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1.
J Dairy Sci ; 96(7): 4258-68, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23684041

ABSTRACT

The dairy protein ß-lactoglobulin (BLG) is known to bind fatty acids such as the salt of the essential longchain fatty acid linoleic acid (cis,cis-9,12-octadecadienoic acid, n-6, 18:2). The aim of the current study was to investigate how bovine BLG-linoleate complexes, of various stoichiometry, affect the enzymatic digestion of BLG and the intracellular transport of linoleate into enterocyte-like monolayers. Duodenal and gastric digestions of the complexes indicated that BLG was hydrolyzed more rapidly when complexed with linoleate. Digested as well as undigested BLG-linoleate complexes reduced intracellular linoleate transport as compared with free linoleate. To investigate whether enteroendocrine cells perceive linoleate differently when part of a complex, the ability of linoleate to increase production or secretion of the enteroendocrine satiety hormone, cholecystokinin, was measured. Cholecystokinin mRNA levels were different when linoleate was presented to the cells alone or as part of a protein complex. In conclusion, understanding interactions between linoleate and BLG could help to formulate foods with targeted fatty acid bioaccessibility and, therefore, aid in the development of food matrices with optimal bioactive efficacy.


Subject(s)
Digestion , Fatty Acids/pharmacokinetics , Lactoglobulins/physiology , Linoleic Acid/pharmacokinetics , Milk/chemistry , Animals , Biological Transport , Caco-2 Cells/metabolism , Cattle , Cholecystokinin/genetics , Cholecystokinin/metabolism , Epithelial Cells/metabolism , Humans , In Vitro Techniques , Linoleic Acid/metabolism , RNA, Messenger/analysis
2.
J Immunol ; 183(7): 4322-8, 2009 Oct 01.
Article in English | MEDLINE | ID: mdl-19734215

ABSTRACT

The ontogeny of the immune system and the effect thereon by type of infant feeding is incompletely understood. We analyzed frequencies and composition of immune cells in blood of breastfed (BF) and formula-fed (FF) infants at 1.5, 4, and 6 mo of age. Three formulas with the same protein concentration but with varying levels of alpha-lactalbumin and caseinoglycomacropeptide were compared. Twenty-nine exclusively BF infants served as reference, and 17 infants in each formula group completed the study. Whole blood and PBMCs were analyzed by flow cytometry and immunoflow cytometry, respectively. Leukocyte count of BF infants increased with time due to increased frequency of neutrophils. Lymphocyte count was high at 1.5 mo and was unchanged over time, as were the relative proportions of CD4+ alphabetaT cells, CD8+ alphabetaT cells, B cells, NK cells, and gammadeltaT cells. Most CD45R0+CD3+ cells were HLA-DR- and hence memory cells. Compared with breastfeeding, formula feeding resulted in a significant decrease in proportion of NK cells, but a significant increase in naive CD4+ alphabetaT cells and an elevated CD4-to-CD8 ratio, that is, 3.3 in the combined FF groups compared with 2.6 in the BF group. No significant differences were found between the three groups of FF infants. In conclusion, blood cells of lymphoid lineage did not change significantly in frequencies or composition from 1.5 to 6 mo of age in BF infants. In contrast, FF infants displayed an ongoing maturation of adaptive immunity cells and a delayed recruitment of innate immunity cells as compared with BF infants.


Subject(s)
Breast Feeding , Infant Formula , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/metabolism , Caseins/administration & dosage , Down-Regulation/immunology , Female , Follow-Up Studies , Glycopeptides/administration & dosage , Glycopeptides/physiology , Humans , Immunity, Innate , Immunophenotyping , Infant , Lactalbumin/administration & dosage , Lactalbumin/biosynthesis , Lactalbumin/pharmacology , Lactoglobulins/administration & dosage , Lactoglobulins/antagonists & inhibitors , Lactoglobulins/physiology , Leukocytes, Mononuclear/cytology , Lymphocyte Subsets/cytology , Lymphocyte Subsets/immunology , Lymphocyte Subsets/metabolism , Prospective Studies , Up-Regulation/immunology
3.
Eur Phys J E Soft Matter ; 30(2): 165-74, 2009 Oct.
Article in English | MEDLINE | ID: mdl-19267243

