Mid-infrared prediction of lactoferrin content in bovine milk: potential indicator of mastitis

Lactoferrin (LTF) is a milk glycoprotein favorably associated with the immune system of dairy cows. Somatic cell count is often used as an indicator of mastitis in dairy cows, but knowledge on the milk LTF content could aid in mastitis detection. An inexpensive, rapid and robust method to predict milk LTF is required. The aim of this study was to develop an equation to quantify the LTF content in bovine milk using mid-infrared (MIR) spectrometry. LTF was quantified by enzyme-linked immunosorbent assay (ELISA), and all milk samples were analyzed by MIR. After discarding samples with a coefficient of variation between 2 ELISA measurements of more than 5% and the spectral outliers, the calibration set consisted of 2499 samples from Belgium (n = 110), Ireland (n = 1658) and Scotland (n = 731). Six statistical methods were evaluated to develop the LTF equation. The best method yielded a cross-validation coefficient of determination for LTF of 0.71 and a cross-validation standard error of 50.55 mg/l of milk. An external validation was undertaken using an additional dataset containing 274 Walloon samples. The validation coefficient of determination was 0.60. To assess the usefulness of the MIR predicted LTF, four logistic regressions using somatic cell score (SCS) and MIR LTF were developed to predict the presence of mastitis. The dataset used to build the logistic regressions consisted of 275 mastitis records and 13 507 MIR data collected in 18 Walloon herds. The LTF and the interaction SCS × LTF effects were significant (P < 0.001 and P = 0.02, respectively). When only the predicted LTF was included in the model, the prediction of the presence of mastitis was not accurate despite a moderate correlation between SCS and LTF (r = 0.54). The specificity and the sensitivity of models were assessed using Walloon data (i.e. internal validation) and data collected from a research herd at the University of Wisconsin – Madison (i.e. 5886 Wisconsin MIR records related to 93 mastistis events – external validation). Model specificity was better when LTF was included in the regression along with SCS when compared with SCS alone. Correct classification of non-mastitis records was 95.44% and 92.05% from Wisconsin and Walloon data, respectively. The same conclusion was formulated from the Hosmer and Lemeshow test. In conclusion, this study confirms the possibility to quantify an LTF indicator from milk MIR spectra. It suggests the usefulness of this indicator associated to SCS to detect the presence of mastitis. Moreover, the knowledge of milk LTF could also improve the milk nutritional quality.     

The mammalian neuroendocrine hormone norepinephrine supplies iron for bacterial growth in the presence of transferrin or lactoferrin

Norepinephrine stimulates the growth of a range of bacterial species in nutritionally poor SAPI minimal salts medium containing 30% serum. Addition of size-fractionated serum components to SAPI medium indicated that transferrin was required for norepinephrine stimulation of growth of Escherichia coli. Since bacteriostasis by serum is primarily due to the iron-withholding capacity of transferrin, we considered the possibility that norepinephrine can overcome this effect by supplying transferrin-bound iron for growth. Incubation with concentrations of norepinephrine that stimulated bacterial growth in serum-SAPI medium resulted in loss of bound iron from iron-saturated transferrin, as indicated by the appearance of monoferric and apo- isoforms upon electrophoresis in denaturing gels. Norepinephrine also caused the loss of iron from lactoferrin. The pharmacologically inactive metabolite norepinephrine 3-O-sulfate, by contrast, did not result in iron loss from transferrin or lactoferrin and did not stimulate bacterial growth in serum-SAPI medium. Norepinephrine formed stable complexes with transferrin, lactoferrin, and serum albumin. Norepinephrine-transferrin and norepinephrine-lactoferrin complexes, but not norepinephrine-apotransferrin or norepinephrine-albumin complexes, stimulated bacterial growth in serum-SAPI medium in the absence of additional norepinephrine. Norepinephrine-stimulated growth in medium containing (55)Fe complexed with transferrin or lactoferrin resulted in uptake of radioactivity by bacterial cells. Moreover, norepinephrine-stimulated growth in medium containing [(3)H]norepinephrine indicated concomitant uptake of norepinephrine. In each case, addition of excess iron did not affect growth but significantly reduced levels of radioactivity ((55)Fe or (3)H) associated with bacterial cells. A role for catecholamine-mediated iron supply in the pathophysiology of infectious diseases is proposed.     

