Immunochemical determination of human lactoferrin

The immunochemically pure preparation of lactoferrin was isolated from human colostrum and used for the immunization of animals with a view of obtaining antiserum, and also as a reference preparation for the determination of the content of lactoferrin in the standard. The monospecific antiserum to lactoferrin, obtained as the result of this procedure, was used for the determination of the content of lactoferrin in samples of human milk by the method of radial immunodiffusion. Through the content of lactoferrin in human milk showed considerable fluctuations, its level essentially decreased on the second week of lactation. In cases of the microbial contamination of milk the tendency towards an increase in the content of lactoferrin was observed irrespective of the time of lactation.     

Rat intestinal epithelial cells modify the reactivity of bovine milk lactoferrin to concanavalin A

Bovine milk lactoferrin suppressed proliferation of concanavalin A-stimulated rat spleen lymphocytes by absorbing mitogenic lectin activity. Culture media, conditioned by incubating allogeneic intestinal epithelial villus and crypt cells with or without lactoferrin, also suppressed the proliferation. Villus cells absorbed lactoferrin during preparation of conditioned medium and the medium lost a lactoferrin-dependent lymphocyte proliferation-suppressing activity. Although crypt cells did not absorb lactoferrin, its conditioned medium lost the activity. These conditioned media did not alter proliferation of lymphocytes stimulated with 12-O-tetradecanoylphorbol-13-acetate plus ionomycin. Serum proteins, albumin and transferrin, did not substitute for milk lactoferrin. Thus, intestinal epithelial cells modified the reactivity of milk lactoferrin to concanavalin A.     

Interaction of calmodulin with lactoferrin.

Calmodulin, as a major intracellular calcium-binding protein, regulates many Ca(2+)-dependent enzymes and plays an important role in a wide spectrum of cellular functions of the eukaryotes. Interaction between calmodulin and human lactoferrin, a 78 kDa protein with antibacterial properties, was found in the presence of Ca2+ using (i) a method for the detection of calmodulin binding proteins with biotinylated calmodulin, (ii) affinity chromatography on an agarose-calmodulin column with subsequent detection by an enzyme-linked immunosorbent assay (ELISA). The binding of calmodulin to lactoferrin blocked the ability of lactoferrin to agglutinate Micrococcus lysodeikticus.     

The N-linked oligosaccharides of human lactoferrin are not required for binding to bacterial lactoferrin receptors.

The oligosaccharides of human lactoferrin were enzymatically removed with glycopeptidase F, resulting in a preparation containing partial and fully deglycosylated human lactoferrin. The derivatives were separated by Concanavalin A affinity chromatography and compared with native human lactoferrin with respect to their ability to bind to bacterial receptors. Competitive binding experiments demonstrated that the lactoferrin derivatives were equally capable as native lactoferrin in binding to receptors of Neisseria meningitidis, Neisseria gonorrhoeae, and Moraxella catarrhalis. This result indicates that the oligosaccharides on human lactoferrin are not essential for binding to the bacterial receptors.     

Discovery of a Homolog of Siderophilin in a Plant

Members belonging to the siderophilin family are iron-binding and iron-transporting proteins, which includes transferrin and lactoferrin. They have only been found in animals previously. If siderophilin could be found in and isolated from a plant, its production and subsequent extensive application could be increased. The present study is the first to report the discovery of a homolog of siderophilin in a plant. In order to purify antifreeze proteins from Ammopiptanthus mongolicus （Maxim.） Cheng f., the authors processed the proteins from the leaves using techniques such as column chromatography using DEAE-Cellulose-52, gel filtration via Sephacryl S-100 HR medium, hydrophobic interaction chromatography, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Mass spectroscopy was performed on the three proteins purified and the sequence of one of the proteins （containing 32 amino acids） was found to have 97% homology with the corresponding part of one type of human lactoferrin. Moreover, one of the two peptides belongs to an iron-binding domain. So, it is possible that siderophilin also exists in plants and plays a role as an antibacterial and antifungal, among other actions.     