ABSTRACT

Understanding and manipulating the interfacial mechanisms that control human digestion of food emulsions is a crucial step towards improved control of dietary intake. This article reports initial studies on the effects of the physiological conditions within the stomach on the properties of the film formed by the milk protein (ß-lactoglobulin) at the air-water interface. Atomic force microscopy (AFM), surface tension and surface rheology techniques were used to visualize and examine the effect of gastric conditions on the network structure. The effects of changes in temperature, pH and ionic strength on a preformed interfacial structure were characterized in order to simulate the actual digestion process. Changes in ionic strength had little effect on the surface properties. In isolation, acidification reduced both the dilatational and the surface shear modulus, mainly due to strong repulsive electrostatic interactions within the surface layer and raising the temperature to body temperature accelerated the rearrangements within the surface layer, resulting in a decrease of the dilatational response and an increase of surface pressure. Together pH and temperature display an unexpected synergism, independent of the ionic strength. Thus, exposure of a pre-formed interfacial ß-lactoglobulin film to simulated gastric conditions reduced the surface dilatational modulus and surface shear moduli. This is attributed to a weakening of the surface network in which the surface rearrangements of the protein prior to exposure to gastric conditions might play a crucial role.


Subject(s)
Digestion , Gastric Mucosa/metabolism , Lactoglobulins/physiology , Emulsions , Gastrointestinal Contents/chemistry , Humans , Hydrogen-Ion Concentration , Lactoglobulins/chemistry , Lactoglobulins/ultrastructure , Microscopy, Atomic Force/methods , Osmolar Concentration , Rheology/methods , Shear Strength , Stomach/chemistry , Surface Tension , Temperature
4.
Amino Acids ; 35(2): 257-65, 2008 Aug.
Article in English | MEDLINE | ID: mdl-17726638

ABSTRACT

Beta-lactoglobulin (beta-Lg) is currently an important source of biologically active peptides. These peptides are inactive within the sequence of the precursor protein, but they can be released by in vivo or in vitro enzymatic proteolysis. Once released, these peptides play important roles in the human health, including antihypertensive, antioxidant and antimicrobial activities as well as opioid-like features and ability to decrease the body-cholesterol levels. Bioactive peptides derived from beta-Lg are currently a point of intensive research. Their structure, biological significance and mechanism of action are briefly presented and discussed in this review.


Subject(s)
Lactoglobulins/chemistry , Peptide Fragments/chemistry , Amino Acid Sequence , Angiotensin-Converting Enzyme Inhibitors/chemistry , Angiotensin-Converting Enzyme Inhibitors/therapeutic use , Animals , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Antihypertensive Agents/chemistry , Antihypertensive Agents/therapeutic use , Antioxidants/chemistry , Antioxidants/pharmacology , Bacteria/drug effects , Humans , Hypertension/drug therapy , Lactoglobulins/pharmacology , Lactoglobulins/physiology , Molecular Sequence Data , Peptide Fragments/pharmacology , Peptide Fragments/physiology , Reactive Oxygen Species/metabolism
5.
J Immunol ; 179(1): 372-81, 2007 Jul 01.
Article in English | MEDLINE | ID: mdl-17579058

ABSTRACT

We analyzed the autologous NK cell interaction with gliadin-presenting dendritic cells. Gliadin is the known Ag priming the celiac disease (CD) pathogenesis. We demonstrate that gliadin prevents immature dendritic cells (iDCs) elimination by NK cells. Furthermore, cooperation between human NK cells-iDCs and T cells increases IFN-gamma production of anti-gliadin immune response. Gliadin fractions were analyzed for their capability to stabilize HLA-E molecules. The alpha and omega fractions conferred the protection from NK cell lysis to iDCs and increased their HLA-E expression. Gliadin pancreatic enzyme digest and a peptide derived from gliadin alpha increased HLA-E levels on murine RMA-S/HLA-E-transfected cells. Analysis of HLA-E expression in the small intestinal mucosa of gluten-containing diet celiac patients and organ culture experiments confirmed the in vitro data.


