Delivery of drugs, proteins and genes into cells using transferrin as a ligand for receptor-mediated endocytosis

Transferrin, an iron-transporting serum glycoprotein, is efficiently taken up into cells by the process of receptor-mediated endocytosis. Transferrin receptors are found on the surface of most proliferating cells, in elevated numbers on erythroblasts and on many kinds of tumors. The efficient cellular mechanism for uptake of transferrin has been subverted for the delivery of low-molecular-weight drugs, protein toxins, and liposomes by linkage of these agents to transferrin or to anti-transferrin receptor antibodies. Linkage may be via chemical conjugation procedures or by the generation of chimeric fusion proteins. Transferrin conjugated to DNA-binding compounds (e.g. polycations or intercalating agents) has been successfully used for the import of DNA molecules into cells. High-level gene expression is obtained only if endosome-disruptive agents such as influenza hemagglutinin peptides or adenovirus particles are included which release the DNA complex from intracellular vesicles into the cytoplasm

Iron Deficiency and Neuroendocrine Regulators of Basal Metabolism, Body Composition and Energy Expenditure in Rats

Although dietary iron is a determinant of iron status in animals, body fat mass has been reported to have an inverse association with iron status in human studies. The goal of this study was to determine the relationship between Fe homeostasis, body composition, energy expenditure and neuroendocrine regulators for severe Fe-deficiency anaemia. Forty male Wistar albino rats recently weaned were divided at random into two groups: the control group was fed the basal diet, AIN-93G diet (normal-Fe) and the anaemic group received a low-Fe diet for 40 days. Neuroendocrine parameters that regulate basal metabolism and appetite (thyroid hormones, ghrelin, glucose-dependent insulinotropic polypeptide (GIP), glucagon, insulin, adrenocorticotropic hormone and corticosterone), body composition, respiratory volumes, energy expenditure, haematological and biochemical were assessed. Total body fat was lower, whereas lean mass, free and total water were higher in the anemic group. O2 consumption, CO2 production, energy expenditure (EE) and respiratory quotient (RQ) were lower in the Fe-deficient animals. Triiodothyronine and thyroxine hormones decreased, while thyroid-stimulating hormone increased in the anemic group. Circulating levels of ghrelin were lower in the anemic group, while GIP, glucagon, insulin, corticosterone and adrenocorticotropic hormone levels were higher. Fe-deficiency impairs weight gain in the rats, with marked reductions in lean mass and body fat, indicating lower energy stores.This study was supported by the Excellence Project (P11-AGR-7648) from the Regional Government of Andalusia

The Bioavailability of Iron from Meat, Spinach, Soy Protein Isolate, Meat:Spinach and Meat:SPI Mixtures Fed to Anemic and Healthy Rats

Two Experiments were conducted to investigate the effect of iron status and iron source on iron bioavailability using the rat model. Beef was proportionally mixed with spinach (Experiment I) or soy protein isolate (Experiment II) to test if meat enhances the absorption of iron from spinach or SPI. Five diets with the iron from meat:spinach (or SPI) ratios of 100:0, 75:25, 50:50, 25:75 and 0:100 were prepared and fed to rats. FeS04 was used as a reference. Two trials of rats were used in Experiment I. In trial I, half of the rats were made severely iron deficient (Hb 6.3 g/dl) and the other half mildly iron deficient (Hb 8.8 g/dl). In trial II, half of the rats were depleted to severe iron deficiency and the remainder were not depleted (Hb 11.3 g/dl). Hemoglobin regeneration efficiency (HRE), apparent absorption and 59Fe absorption were compared. The HREs were 41, 53 and 36% (spinach); 42, 51,and 44% (beef) and 73, 66 and 46% (FeS04) in severely, mildly iron-depleted and healthy rats and 56 and 42% (SPI) in iron-depleted and healthy rats, respectively. Iron depletion stimulated iron absorption. This effect was very significant for rats fed a FeS04 supplemented diet and lessened for rats fed diets containing beef, spinach or SPI as the iron source. Spinach and soy protein isolate were good iron sources both having similar iron bioavailabilities as beef. Beef ~as a good iron source, as noted in other reports; however, enhancement of meat on absorption of iron from spinach and SPI did not occur. Hemoglobin regeneration efficiency is a simple, accurate and definitive method for measuring effects of iron source and iron status on iron bioavailability. Apparent absorption gives similar values to HRE. However, it requires meticulous iron analysis and has more potential for errors. Extrinsic radioisotope tagging is an easy method, but there is evidence of possible incomplete exchange of radio tracer with iron in food. The different absorption pattern between FeS04 and food iron suggests a third iron pool, highly soluble iron salt, in addition to heme and nonheme iron complex pools