Efficient gene transfer by transferrin lipoplexes in the presence of serum

Cationic lipids are being used increasingly as reagents for gene delivery both in vitro and in vivo. One of the limitations to the application of cationic lipid-DNA complexes (lipoplexes) in vivo is the inhibition of gene delivery by serum. In this study, we have shown that transferrin (Tf)-lipoplexes, which had transferrin adsorbed at their surface via electrostatic interactions, are much more effective than plain lipoplexes in transfecting cells in the presence of relatively high concentrations (up to 60%) of fetal bovine serum (FBS). Serum even enhanced transfection by Tf-lipoplexes composed of 1,2-dioleoyl-3-(trimethylammonium) propane (DOTAP)/dioleoylphosphatidylethanolamine (DOPE)/pCMVLacZ at high lipid/DNA (+/-) charge ratios, and inhibited lipofection for those with low charge ratios when they were added to the cells immediately after the preparation of complexes. The effect of serum on lipofection was dose-dependent. Preincubation of the complexes at 20 degrees C for 6 h led to serum resistance, even for the negatively charged transferrin-lipoplexes. A similar tendency was observed for DOTAP/cholesterol and DOTAP/DOPE/cholesterol liposomes. The percentage of cells transfected, measured by beta-galactosidase expression, also increased with the serum concentration. Cell viability was not affected significantly when the cells were incubated with the complexes for 4 h at 37 degrees C, followed by a 48-h incubation. Our findings extend the scope of previous studies where transferrin-lipoplexes were used to introduce DNA into cells, rendering these complexes and their future derivatives potential alternatives to viral vectors for gene delivery in vivo.     

Characterization of a single nucleotide polymorphism in the coding sequence of the bovine transferrin gene.

A single nucleotide polymorphism was identified in the coding sequence of the bovine transferrin gene. Two alleles (SSCP1 and SSCP2) were detected by SSCP analysis and the mutation point was identified and confirmed by direct sequencing of the PCR products. The relationship between protein and DNA polymorphism was established. Protein variants A, D1 and E correspond to SSCP allele 1 and variant D2 corresponds to SSCP allele 2. DNA sequences from genotypes AA, AE, AD2, D1E, D2E and D2D2 reveal an A/G substitution at position 1455 of the cDNA which causes a Gly/Glu substitution which could be responsible for the mobility difference between D1 and D2 variants. Because of the number of variants, this suggests that other SNPs exist in the bovine transferrin gene. A linkage analysis between the SSCPs and two microsatellites (UWCA46 and CSSM019) mapped the transferrin gene to BTA1. Two-point analysis revealed a tight linkage within the transferrin protein variants and the SSCPs.     

Specificity of the lactoferrin and transferrin receptors in Neisseria gonorrhoeae

Lactoferrin (LF) and transferrin (TF) are postulated to be important physiological sources of iron for Neisseria gonorrhoeae. A dot binding assay involving the use of gonococcal total membranes derived from cells grown in iron-limited conditions demonstrated the presence of separate receptors for LF and TF. The ligand and functional specificities of these receptors were examined in competition-binding and growth experiments. The results indicate that the LF and TF receptors are highly specific for the human protein, suggesting that this property may be partially responsible for conferring the human host specificity of N. gonorrhoeae.     

Enhancement of human T-lymphocyte growth by human transferrin in the presence of fetal bovine serum

All dividing cells require transferrin as a growth factor. During in vitro culture of human lymphocytes, transferrin is usually supplied in the form of serum, either synergic or xenogenic (usually fetal bovine serum (FBS)). In the present work the growth of certain human T-cell lines was examined; these lines were derived from the synovium of rheumatoid arthritis patients and maintained in 10% FBS and 1% synovial fluid. Their growth especially at limiting dilutions was found to be strongly dependent on the presence of synovial fluid at low concentration (0.05-0.1%) in culture medium containing 10% FBS. Further studies indicated that this effect of synovial fluid was duplicated by human serum or plasma, and was due to the presence of human transferrin. A significant effect on T-cell growth was observed using 2 micrograms/ml human transferrin with optimal growth at 10-20 micrograms/ml. This requirement for human transferrin was not a peculiarity of the synovium-derived T-cell lines, but was observed with all T-cell lines tested irrespective of phenotype or function. These observations suggest that bovine transferrin is inadequate for T-cell growth, and that the growth enhancing properties of FBS do not primarily reflect the provision of transferrin. Since some T cells have recently been shown to be capable of secreting transferrin upon activation, endogenous synthesis of transferrin may be an important factor in the in vitro growth of T cells so that such cells would be selected when FBS is the source of serum used to grow human T-cell lines or clones.     