Subject(s)
Cell Communication/immunology , Cell Membrane/metabolism , Dendritic Cells/metabolism , Gliadin/pharmacology , HLA Antigens/metabolism , Histocompatibility Antigens Class I/metabolism , Killer Cells, Natural/metabolism , Adolescent , Adult , Animals , CD4-Positive T-Lymphocytes/drug effects , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , Cell Communication/drug effects , Cell Differentiation/immunology , Cell Line , Cell Membrane/drug effects , Cell Membrane/immunology , Child , Cytotoxicity, Immunologic/drug effects , Dendritic Cells/drug effects , Dendritic Cells/immunology , Epitopes, T-Lymphocyte/immunology , Epitopes, T-Lymphocyte/metabolism , Female , HLA Antigens/biosynthesis , Histocompatibility Antigens Class I/biosynthesis , Humans , Immunophenotyping , Intestinal Mucosa/cytology , Intestinal Mucosa/drug effects , Intestinal Mucosa/immunology , Killer Cells, Natural/drug effects , Killer Cells, Natural/immunology , Lactoglobulins/physiology , Male , Mice , Organ Culture Techniques , Peptides/physiology , HLA-E Antigens
6.
J Mol Biol ; 361(2): 216-30, 2006 Aug 11.
Article in English | MEDLINE | ID: mdl-16859709

ABSTRACT

Although positioned nucleosomes are known to play a direct, localised role in regulating access to DNA sequence, they also have the potential, through their long-range distribution, to affect the detailed structure of the higher-order chromatin fibre. To investigate this possibility, we firstly mapped, in vitro, the sequence-dependent positions that the core histone octamer adopts when reconstituted onto DNA containing the ovine beta-lactoglobulin gene. These positioning sites are discussed in terms of their relative affinity for the histone octamer, their locations with respect to the gene sequence and their periodic distribution throughout the gene region. Secondly, we mapped, in vivo, the sites that nucleosomes occupy on the same sequence in liver nuclei, where the gene is transcriptionally inactive. Although the sequence is largely packaged into regularly spaced nucleosomes, reflecting a fibre of uniform higher-order structure, this organisation is disrupted by a number of unusual chromatin structures in a region stretching from the second to the third introns of the gene. A comparison of the in vitro and in vivo nucleosome positioning data shows that they are qualitatively and quantitatively related, suggesting that the structure of the higher-order chromatin fibre containing the beta-lactoglobulin gene is determined, in part, by the long-range organisation of the non-coding sequences within which the gene is embedded.


Subject(s)
Histones/metabolism , Lactoglobulins/physiology , Liver/metabolism , Nucleosomes/physiology , Animals , Cell Nucleus/metabolism , Chromatin , DNA/metabolism , Histones/chemistry , In Vitro Techniques , Nucleosomes/ultrastructure , Plasmids , Sheep, Domestic
7.
Biochim Biophys Acta ; 1752(2): 154-65, 2005 Sep 25.
Article in English | MEDLINE | ID: mdl-16143573

ABSTRACT

beta-Lactoglobulin (beta-LG) denatured with 6 M guanidine hydrochloride (GdnHCl) containing a reducing agent and subsequently dialysed against phosphate-buffered saline (PBS) resulted in incomplete refolding of this protein despite the fact that the biological activity for retinol-binding was recovered to almost the same degree as that of the native molecule [Hattori, M., Ametani, A., Katakura, Y., Shimizu, M., Kaminogawa, S. J., Biol. Chem. 268 (1993) 22414-22419]. The enzyme probe method, evaluation of hydrophilicity values, in-gel mobility on SDS-PAGE, and evaluation of disulfide bonds with the Ellman method showed exposure of the hydrophobic region(s) and incorrect disulfide bond formation in such dialyzed beta-LG molecules. We reveal in this present work that complete refolding could be attained by diluting denatured beta-LG with PBS containing a reducing agent, before slow reoxidation of the sulfhydryl groups upon dialysis for gradient removal of the reducing agent in 6 steps. Complete renaturation was confirmed by analyzing the retinol-binding activity, CD spectra, intrinsic fluorescence, binding ability of monoclonal antibodies (mAbs), and SDS-PAGE. Step-by-step disulfide bond formation was considered to be critical for the complete refolding of denatured beta-LG. Our method can contribute to establish a procedure for complete refolding of useful recombinant proteins in vitro without such biological aids as chaperones.