Molecular Cloning and Expression of Yak (Bos grunniens) Lactoferrin cDNA in Pichia pastoris

cDNA encoding lactoferrin from yak was isolated by RT-PCR and then sequenced. The cloned cDNA (2127 bp) encodes a 709 amino acid precursor molecule of yak lactoferrin with a signal peptide of 19 amino acids. The yak lactoferrin cDNA was expressed in Pichia pastoris. The recombinant protein, purified by Ni-NTA affinity column, had a molecular weight of 76 kDa and reacted with an antibody raised against native bovine lactoferrin. The iron-binding behavior and antimicrobial activity of the purified protein indicated that it was correctly folded and functional.     

Arginine 25 and Arginine 28 of lactoferrin are critical for effective heparin neutralization in blood

The interaction of lactoferrin with endogenous heparin-like molecules modulates glycosaminoglycan-mediated biological processes. We performed site-specific mutagenesis and expressed recombinant lactoferrin and lactoferrin mutants by the baculovirus insect cell expression system. Five basic residues at the lactoferrin N terminus; Arg 5, Arg 25, Arg 28, Lys 29, and Arg 31, were individually replaced by alanines. Heparin chromatography on fast-performance liquid chromatography system showed that the NaCl concentrations corresponding to the peak of each eluted recombinant protein from the column were 665, 620, 540, 550, 630, or 650 mM for wild-type recombinant lactoferrin, Arg 5, Arg 25, Arg 28, Lys 29, or Arg 31 recombinant lactoferrin mutant, respectively. We compared the ability of each mutated lactoferrin derivative to neutralize glycosaminoglycans in the thrombin serpin inhibition assays. In comparison to wild-type recombinant lactoferrin, all the mutants showed decreased ability to neutralize glycosaminoglycan in a dose-dependent manner. The mutations of lactoferrin at Arg 25 and Arg 28 demonstrated the most striking decrease in lactoferrin's ability to neutralize various glycosaminoglycans in both enzymatic and plasma clotting-based experiments. Therefore, our results suggest that Arg 25 and Arg 28 are the critical basic residues at the lactoferrin N terminus responsible for heparin binding. The other basic residues on the N terminus, Arg 5, Lys 29, and Arg 31, also contribute to heparin binding by presenting an additional cationic motif.     

Identification of a lactoferrin-binding protein in Prevotella nigrescens

A 40-kDa lactoferrin-binding protein was identified in a strain of Prevotella nigrescens isolated from a patient with periodontitis. The protein was purified by affinity column chromatography using a Sepharose–lactoferrin column and detergent-solubilized membranes. The N-terminal sequence revealed no apparent similarities with any other sequenced bacterial protein. The native conformation of the 40-kDa protein was a condition to bind either iron-free or iron-saturated lactoferrin. A possible function of this Lf-binding protein could be related with an iron acquisition mechanism in P. nigrescens.     

A simplified method for the rapid preparation of peroxidase-anti-peroxidase (PAP) complexes

Summary A modified method is described for the rapid production of peroxidase-antiperoxidase complexes to be used in immunocytochemistry. In this method anti-peroxidase antibodies are precipitated from crude serum with peroxidase at equivalence and subsequently resolubilized at low pH with excess peroxidase. The complexes are isolated from unbound immunoglobulin and peroxidase by gelfiltration. The method combines the advantages of both previously described preparation procedures. The resulting PAP-complex, when tested in indirect immunocytochemistry, is comparable to that obtained in established preparation procedures.Supported by a grant from the Queen Wilhelmina Cancer Foundation (grant no. PA 77/52)     

Production of recombinant bovine lactoferrin N-lobe in insect cells and its antimicrobial activity

Lactoferrin is a multifunctional, iron-binding glycoprotein found in physiological fluids of mammals. In the present study, a gene encoding the N-terminal half (N-lobe) of bovine lactoferrin was cloned and expressed in cultured insect cells using a baculovirus expression system. One mutation was found in the lactoferrin N-lobe gene, but it resulted in no amino acid substitution. The recombinant lactoferrin N-lobe was secreted into the culture medium and partially purified by means of an immobilized heparin column. The recombinant lactoferrin N-lobe secreted was not glycosylated, but it possessed antimicrobial activity toward Escherichia coli O111. The recombinant product synthesized and accumulated in the host cells exhibited greater electrophoretic mobility on SDS-PAGE than the secreted product and showed no potency to inhibit the growth of bacteria. It is thought that the product accumulated intracellularly lacks antimicrobial ability due to its degradation in the host cells or due to disruption of the active conformation.     