Characterization of the human transferrin and lactoferrin receptors in Haemophilus influenzae

The expression of human transferrin and lactoferrin binding activity in Haemophilus influenzae, detected by a binding assay using human transferrin or lactoferrin conjugated to peroxidase, was regulated by the level of available iron in the medium. Transferrin binding activity was present in all H. influenzae isolates tested but not detected in other Haemophilus species or in species of Pasteurella or Actinobacillus. Lactoferrin binding activity was only detected in 1/15 H. influenzae isolates tested. The transferrin and lactoferrin receptors were shown to be specific for the respective human proteins by means of a competition binding assay. Competition binding assays also showed that iron-loaded transferrin was more effective at blocking the transferrin receptor than apotransferrin, but no differences in receptor blocking were observed between iron-loaded lactoferrin and apolactoferrin.     

Study on the polymorphism of bovine lactoferrin gene and its relationship with mastitis

Mastitis is a major cause of economic loss to the dairy industry. Lactoferrin (Lf) is known to contribute to resistance against bacterial infections. Hence, we decided to characterize the relevance between mastitis resistance and the variants of Lf gene. By using PCR-SSCP, five fragments within 5' region and all exons of bovine lactoferrin gene were amplified and identified the nucleotide diversity. For the five segments within the 5'-region: Lf5'-1, Lf5'-2, Lf5'-3, Lf5'-4, and Lf5'-5 from upstream to downstream, we found that three had base variation. Totally, mutations were observed in Lf5'-1, Lf5'-3, and Lf5'-5, exons 4, 8, 9, 11, 15, and intron 4. We analyzed the effects of all mutated loci on milk production traits with least squares method.     

Synthesis of transferrin and transferrin mRNA in bovine Sertoli cells in culture and in vivo: sequence of partial cDNA clone for bovine transferrin

Techniques were developed for generating enriched cultures of bovine Sertoli cells and indifferent supporting cells (immature Sertoli cells). The [35S]methionine and [35S]sulfate-labeled proteins secreted by cultured cells were analyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and fluorography. The electrophoretic pattern of the major Sertoli cell-secreted proteins was distinct from that of the major proteins secreted by cultured peritubular cells (the predominant contaminating cell type). Five major polypeptides ranging in molecular mass from 22 kDa to 77 kDa were resolved by 2D-PAGE in reducing conditions and were assigned numbers for reference purposes. Polypeptides 1 and 2 appeared to be analogous to two rat Sertoli cell-secreted proteins, sulfated glycoprotein-1 and sulfated glycoprotein-2, because of similar molecular mass, isoelectric point, subunit composition, sulfation, and sialation characteristics. Transferrin was detected in conditioned medium by immunoprecipitation using an antibody to bovine serum transferrin. Cultured Sertoli cells isolated from prepubertal bulls secreted higher levels of transferrin than did cells isolated from infant bulls. An 850 bp cDNA corresponding to the 3' portion of bovine transferrin mRNA was cloned and sequenced. Transferrin message was shown to be present in testicular tissue isolated from infant and prepubertal bulls and it increased as bulls matured. Levels of testicular transferrin mRNA were subsequently shown to correlate with daily sperm production in yearling beef bulls.     

Lactoferrampin: a novel antimicrobial peptide in the N1-domain of bovine lactoferrin

The antimicrobial activity of bovine lactoferrin is attributed to lactoferricin, situated in the N1-domain. Based on common features of antimicrobial peptides, a second putative antimicrobial domain was identified in the N1-domain of lactoferrin, designated lactoferrampin. This novel peptide exhibited candidacidal activity, which was substantially higher than the activity of lactoferrin. Furthermore, lactoferrampin was active against Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa, but not against the fermenting bacteria Actinomyces naeslundii, Porphyromonas gingivalis, Streptococcus mutans and Streptococcus sanguis. Notably, lactoferrampin is located in the N1-domain in close proximity to lactoferricin, which plays a crucial role in membrane-mediated activities of lactoferrin.     