Subject(s)
Cattle/metabolism , Disulfides/metabolism , Lactoglobulins/metabolism , Models, Molecular , Protein Folding , Animals , Antibodies, Monoclonal , Dithionitrobenzoic Acid , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Guanidine , Lactoglobulins/physiology , Protein Binding , Protein Denaturation/physiology , Spectrometry, Fluorescence
8.
J Dairy Sci ; 87(4): 785-96, 2004 Apr.
Article in English | MEDLINE | ID: mdl-15259212

ABSTRACT

beta-Lactoglobulin (beta-LG) is the major whey protein of ruminant species and is also present in the milks of many, but not all, other species. Its amino-acid sequence and 3-dimensional structure show that it is a lipocalin, a widely diverse family, most of which bind small hydrophobic ligands and thus may act as specific transporters, as does serum retinol binding protein. Bovine beta-LG binds a wide range of ligands, but this may not be the reason for its presence in milk. In reviewing the structure and physicochemical properties of the protein, we present the structures of the ligands cholesterol (at a resolution of 2.0A, R = 0.221; Rfree = 0.295) and vitamin D2 (at a resolution of 2.4A, R = 0.212; Rfree = 0.297) each bound to the central binding cavity of bovine beta-LG at pH 7.3. Neither ligand is fully visible in the electron density maps, and the less well-ordered regions are the polar end groups at the mouth of the binding site. In a separate experiment, a mercury ion was bound to the free Cys121 (at a resolution of 2.2A, R = 0.218; Rfree = 0.288) in a way that transmitted a small structural change through Asp137 via Arg148 to the dimer interface. It is not clear if the known dissociation that arises from the reaction of beta-LG with HgCl2 results from this perturbation. In reviewing the structural studies that reveal the ligand binding sites for long-chain fatty acids, retinoids, and steroids, only the central location, common to all lipocalins so far examined, is occupied under the conditions used. We find that there is no crystallographic evidence of another ligand binding site in our crystals grown in approximately 1.3 M citrate, although low ionic strength studies in solution indicate the possible presence of at least one other low affinity site. The apparent ability of the binding site to accommodate a wide range of ligands may point to a possible physiological function. However, by considering the lipocalin family in general, and the species distribution of beta-LG in particular, some speculation as to the physiological function can be made. beta-Lactoglobulin has been reported as being implicated, inter alia, in hydrophobic ligand transport and uptake, enzyme regulation, and the neonatal acquisition of passive immunity. However, these functions do not appear to be consistent between species. Sequence comparisons among members of the lipocalin family reveal that glycodelin, found in the human endometrium during early pregnancy, is the most closely related to beta-LG. Although the function of glycodelin is also unknown, it appears to have effects on the immune system and/or to be involved in differentiation. It is proposed that beta-LG, over-expressed in the lactating mammary gland of many, but not all, species, is primarily an important source of amino acids for the offspring of those animals that produce it, but that this function arose by gene duplication from the physiologically essential glycodelin. The other functions that have been associated with beta-LG in the neonate are, therefore, fortuitous.


