Identification of lactoferrin-binding proteins in bovine mastitis-causing Streptococcus uberis

All strains of Streptococcus uberis evaluated bound to lactoferrin (Lf) in milk as detected by polyacrylamide gel electrophoresis and Western blotting. A biotin-avidin-based microplate binding assay and ELISA also revealed that these bacterial strains bound to purified Lf. Binding of bacteria of Lf was not inhibited by mannose and galactose, indicating that glycosidic domains of the Lf molecule were not involved in binding. Lf binding was also unaffected by bovine transferrin. Western blot analysis demonstrated that there were at least two bacterial proteins involved in Lf-binding. Lf binding by S. uberis could enable this bacterium to acquire iron necessary for its growth.     

Effect of dietary bovine lactoferrin on performance and antioxidant status of piglets

The experiment investigated the effect of dietary bovine lactoferrin (bLF) on growth performance and antioxidant systems of piglets. Duroc–Landrace–Yorkshire crossbred female piglets (n = 120, 35 days of age, live-weight 9.70 ± 0.71 kg) were fed a diet containing 0, 1250 or 2500 mg/kg bLF for 30 days. After completion of the feeding experiment, eight animals from each treatment were randomly selected to determine malondialdehyde (MDA) and total antioxidant capacity (TAOC), copper–zinc–superoxide dismutase (CuZnSOD), glutathione peroxidase (GPx), catalase (CAT) activities in serum and longissimus muscle, and CuZnSOD, GPx and CAT mRNA levels in longissimus muscle. Results showed that supplementation with bLF improved average daily gain (ADG), average daily feed intake (ADFI) and reduced diarrhea ratios of piglets with the linear and quadratic polynomial contrasts being significant (P<0.01). The highest weight gain and the best feed conversion occurred at 2500 mg/kg bLF. Serum and longissimus muscle CuZnSOD and GPx, and serum TAOC linearly and quadraticly increased (P<0.01), serum CAT and longissimus muscle CAT and TAOC linearly increased (P<0.01), whereas MDA concentrations in serum and longissimus muscle linearly decreased (P<0.01) as dietary bLF supplementation increased. Increasing dietary bLF levels improved (P<0.01) mRNA expression of CuZnSOD and CAT in longissimus muscle of piglets. The results indicated that exogenous antioxidant bLF was effective in improving growth performance and enhancing antioxidant enzymes activity and mRNA levels of piglets.     

The effects of lactoferrin in a rat model of catecholamine cardiotoxicity

Lactoferrin is recently under intense investigation because of its proposed several pharmacologically positive effects. Based on its iron-binding properties and its physiological presence in the human body, it may have a significant impact on pathological conditions associated with iron-catalysed reactive oxygen species (ROS). Its effect on a catecholamine model of myocardial injury, which shares several pathophysiological features with acute myocardial infarction (AMI) in humans, was examined. Male Wistar rats were randomly divided into four groups according to the received medication: control (saline), isoprenaline (ISO, 100 mg kg⁻¹ s.c.), bovine lactoferrin (La, 50 mg kg⁻¹ i.v.) or a combination of La + ISO in the above-mentioned doses. After 24 h, haemodynamic functional parameters were measured, a sample of blood was withdrawn and the heart was removed for analysis of various parameters. Lactoferrin premedication reduced some impairment caused by ISO (e.g. a stroke volume decrease, an increase in peripheral resistance and calcium overload). These positive effects were likely to have been mediated by the positive inotropic effect of lactoferrin and by inhibition of ROS formation due to chelation of free iron. The failure of lactoferrin to provide higher protection seems to be associated with the complexity of catecholamine cardiotoxicity and with its hydrophilic character.     

Development and evaluation of luminescence-based sandwich assay for plasma lactoferrin as a marker for sepsis and bacterial infections in paediatric medicine

An immunoluminometric assay for plasma lactoferrin has been developed and used to study the levels in children and neonates with viral and bacterial infections. The reference range for plasma lactoferrin was 50–250 μg/l. Lactoferrin levels were significantly higher in patients with bacterial versus viral infections.     

Formation of Multilayers at the Interface of Oil-in-Water Emulsion via Interactions between Lactoferrin and β-Lactoglobulin

The interactions between negatively charged β-lactoglobulin and the positively charged lactoferrin at the droplet surface to form a multi-protein surface layer were examined. Addition of lactoferrin to the aqueous phase of emulsions formed with β-lactoglobulin at pH 7.0 caused an increase in the ζ-potential of emulsion droplets, and the ζ-potential became positive as the concentration of added lactoferrin was higher than 1% in the system. It is found that lactoferrin binds to adsorbed β-lactoglobulin at droplet surface probably via electrostatic interactions. The amount of lactoferrin at interface increased with increasing the concentration of added lactoferrin, but it decreased with a decrease in the pH. No lactoferrin was observed at interface at pH 3 and 4. By contrast, when β-lactoglobulin was added in the emulsions formed with lactoferrin at pH 7.0, the ζ-potential of emulsions changed from positive to negative as the concentration of added β-lactoglobulin increased. The amount of β-lactoglobulin at surface increased correspondingly with increasing the concentration of added β-lactoglobulin. However, in this case, β-lactoglobulin remained bound at interface even at pH 3 and 4 where both lactoferrin and β-lactoglobulin are positively charged. The association of lactoferrin or β-lactoglobulin with the surface proteins that have oppositely charge is probably mainly through electrostatic interactions between the two proteins. It appears that alternative layers of these proteins could be created at the droplet surface.     