Subdivision in an ancestral species creates asymmetry in gene trees

We consider gene trees in three species for which the species tree is known. We show that population subdivision in ancestral species can lead to asymmetry in the frequencies of the two gene trees not concordant with the species tree and, if subdivision is extreme, cause the one of the nonconcordant gene trees to be more probable than the concordant gene tree. Although published data for the human-chimp-gorilla clade and for three species of Drosophila show asymmetry consistent with our model, sequencing error could also account for observed patterns. We show that substantial levels of persistent ancestral subdivision are needed to account for the observed levels of asymmetry found in these two studies.     

N-glycosylation potential of maize: the human lactoferrin used as a model

In order to determine the N-glycosylation potential of maize, a monocotyledon expression system for the production of recombinant glycoproteins, human lactoferrin was used as a model. The human lactoferrin coding sequence was inserted into the pUC18 plasmid under control of the wheat glutenin promoter. Maize was stably transformed and recombinant lactoferrin was purified from the fourth generation seeds. Glycosylation was analysed by gas chromatography, lectin detection, glycosidase digestions and mass spectrometry. The results indicated that both N-glycosylation sites of recombinant lactoferrin are mainly substituted by typical plant paucimannose-type glycans, with 1,2-xylose and 1,3-linked fucose at the proximal N-acetylglucosamine, and that complex-type glycans with Lewis^a determinants are not present in maize recombinant lactoferrin.     

Affinity enrichment of bovine lactoferrin in whey

Bovine lactoferrin was enriched in various whey samples by affinity chromatography using immobilized gangliosides. Bovine gangliosides were isolated from fresh buttermilk using a combination of ultrafiltration and organic extraction. Isolated gangliosides were covalently immobilized onto controlled-pore glass beads. The immobilized matrix contained 66 micrograms of gangliosides per gram of beads. After loading the matrix with reconstituted whey protein isolate (WPI) or whey protein concentrate (WPC), the matrix was washed with sodium phosphate buffer (pH 7) followed by sodium acetate buffer (pH 4) before elution of lactoferrin with 1 M NaCl in sodium acetate buffer. From the intensities of the protein bands in SDS-PAGE, lactoferrin constituted a minimum of 40% of the total protein in the salt eluted sample. WPI, pretrated by heating and ultrafiltration, showed the highest lactoferrin purity among protein sources, while WPI (10% wt/vol) showed the highest recovery. These results show that immobilized gangliosides can be used to enrich the lactoferrin content of whey.     

Comparative analysis of the transferrin and lactoferrin binding proteins in the family Neisseriaceae

Intact cells of several bacterial species were tested for their ability to bind human transferrin and lactoferrin by a solid-phase binding assay using horseradish peroxidase conjugated transferrin and lactoferrin. The ability to bind lactoferrin was detected in all isolates of Neisseria and Branhamella catarrhalis but not in isolates of Escherichia coli or Pseudomonas aeruginosa. Transferrin-binding activity was similarly detected in most isolates of Neisseria and Branhamella but not in E. coli or P. aeruginosa. The expression of transferrin- and lactoferrin-binding activity was induced by addition of ethylenediamine di-o-phenylacetic acid and reversed by excess FeCl3, indicating regulation by the level of available iron in the medium. The transferrin receptor was specific for human transferrin and the lactoferrin receptor had a high degree of specificity for human lactoferrin in all species tested. The transferrin- and lactoferrin-binding proteins were identified after affinity isolation using biotinylated human transferrin or lactoferrin and streptavidin-agarose. The lactoferrin-binding protein was identified as a 105-kilodalton protein in all species tested. Affinity isolation with biotinylated transferrin yielded two or more proteins in all species tested. A high molecular mass protein was observed in all isolates, and was of similar size (approximately 98 kilodaltons) in all species of Neisseria but was larger (105 kilodaltons) in B. catarrhalis.