Subject(s)
Lactoglobulins/chemistry , Lactoglobulins/physiology , Animals , Binding Sites , Cholesterol/chemistry , Cholesterol/metabolism , Crystallization , Cysteine/chemistry , Ergocalciferols/chemistry , Ergocalciferols/metabolism , Ligands , Mercury/chemistry , Models, Molecular , Molecular Structure , Protein Binding
9.
Article in English | MEDLINE | ID: mdl-15954657

ABSTRACT

The effect of environmental factors (protein concentration, pH, ionic strength) on emulsification properties of beta-lactoglobulin (beta-Lg) was analysed. The protein was obtained from phospholipoprotein-free whey 25-fold concentrated by ultrafiltration (10 kDa cut off) after precipitation of alpha-lactalbumin (alpha-La) with 6% trichloroacetic acid. The Sauter diameter D(vs) of oil droplets in the emulsion stabilised by beta-Lg was found to increase with increases in the protein concentration in the range of 0.5 to 2.0% and medium acidity in the pH range of 5.0 to 7.0. An increase in ionic strength in the range of 0 to 100 mM caused that the emulsification properties of the protein worsened as compared with control emulsion.


Subject(s)
Emulsions/analysis , Lactoglobulins/physiology , Milk Proteins/chemistry , Cheese , Hydrogen-Ion Concentration , Lactoglobulins/isolation & purification , Osmolar Concentration , Particle Size , Solubility , Ultracentrifugation/methods , Whey Proteins
10.
Biochemistry ; 37(40): 14014-23, 1998 Oct 06.
Article in English | MEDLINE | ID: mdl-9760236

ABSTRACT

The structures of the trigonal crystal form of bovine beta-lactoglobulin variant A at pH 6.2, 7.1, and 8.2 have been determined by X-ray diffraction methods at a resolution of 2.56, 2. 24, and 2.49 A, respectively. The corresponding values for R (Rfree) are 0.192 (0.240), 0.234 (0.279), and 0.232 (0.277). The C and N termini as well as two disulfide bonds are clearly defined in these models. The glutamate side chain of residue 89 is buried at pH 6.2 and becomes exposed at pH 7.1 and 8.2. This conformational change, involving the loop 85-90, provides a structural basis for a variety of pH-dependent chemical, physical, and spectroscopic phenomena, collectively known as the Tanford transition.


Subject(s)
Lactoglobulins/chemistry , Protein Conformation , Animals , Cattle , Computer Simulation , Crystallography, X-Ray , Electrons , Female , Hydrogen-Ion Concentration , Intestinal Absorption , Lactoglobulins/physiology , Ligands , Models, Molecular , Protein Binding , Protein Structure, Secondary , Structure-Activity Relationship
11.
Chem Biol Interact ; 100(3): 277-88, 1996 May 06.
Article in English | MEDLINE | ID: mdl-8653809

ABSTRACT

The effect of some milk components on the cellular uptake of cadmium has been studied using a human intestinal cell line (Caco-2). Cadmium uptake by Caco-2 cells increased with the concentration of this metal in the culture medium, in a saturable way. These cells were exposed to different concentrations of cadmium and the synthesis of metallothionein was studied by a cadmium-saturation method. The levels of metallothionein increased with the cadmium concentration in the medium up to 20 microM of metal. Supplementation of the culture medium with 10% bovine milk caused a 25% decrease in the uptake of cadmium with respect to that internalized by the cells maintained in the culture medium alone. However, the uptake of cadmium from the medium supplemented with 10% human milk was similar to that with serum-free medium. beta-Lactoglobulin interacted with cadmium when studied by equilibrium dialysis, showing a stoichiometric binding constant of 5 x 10(4) l/mol. Interaction of lactoferrin with cadmium, however, was negligible. When Caco-2 cells were incubated in culture medium containing lactoferrin, cadmium uptake decreased with respect to that observed incubating the cells in a medium containing beta-lactoglobulin or in the free-protein medium. The inhibitory effect of lactoferrin on the uptake of cadmium might be due to a reduction of the cell surface charge, through its binding to the membrane.


