On the other hand, 2,140 genes were up-regulated and 2,244 genes were down-regulated by CHP treatment. The results of the microarray analysis revealed that up-regulation of IL-2, IL12a, and leptin receptor and down-regulation of PIK3 played important physiological roles in the onset of T2DM. In conclusion, we hypothesize that CHP accelerates alterations of gene expression in ameliorating diabetes and antagonizes those that induces the disease.