Structure and function of proteins involved in milk allergies

Allergy to milk proteins has been defined as any adverse reaction mediated by immunological mechanisms to one or several of proteins found in milk. The milk allergy has been classified according to the onset of symptoms as immediate or delayed type. The milk allergy seems to be manifested by three major proteins found in milk: α-lactalbumin, β-lactoglobulin and caseins. The structural comparison of allergenic sites in α-lactalbumin and β-lactoglobulin with the structure of lactoferrin has clearly shown that yet another major milk protein lactoferrin also possesses allergenic sites and thus may qualify to be an allergen. The heat treatment of milk proteins considerably reduces their allergenicity.     

Fecal lactoferrin and Clostridium spp. in stools of autistic children

Stools from autistic and healthy children were studied for fecal lactoferrin, Clostridium difficile toxins, Clostridium perfringens enterotoxin and cultured for Clostridium spp. Elevated level of FLA was demonstrated in 24.4% stools, all from boys (31.25%). No toxins were detected. Clostridium spp. was isolated with similar frequency from all samples. C. perfringens were isolated significantly often from the autistic stools, intermediate sensitive strains to penicillin 19%, to clindamycin 11.3%, and to metronidazole 7.5% were detected. Further studies on fecal microflora and inflammatory mediators, with larger groups of patients, are required in order to explain their role in neurological deficits.     

Physicochemical characterization and functional site analysis of lactoferrin gene of Vechur cow

The Bos indicus Vechur breed cow milk is known for its medicinal value and the breed is listed under the category of critically maintained breeds by the Food and Agriculture Organization. The lactoferrin protein in milk is known for its nutritional value. Gene polymorphisms have been reported for Bovine lactoferrin. Mutations in the evolutionarily conserved sites tend to impair protein function and are related with the physicochemical difference between the known variants with 11 SNPs within the wild type. Structural differences are located due to these SNPs that may lead to functional variations. The structural variation is seen primarily in the first 48 residues at 5' end in all the samples modelled. Out of 11 SNPs 5 amino acid variations fall under alpha helix and beta sheet region, this might be of functional significance. This result may provide evidence that the SNPs detected in lactoferrin gene might have potential effects on milk composition. Our result demonstrates one major domain that could be a common binding pocket to all the samples, and important as an active site common to all the breeds that could be utilized for effective drug designing. Moreover, at some SNP positions in Vechur breed, antimicrobial peptides were located indicating importance of those residues for enhanced antimicrobial activity in lactoferrin of Vechur breed. Second binding pocket found in N- lobe region with the three required residues aspartic acid, histidine and tyrosine for iron binding, was considered as major binding site.     

Identification of neutrophil granule glycoproteins as Lewis(x)-containing ligands cleared by the scavenger receptor C-type lectin

The scavenger receptor C-type lectin (SRCL) is a glycan-binding receptor that has the capacity to mediate endocytosis of glycoproteins carrying terminal Lewis(x) groups (Galβ1-4(Fucα1-3)GlcNAc). A screen for glycoprotein ligands for SRCL using affinity chromatography on immobilized SRCL followed by mass spectrometry-based proteomic analysis revealed that soluble glycoproteins from secondary granules of neutrophils, including lactoferrin and matrix metalloproteinases 8 and 9, are major ligands. Binding competition and surface plasmon resonance analysis showed affinities in the low micromolar range. Comparison of SRCL binding to neutrophil and milk lactoferrin indicates that the binding is dependent on cell-specific glycosylation in the neutrophils, as the milk form of the glycoprotein is a much poorer ligand. Binding to neutrophil glycoproteins is fucose-dependent, and mass spectrometry-based glycomic analysis of neutrophil and milk lactoferrin was used to establish a correlation between high affinity binding to SRCL and the presence of multiple clustered terminal Lewis(x) groups on a heterogeneous mixture of branched glycans, some with poly N-acetyllactosamine extensions. The ability of SRCL to mediate uptake of neutrophil lactoferrin was confirmed using fibroblasts transfected with SRCL. The common presence of Lewis(x) groups in granule protein glycans can thus target granule proteins for clearance by SRCL. PCR and immunohistochemical analysis confirm that SRCL is widely expressed on endothelial cells and thus represents a distributed system that could scavenge released neutrophil glycoproteins both locally at sites of inflammation or systemically when they are released in the circulation.     

Induction of lactoferrin and IL-8 release from human neutrophils by tryptic enzymes via proteinase activated receptor-2

Tryptic enzymes such as tryptase, trypsin and thrombin are reportedly able to alter neutrophil behavior. However, little is known of the influence of these proteinases on lactoferrin or IL-8 release from neutrophils. In the present study, we investigated the effects of tryptase, trypsin, thrombin and elastase, and agonist peptides of PAR-1 SFLLR-NH(2) and PAR-2 SLIGKV-NH(2) and tc-LIGRLO-NH(2) on lactoferrin and IL-8 release from highly purified human neutrophils. Flow cytometry shows CD16(+) neutrophils express PAR-1 and PAR-2, but not PAR-3 and PAR-4 proteins. RT-PCR analysis reveals that neutrophils express only PAR-2 genes. Tryptase and trypsin, but not thrombin and elastase, induced significant lactoferrin and IL-8 secretion from neutrophils. SLIGKV-NH(2) and tc-LIGRLO-NH(2), but not SFLLR-NH(2), also stimulated lactoferrin and IL-8 secretion from neutrophils. In conclusion, only a proportion of neutrophils express PAR-1 and/or PAR-2. Tryptase and trypsin-induced lactoferrin and IL-8 secretion from neutrophils most likely occur through activation of PAR-2.