Subject(s)
Caco-2 Cells/metabolism , Cadmium/metabolism , Milk, Human/physiology , Milk/physiology , Animals , Cells, Cultured , Chemical Fractionation , Cytosol/chemistry , Female , Humans , Lactoferrin/physiology , Lactoglobulins/physiology , Metallothionein/analysis , Metallothionein/biosynthesis , Protein Binding
12.
J Dairy Sci ; 78(5): 978-88, 1995 May.
Article in English | MEDLINE | ID: mdl-7622732

ABSTRACT

beta-Lactoglobulin is the major whey protein in the milk of ruminants and some nonruminants, such as pigs and horses. Although beta-lactoglobulin was first isolated 60 yr ago, no function has been definitely ascribed to beta-lactoglobulin. Recent x-ray crystallographic studies have advanced knowledge of the structure of beta-lactoglobulin, which is homologous with that of retinol-binding protein and lipocalycins; the function of these proteins seems to be participation in the transport of small hydrophobic substances. By analogy, this protein has been suggested as having a role as a transporter of fatty acids and retinol. This review reassesses the function of beta-lactoglobulin in light of the large amount of information that has accrued in the last few years. In particular, this review concentrates upon studies of the binding of retinol and fatty acids to beta-lactoglobulin, including the binding constants and number of binding sites, the location of the binding sites, and the influence of chemical modifications in the interaction of the protein with both ligands. This study also describes studies of the influence of beta-lactoglobulin on several biological processes that may be relevant to the possible biological role of this protein.


Subject(s)
Fatty Acids/metabolism , Lactoglobulins/physiology , Vitamin A/metabolism , Animals , Ruminants
13.
Biochim Biophys Acta ; 1123(2): 151-5, 1992 Jan 24.
Article in English | MEDLINE | ID: mdl-1739746

ABSTRACT

The interaction of bovine beta-lactoglobulin with palmitic and oleic acids has been studied by a partition equilibrium method. Bovine beta-lactoglobulin displays only one high affinity binding site for fatty acids whose association constants for palmitic and oleic acids are 4.2 x 10(6) and 2.3 x 10(6) M-1, respectively. However, other binding sites with low affinity are also present. The existence of one high affinity binding site is in accordance with the amount of fatty acids naturally bound to beta-lactoglobulin isolated from milk. The effect of beta-lactoglobulin on ruminant pregastric lipases from a pharyngeal extract has been assayed. The activity of pharyngeal lipase on a triglyceride emulsion is increased about 200%, 250% and 190% in the presence of 10 mg/ml, 20 mg/ml and 40 mg/ml of beta-lactoglobulin, respectively, the last concentration representing that found physiologically in colostrum. Albumin, another ligand-binding protein, increases the activity of this enzyme to a lesser extent and high levels tend to inhibit enzyme action. These results indicate that beta-lactoglobulin could participate in the digestion of milk lipids during the neonatal period by enhancing the activity of pregastric lipase through removal of the fatty acids that inhibit this enzyme.


Subject(s)
Esophagus/enzymology , Lactoglobulins/physiology , Laryngeal Mucosa/enzymology , Lipase/metabolism , Milk/enzymology , Pharynx/enzymology , Animals , Cattle , Fatty Acids/chemistry , Lactoglobulins/pharmacology , Laryngeal Mucosa/drug effects , Lipase/drug effects , Lipolysis , Sheep
14.
Biol Chem Hoppe Seyler ; 371(9): 871-9, 1990 Sep.
Article in English | MEDLINE | ID: mdl-2291812

ABSTRACT

The complete primary structure of the minor beta-lactoglobulin II component from donkey milk is presented. It has been established by amino-acid sequencing and mass-spectrometry analysis of intact protein and peptides obtained after enzymatic and chemical cleavages. The molecular mass and the pI of the protein are calculated to be 18,261 Da and 4.5 respectively. Despite the close structural similarity of the donkey and horse major beta-lactoglobulin I components, their minor beta-lactoglobulin II components show substantial differences in sequence. Most observed exchanges are clustered at residues 78-106 where only 6 amino-acid residues are conserved. The primary structure of donkey beta-lactoglobulin II reveals some unusual features of minor beta-lactoglobulins II and gives new light to the evolution of beta-lactoglobulins and other lipocalins involved in retinol binding or reproductive functions.