El receptor soluble de la transferrina : estudio clínico de un nuevo marcador del metabolismo del hierro

Tesis de la Universidad Complutense de Madrid, Facultad de Farmacia, Departamento de Bioquímica y Biología Molecular II, leída el 21-03-2000El receptor soluble de la transferrina (sTfR), es un nuevo marcador clínico del metabolismo del hierro, cuya utilidad diagnóstica todavía no está bien aclarada. El objetivo de este trabajo es evaluar la relación existente entre la concentración sérica de sTfR, y el metabolismo férrico en condiciones patológicas. Para ello se ha evaluado una serie de 172 pacientes con diversas enfermedades hematológicas, en quienes se realizó un examen hematimétrico completo, parámetros habituales del metabolismo del hierro, y estudiod e médula ósea. Los resultados demuestran que la concentración sérica de sTfR aumenta en situaciones de deficiencia funiconal de hierro tanto en la ferropenia como en el bloqueo del metal en los macrófagos, y también enla hiperplasia eritropoyética. Los niveles del parámetro descienden en la hipoplasia eritropoyética. En pacientes con alteraciones patológicas en la maduración de la serie roja, el comprotamiento del sTfR no es afectado por estos factores en la misma maginitud que en el resto de los pacientes. Este marcador no se comporta como un reactante de fase agusa. El sTfR no es un parámetro sensible ni específico para detectar la feropenia, porque no es un marcador del compartimento de depósito orgánico de hierro. Este parámetro puede ser utilizado como indicador de la deficiencia funcional de hierro, y también del grado de eritropoyesis . Pero si se utiliza como índice de deficiencia, se necesita conocer el grado de actividad eritropoyética del paciente. Y viceversa, su utilización como indicador de eritropoyesis, precisa el conocimiento del status férrico del individuoSección Deptal. de Bioquímica y Biología Molecular (Farmacia)Fac. de FarmaciaTRUEpu

Iron overload across the spectrum of non-transfusion-dependent thalassaemias: role of erythropoiesis, splenectomy and transfusions

Non-transfusion-dependent thalassaemias (NTDT) encompass a spectrum of anaemias rarely requiring blood transfusions. Increased iron absorption, driven by hepcidin suppression secondary to erythron expansion, initially causes intrahepatic iron overload. We examined iron metabolism biomarkers in 166 NTDT patients with β thalassaemia intermedia (n = 95), haemoglobin (Hb) E/β thalassaemia (n = 49) and Hb H syndromes (n = 22). Liver iron concentration (LIC), serum ferritin (SF), transferrin saturation (TfSat) and non-transferrin-bound iron (NTBI) were elevated and correlated across diagnostic subgroups. NTBI correlated with soluble transferrin receptor (sTfR), labile plasma iron (LPI) and nucleated red blood cells (NRBCs), with elevations generally confined to previously transfused patients. Splenectomised patients had higher NTBI, TfSat, NRBCs and SF relative to LIC, than non-splenectomised patients. LPI elevations were confined to patients with saturated transferrin. Erythron expansion biomarkers (sTfR, growth differentiation factor-15, NRBCs) correlated with each other and with iron overload biomarkers, particularly in Hb H patients. Plasma hepcidin was similar across subgroups, increased with >20 prior transfusions, and correlated inversely with TfSat, NTBI, LPI and NRBCs. Hepcidin/SF ratios were low, consistent with hepcidin suppression relative to iron overload. Increased NTBI and, by implication, risk of extra-hepatic iron distribution are more likely in previously transfused, splenectomised and iron-overloaded NTDT patients with TfSat >70%

Bifidobacterium xylocopae sp. nov. and Bifidobacterium aemilianum sp. nov., from the carpenter bee (Xylocopa violacea) digestive tract

Social bees harbor a community of gut mutualistic bacteria, among which bifidobacteria occupy an important niche. Recently, four novel species have been isolated from guts of different bumblebees, thus allowing to suppose that a core bifidobacterial population may be present in wild solitary bees. To date there is sparse information about bifidobacteria in solitary bees such as Xylocopa and Osmia spp., this study is therefore focused on the isolation and characterization of bifidobacterial strains from solitary bees, in particular carpenter bee (Xylocopa violacea), builder bee (Osmia cornuta), and red mason bee (Osmia rufa). Among the isolates from Osmia spp. no new species have been detected whereas among Xylocopa isolates four strains (XV2, XV4, XV10, XV16) belonging to putative new species were found. Isolated strains are Gram-positive, lactate- and acetate-producing and possess the fructose-6-phosphate phosphoketolase enzyme. Full genome sequencing and genome annotation were performed for XV2 and XV10. Phylogenetic relationships were determined using partial and complete 16S rRNA sequences and hsp60 restriction analysis that confirmed the belonging of the new strains to Bifidobacterium genus and the relatedness of the strains XV2 and XV10 with XV16 and XV4, respectively. Phenotypic tests were performed for the proposed type strains, reference strains and their closest neighbor in the phylogenetic tree. The results support the proposal of two novel species Bifidobacterium xylocopae sp. nov. whose type strain is XV2 (=DSM 104955T = LMG 30142T), reference strain XV16 and Bifidobacterium aemilianum sp. nov. whose type strain is XV10 (=DSM 104956T = LMG 30143T), reference strain XV4