A simple and inexpensive on-column frit fabrication method for fused-silica capillaries for increased capacity and versatility in LC-MS/MS applications

A modified sol-gel method for a one-step on-column frit preparation for fused-silica capillaries and its utility for peptide separation in LC-MS/MS is described. This method is inexpensive, reproducible, and does not require specialized equipments. Because the frit fabrication process does not damage polyimide coating, the frit-fabricated column can be tightly connected on-line for high pressure LC. These columns can replace any capillary liquid transfer tubing without any specialized connections up-stream of a spray tip column. Therefore multiple columns with different phases can be connected in series for one- or multiple-dimensional chromatography.     

Purification of C-reactive protein

A concise method was designed for preparation of C-reactive protein (CRP) from pleural effusion. By addition of L-alpha-lecithin to the pleural effusion in the presence of calcium ions, a flocculence of the CRP-lecithin complex formed. Subsequent treatment of the CRP-lecithin complex with chloroform and sodium citrate buffer enabled extraction of the CRP in the buffer layer. This extracted CRP was further purified by sequential treatment on column chromatography of DEAE cellulose (DE52) and gel filtration using Sephacryl S-300. The isolated protein was proved to be native CRP with a high degree of purity, as determined by electrophoretical and immunological analysis. The yield was 41.8% recovery from the starting material. E1%(280) of the CRP preparation was estimated to be 18.75.     

Applicability of using acrylic resins in post-embedding ultrastructural immunolabelling of human neutrophil granule proteins

Summary In this study, quantitative assessments were carried out, (1) by light microscopy during tissue preparation for electron microscopy and (2) by electron microscopy after on-grid immunogold staining, to determine the suitability of using LR White and Lowicryl K4M thin sections to identify lactoferrin and elastase in the granules of human neutrophil leucocytes. Quantitative assessment of the effect of fixation, dehydration and embedding on the preservation of antigenicity during tissue preparation for electron microscopy, using light microscopic peroxidase anti-peroxidase immunocytochemistry, enabled the selection of preparation conditions that adequately preserved both antigenicity and ultrastructure. OsO₄ post-fixation, following primary aldehyde fixation, improved the retention of antigenicity during dehydration and embedding and the preservation of fine structure. Partial rather than complete dehydration retained more of the antigenicity. The efficiency, sensitivity and resolution of immunolabelling and the ultrastructure and quality of sections achieved after embedding in LR White were superior to those obtained after embedding in Lowicryl K4M. Consequently room temperature embedding in LR White following double fixation and partial dehydration is a better and more reliable preparation technique than low-temperature embedding in Lowicryl K4M following single fixation and partial dehydration for localizing lactoferrin and elastase to the specific and primary granules respectively in human neutrophilic granulocytes by the on-grid immunogold staining method.     

Fractionation and recovery of whey proteins by hydrophobic interaction chromatography

A method for the recovery and fractionation of whey proteins from a whey protein concentrate (80%, w/w) by hydrophobic interaction chromatography is proposed. Standard proteins and WPC 80 dissolved in phosphate buffer with ammonium sulfate 1 M were loaded in a HiPrep Octyl Sepharose FF column coupled to a fast protein liquid chromatography (FPLC) system and eluted by decreasing the ionic strength of the buffer using a salt gradient. The results showed that the most hydrophobic protein from whey is α-lactalbumin and the less hydrophobic is lactoferrin. It was possible to recover 45.2% of β-lactoglobulin using the HiPrep Octyl Sepharose FF column from the whey protein concentrate mixture with 99.6% purity on total protein basis.     

Comparison between procedures using SDS for shotgun proteomic analyses of complex samples

Filter-aided sample preparation (FASP) and a new sample preparation method using a modified commercial SDS removal spin column are quantitatively compared in terms of their performance for shotgun proteomic experiments in three complex proteomic samples: a Saccharomyces cerevisiae lysate (insoluble fraction), a Caenorhabditis elegans lysate (soluble fraction), and a human embryonic kidney cell line (HEK293T). The characteristics and total number of peptides and proteins identified are compared between the two procedures. The SDS spin column procedure affords a conservative fourfold improvement in throughput, is more reproducible, less expensive (i.e. requires less materials), and identifies between 30 and 107% more peptides at q≤0.01, than the FASP procedure. The peptides identified by SDS spin column are more hydrophobic than species identified by the FASP procedure as indicated by the distribution of GRAVY scores. Ultimately, these improvements correlate to as great as a 50% increase in protein identifications with two or more peptides.     