Subject(s)
Lactoglobulins/chemistry , Milk/chemistry , Amino Acid Sequence , Animals , Biological Evolution , Humans , Lactoglobulins/classification , Lactoglobulins/physiology , Molecular Sequence Data , Perissodactyla , Sequence Homology, Nucleic Acid
15.
Pediatr Res ; 27(5): 525-8, 1990 May.
Article in English | MEDLINE | ID: mdl-2345681

ABSTRACT

Lactoferrin has been identified as a factor in human colostrum that accounts for increased incorporation of thymidine into the DNA in an in vitro rat crypt enterocyte bioassay. We have examined lactoferrin-stimulated thymidine incorporation by comparing the effects of iron-free lactoferrin (apolactoferrin) with those of iron-saturated lactoferrin (diferric lactoferrin) under conditions that inhibit the transfer of iron between these iron-binding proteins in the bioassay system. In addition, we have compared the dose-response relationships of diferric lactoferrin and apolactoferrin. The results demonstrated that lactoferrin, independent of iron-binding states, promoted the incorporation of thymidine into the DNA of rat crypt enterocytes. These observations suggest a previously unreported nutritional role for lactoferrin that is independent of its iron-binding capacity.


Subject(s)
Apoproteins/pharmacology , DNA/biosynthesis , Intestines/drug effects , Iron/physiology , Lactoferrin/pharmacology , Lactoferrin/physiology , Lactoglobulins/pharmacology , Lactoglobulins/physiology , Thymidine/metabolism , Animals , Cells, Cultured , Humans , Intestinal Mucosa/metabolism , Iron Radioisotopes , Lactoferrin/isolation & purification , Male , Rats , Rats, Inbred Strains
16.
J Exp Med ; 170(2): 415-29, 1989 Aug 01.
Article in English | MEDLINE | ID: mdl-2754391

ABSTRACT

Lactoferrin (Lf), the major iron-binding component of milk, also a major constituent of the specific granules of neutrophils involved in antimicrobial activity and a glycoprotein thought to play a role in regulatory functions in the hematopoietic system as well as other physiologic activities, is shown to occur in three isoforms. One, Lf-alpha, binds iron; the other two, Lf-beta and Lf-gamma, express potent RNase activity, but do not bind iron. The three isoforms are very similar or identical in Mr, pI, partial proteolytic peptide patterns, NH2-terminal amino acid sequence, and reactivity with mAbs and polyclonal antisera against the RNase and Lf, respectively. The finding of structurally similar but enzymatically distinct forms of Lf may be related to the diverse functions of the molecule.


Subject(s)
Iron/metabolism , Lactoferrin/physiology , Lactoglobulins/physiology , Milk, Human/enzymology , Ribonucleases/metabolism , Antibodies, Monoclonal/immunology , Blotting, Western , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Humans , Lactoferrin/isolation & purification , Molecular Weight , Peptide Mapping
17.
Z Gesamte Inn Med ; 44(12): 345-50, 1989 Jun 15.
Article in German | MEDLINE | ID: mdl-2669392

ABSTRACT

A survey of recent knowledge on structure and importance of lactoferrin and ferritin is given. Lactoferrin is a symmetrically constructed glycoprotein which appears in the milk and in the body fluids and develops a bacteriostatic efficiency on account of the ability to Fe-binding together with other factors such as the IgA. In a particularly high concentration it is contained in the colostral milk of the woman. In the milk of the cattle the content is small. In the neutrophil granulocytes it is of importance for the functional ability in the phagocytosis. The ferritins are spherically constructed molecules which possess channels through which Fe-ions (up to 4,500 pro molecule) can be taken up or taken off. The ferritin content in the serum is correlated with the Fe-content of the liver and is dependent upon age as well as the Fe-supply. It decreases in Fe-deficiency: it increases in iron overload, in infectious diseases, in inflammation as well as in tumour development.