Lactoferrin's anti-cancer properties. Safety, selectivity, and wide range of action

Despite recent advances in cancer therapy, current treatments, including radiotherapy, chemotherapy, and immunotherapy, although beneficial, present attendant side effects and long-term sequelae, usually more or less affecting quality of life of the patients. Indeed, except for most of the immunotherapeutic agents, the complete lack of selectivity between normal and cancer cells for radio- and chemotherapy can make them potential antagonists of the host anti-cancer self-defense over time. Recently, the use of nutraceuticals as natural compounds corroborating anti-cancer standard therapy is emerging as a promising tool for their relative abundance, bioavailability, safety, low-cost effectiveness, and immuno-compatibility with the host. In this review, we outlined the anti-cancer properties of Lactoferrin (Lf), an iron-binding glycoprotein of the innate immune defense. Lf shows high bioavailability after oral administration, high selectivity toward cancer cells, and a wide range of molecular targets controlling tumor proliferation, survival, migration, invasion, and metastasization. Of note, Lf is able to promote or inhibit cell proliferation and migration depending on whether it acts upon normal or cancerous cells, respectively. Importantly, Lf administration is highly tolerated and does not present significant adverse effects. Moreover, Lf can prevent development or inhibit cancer growth by boosting adaptive immune response. Finally, Lf was recently found to be an ideal carrier for chemotherapeutics, even for the treatment of brain tumors due to its ability to cross the blood-brain barrier, thus globally appearing as a promising tool for cancer prevention and treatment, especially in combination therapies

Human platelets mediate iron release from transferrin by adenine nucleotide-dependent and -independent mechanisms

We assessed the ability of platelet sonicates and mediators secreted by unstimulated and thrombin-stimulated platelets to facilitate the release of iron from transferrin. Platelet sonicates and platelet conditioned media potentiated the release of iron from transferrin. The rate of release of iron was dependent on the pH of the reaction and amount of platelet sample added. Conditioned media from thrombin-stimulated platelets was more effective in mediating the release of iron from transferrin than was conditioned media from unstimulated cells. The rate of iron released from transferrin following addition of ATP and ADP in amounts equivalent to that present in platelet conditioned media was significantly less than the rate of iron released following the addition of conditioned media from platelets. Depletion of ATP and ADP in platelet conditioned media by incubation with apyrase only partially inhibited their ability to enhance the rate of iron release from transferrin. These observations indicate that platelets enhance the release of iron from transferrin by adenine nucleotide-dependent and -independent mechanisms. These observations are consistent with the hypothesis that platelets promote oxidant-induced tissue injury at sights of inflammation secondary to their ability to enhance the local release of iron from transferrin.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/28084/1/0000530.pd

FRAMEWORK ESTABLISHMENT OF TRANSGENIC PLANTAE FACILITATING SYSTEMIC DELIVERY OF GLUCAGON-LIKE PEPTIDE-1 (GLP-1)

Human serum transferrin (hsTf) is well characterized for its ability to transport fused therapeutics throughout the body upon oral administration. Glucagon-like peptide-1 (GLP-1) is one such therapeutic, effective against Type II diabetes that could benefit from such a fusion strategy. However, therapeutic use of hsTf and GLP-1 is limited by their generation using current recombinant protein production platforms. This project assesses the viability of accumulating hsTf and GLP-1 independently in transgenic plants as a novel source for the generation o f both recombinant proteins. Here, nuclear-transformed tobacco plants stably accumulating either hsTf or a synthetic GLP-1 decamer have been developed. Plant-derived hsTf was shown to reversibly bind iron in vitro and the biological activity of GLP-1 was confirmed by its ability to stimulate insulin secretion from a pancreatic cell-line in vitro. Overall, results warrant future hsTf-GLP-1 genetic fusions in transgenic plants intended for enhanced bioavailability of GLP-1 upon oral administratio