Improved sample preparation for the testosterone hydroxylation assay using disposable extraction columns

The preparation of samples for injection into a high-performance liquid chromatography from assay mixtures for the determination of cytochrome P-450-dependent testosterone hydroxylation has been substantially facilitated. By replacing the multiple cumbersome extraction steps of the conventional method with a single column extraction the time for sample preparation was reduced from hours to minutes. The new procedure also yields better recoveries for most of the testosterone metabolites than the original protocol. The use of extraction columns for sample preparation allows the simultaneous treatment of a large number of samples or even the automation of the whole assay procedure. The modified procedure is a straightforward, easy-to-perform method that should greatly facilitate the implementation of the testosterone hydroxylation assay for sharply discriminating between many individual cytochrome P-450 species in routine enzyme diagnostics.     

Purification and characterisation of an inducible beta-galactosidase from Corynebacterium murisepticum

The beta-galactosidase (EC 3.2.1.32) of Corynebacterium murisepticum (inducible by lactose and galactose) was purified by successive column chromatography on Sephadex G-200, DEAE-Sephadex A-50 and DEAE-cellulose (DE52). The enzyme was found to be a dimer of identical subunits of molecular mass 100,000 daltons. The Km values of the enzyme for the substrates lactose and o-nitrophenyl-beta-D-galactopyranoside (ONPG) are 16.7 mM and 4.4 mM, respectively, indicating, its low affinity for the substrates. The Ouchterlony immunodiffusion method exhibited immunological homogeneity of the enzyme preparation. The catalytic site of the enzyme does not take part in antigen-antibody reaction.     

Maternal serum-derived exosomal lactoferrin as a marker in detecting and predicting ventricular septal defect in fetuses

Among different types of congenital heart diseases, ventricular septal defect is the most frequently diagnosed type and is frequently missed in early prenatal screening programs. Herein, we explored the role of maternal serum-derived exosomes in detecting and predicting ventricular septal defect in fetuses in the early stage of pregnancy. A total of 104 pregnant women consisting of 52 ventricular septal defect cases and 52 healthy controls were recruited. TMT/iTRAQ proteomic analysis uncovered 15 maternal serum exosomal proteins, which showed differential expression between ventricular septal defect and control groups. Among these, four down-regulated proteins, lactoferrin, SBSN, DCD, and MBD3, were validated by Western blot. The protein lactoferrin was additionally verified by ELISA which was able to distinguish ventricular septal defects from controls with area under the ROC curve (AUC) 0.804 (p < 0.001). Our findings reveal that lactoferrin in maternal serum-derived exosomes may be a potential biomarker for non-invasive prenatal diagnosis of fetal ventricular septal defects.     

抗菌肽Lactoferricin生物学功能及其应用研究进展

概述了乳铁蛋白活性多肽(lactofericin)所具有的广谱抗菌、抗寄生虫、抗病毒、抗癌、抗氧化等多种生物学活性,讨论了lactnferricin的制备方法,并对lactnferricin作为饲料添加剂的应用前景作了初步探讨。     

Emulsions Based on the Interactions Between Lactoferrin and Chitosans

In this work, purification of lactoferrin from whey was performed with high recovery rate. Lactoferrin was then exploited in the preparation of food emulsions. Two tertiary emulsions, formed by olive oil, lecithin, chitosan, and lactoferrin, were compared: both the emulsions showed similar turbidity and stability. In the secondary emulsion formed by oil/lecithin/chitosan, the pH was increased to 9 before addition of lactoferrin. Then, lactoferrin was added, and the pH was stabilized above pH 9. Lactoferrin was found in amounts of 1 to 2.5 mg/ml in the multiple experiments. A fraction of the added lactoferrin was also present in a milky layer above the emulsion layer. This was, to our knowledge, the first study of emulsions made exploiting the interactions between lactoferrin and chitosan. It was noted that chitosan droplets remained soluble, although the hydrocolloid solubility occurs at pH lower than 5.9. These results showed the feasibility of manufacturing lactoferrin-based emulsions as functional foods.