Subject(s)
Ferritins/physiology , Infections/immunology , Lactoferrin/physiology , Lactoglobulins/physiology , Animals , Humans , Milk, Human/immunology , Phagocytosis , Structure-Activity Relationship , Transferrin/physiology
18.
Eur J Biochem ; 179(2): 481-7, 1989 Feb 01.
Article in English | MEDLINE | ID: mdl-2537213

ABSTRACT

In the resting rate, the human peripheral blood lymphocytes did not show detectable surface and intracellular receptors for human lactotransferrin. However, both types of lactotransferrin receptors were expressed during stimulation of lymphocytes with phytohemagglutinin. The appearance of receptors was time-dependent and the number of receptors reached a plateau after at least two days of mitogen stimulation. These results suggest that the presence of surface receptors on mitogen-stimulated lymphocytes is not consecutive to a modification of subcellular distribution but to an induction of biosynthesis of the receptors. As measured by incorporation of [3H]thymidine into DNA, addition of human lactotransferrin in a serum-free medium increased the proliferative activity of phytohemagglutinin-stimulated lymphocytes. Optimal enhancement of [3H]thymidine incorporation was obtained by adding 30% iron-saturated lactotransferrin at a concentration of 0.17 microM. Therefore, the role of lactotransferrin in the response of lymphocytes to mitogen stimulation appears to be similar to that previously described for serotransferrin. The lactotransferrin receptor was visualized using 125I-labeled lactotransferrin on nitrocellulose paper after electroblotting of the Triton X-100 extract of the phytohemagglutinin-stimulated lymphocytes as two protein bands of 100 and 110 kDa molecular mass. Purification of the lactotransferrin receptor from the Triton-X-100-soluble extract of stimulated lymphocytes was performed by antiligand-affinity chromatography. The binding of lactotransferrin to the purified receptors was reversible and dependent on concentration and pH.


Subject(s)
Lactoferrin/physiology , Lactoglobulins/physiology , Lymphocyte Activation , Lymphocytes/metabolism , Receptors, Cell Surface/isolation & purification , Cell Membrane/metabolism , Chromatography, Affinity/methods , Electrophoresis, Polyacrylamide Gel , Humans , Kinetics , Lymphocyte Activation/drug effects , Mitogens , Phytohemagglutinins/pharmacology , Solubility
19.
Blood Cells ; 15(2): 371-85, 1989.
Article in English | MEDLINE | ID: mdl-2670003

ABSTRACT

A physiologic role for lactoferrin (Lf) has been implicated by (1) its antibacterial effect and (2) its involvement as a negative-feedback regulator for colony stimulating factor (CSF) and, therefore, granulocyte production. The isolation and purification of endotoxin-free, species-specific mouse and human Lf have enabled a study of the role of Lf both in vitro and in vivo. Injection of Salmonella typhimurium or LPS into mice resulted in a dose-dependent increase in plasma Lf. Treating normal and neutropenic mice with LPS showed that the plasma Lf level was directly related to the number of granulocytes found in the peripheral blood. The effect of neutropenia did not inhibit release of Lf. By incubating mouse bone marrow and adherent peritoneal cells with 0.1 microM mouse or human Lf in the absence or presence of the prostaglandin synthesis inhibitor, indomethacin (1.0 microM), no evidence could be obtained in support of a negative-feedback regulation of CSF. In fact, rather than an inhibition of CSF, the production of the latter was found to be stimulated from both cell types. Injection of endotoxin-free, mouse Lf (2 mg/animal) into mice at concentrations in the same order of magnitude as that found during bacterial infection, resulted in an increase in CSF production by 12 hours and prior to the increase in bone marrow granulocyte-macrophage progenitor cells (GM-CFC) at 48 hours. The results do not support a negative-feedback regulation of CSF by macrophages. Instead, they can be incorporated into a "demand signal" model for CSF production by macrophages.


Subject(s)
Colony-Stimulating Factors/biosynthesis , Lactoferrin/physiology , Lactoglobulins/physiology , Animals , Blood Cell Count , Bone Marrow/physiology , Cell Adhesion , Cells, Cultured , Colony-Stimulating Factors/metabolism , Granulocyte-Macrophage Colony-Stimulating Factor , Granulocytes/radiation effects , Growth Substances/metabolism , Lactoferrin/isolation & purification , Lipopolysaccharides/isolation & purification , Lipopolysaccharides/pharmacology , Mice , Mice, Inbred Strains , Neutropenia/blood , Salmonella Infections, Animal/physiopathology , Salmonella typhimurium , Spleen/physiology , Whole-Body Irradiation
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