Molecular cloning, promoter analysis and SNP identification of Italian Nicastrese and Saanen lactoferrin gene

Lactoferrin (Lf) is an iron-binding glycoprotein found in exocrine secretions including milk. High levels of lactoferrin may have a role in the prevention of microbial infection of the mammary gland. In this report we sequenced and characterized goat lactoferrin cDNA and its promoter region in two different breeds of goat. The complete cDNA comprised 2356 nucleotides, including 38bp at the 5'-UTR and 194bp at the 3'-UTR. The open reading frame is 2127bp long and it encodes a mature protein of 689 aminoacids. A total of 19 nucleotide differences, 11 of them being responsible for 8 aminoacid changes, were identified through the comparison with French, Korean and Tibetan goat lactoferrin cDNAs. About 1700bp of the lactoferrin gene promoter were sequenced. Sequence analysis revealed a non-canonical TATA box, multiple SP1/GC elements, and other putative binding sites for transcription factors, such as NF-kappaB, STAT3 and AP2. Two SNPs were identified, one of which would seem to create a new putative AP2 consensus sequence. The presence of an additional AP2 binding site could be associated with quantitative differences of such protein fraction, which could enhance all the activities related to such protein, and improve mammary gland defence against bacterial infections

Genetic variability detected at the lactoferrin locus (LTF) in the Italian Mediterranean river buffalo

Lactoferrin (LTF) is multi-functional protein belonging to the whey protein fractions of the milk. The gene LTF encoding for such protein is considered a potential candidate for body measurement, milk composition and yield. This study reports on the genetic variability at LTF locus in the Italian Mediterranean river buffalo and its possible association with milk yield. Eleven polymorphic sites were found in the DNA fragment spanning the exons 15-16. In particular, the intron 15 was extremely polymorphic with 9 SNPs detected, whereas the remaining 2 SNPs were exonic mutations (g.88G>A at the exon 15 and g.1351G>A at the exon 16) and both synonymous. The genotyping of the informative samples evidenced 3 haplotypes, whose frequencies were 0.6; 0.3 and 0.1 respectively, whereas the analysis of the exonic SNPs showed a perfect condition of linkage disequilibrium (g.88A/g.1351G and g.88G/g.1351A). The association study carried out by using the SNP g.88G>A showed that buffalo LTF gene has no statistically significant influence on daily milk yield. This study adds knowledge to the genetic variability of a species less investigated than the other ruminant species, that may serve as a useful tool for large-scale screening of buffalo populations

THE DIVALENT CATION TRANSPORTER NRAMP IN PARASITE PERKINSUS MARINUS: GENOMIC, MOLECULAR, STRUCTURAL, FUNCTIONAL AND EVOLUTIONARY ASPECTS

Perkinsus marinus, the causative agent of Dermo disease in eastern oyster Crassostrea virginica has been a great hurdle for oyster population restoration. Iron was shown to be an essential element for P. marinus growth and virulence, but iron uptake pathways have not been elucidated. Natural Resistance-associated Macrophage Protein (Nramp), an iron transporter, was considered to be a potential virulence factor in intracellular pathogens. One Nramp homolog (PmNramp1) was reported in P. marinus previously. Two other PmNramp isotypes (PmNramp2 and PmNramp3) were identified through genome mining followed by molecular characterization. The three PmNramp isotypes with distinct gene structures were transcribed in parasite trophozoites cultured in defined medium. Transcripts of a number of P. marinus genes, including PmNramp isotypes, superoxide dismutases (PmSOD), ascorbate peroxidase (PmAPX) and heat shock proteins (PmHSP70 and PmHSP90) were trans-spliced with a trans-splicing leader (SL) highly similar to dinoflagellate SL. No change in transcription level of those genes was detected by real-time quantitative reverse transcription PCR (qRT-PCR), under iron/manganese overload, iron depletion and host hemolymph exposure, indicating a constitutive polycistronic transcription in the parasite. Functional study by yeast complementation assays suggested iron uptake activity by PmNramp1. Prediction of PmNramp1 topology by homologous modeling indicated that PmNramp1 was an integral protein with 12 transmembrane segments (TMS). The central position of the Nramp-specific triplets Asp-Pro-Gly (TMS1) and Met-Pro-His (TMS6) in a three-dimensional (3D) arrangement formed with TMS3 and TMS8 provided the mechanistic basis for iron acquisition via PmNramp1. Site-directed mutagenesis of the residues on the triplets in PmNramp1 caused the lost of complementation activity as iron transporter in yeast. A chimeric protein with PmNramp1 N- and C-termini but PmNramp3 core structure from TMS1 to TMS12 complemented yeast growth, suggesting PmNramp3 an iron transporter. Phylogeny data implied that all the three PmNramp isotypes were archetype Nramp. Protein sequence divergence among PmNramp isotypes was not related to diversification of critical functional elements, which remained constrained by purifying selection. This result was consistent with the function of both PmNramp1 and PmNramp3 as iron transporters in yeast, despite their different evolutionary rate and substitution patterns. Subcellular localization of PmNramp isotypes in P. marinus trophozoites are in progress. PmNramp3 was shown to localize on cell peripheral when the parasite proliferates by binary fission. The data were consistent with the previous observation that iron is important for P. marinus growth. As the first functional study of Nramp homolog in protozoan parasites, the work in the dissertation may serve as the reference for research in other protozoan Nramp and iron transporters

Studies on genetic variants of human plasma transferrin.

PhDThe work presented in this thesis is concerned with the characterisation of human plasma transferrins showing abnormal electrophoretic mobilities on polyacrylamide gels. The work is divided into three studies: (i) a study of transferrin variants detected by nondenaturing polyacrylamide gel electrophoresis; (ii) a study of the plasma concentrations of individuals showing transferrin phenotypes associated with the three most common Tf C alleles, Tf Cl, Tf C2 and Tf C3; and (iii) a study of a reported nondegenerate nucleotide difference in the sequence of the cloned human transferrin gene. In the first study, six transferrin variants (3 Tf Br,,.,,, s and 3 Tf D. 5) showing abnormal electrophoretic mobilities on nondenaturing polyacrylamide gels, and the two Tf C variants, Tf Cl and Tf C2 which occur at polymorphic levels (> 1%) in human populations, were isolated and purified from human plasma. Transferrins were purified by a combination of DEAE Sephacel anion-exchange chromatography, SP. Sephadex cation-exchange chromatography and G-200 gel-filtration chromatography. A series of comparative studies were then carried out on the isolated transferrins to determine whether the six transferrin variants detected in this thesis and the Tf C2 variant, showed similar characteristics to the wild-type Tf Cl. Transferrins were studied for sialic acid content of the two glycan chains, and for molecular weights and isoelectric points of the iron-free (apo) and iron-saturated (holo) transferrin forms. Metal-binding properties were examined by studying the binding of Fe", Cu", Al" and Ga"'. Iron-binding was studied at physiological and endosomal pH (7.5 and 5.5 respectively) using FENTA as the iron donor. Binding of Cue*, Al"' and Ga'* were examined at physiological pH using CUNTA, ALNTA and GANTA respectively. The ability of transferrins to retain bound iron was examined by studying pH-induced iron release over a pH range of 6.0-4.0. Conformational stabilities were determined by studying the iron-binding abilities of apotransferrins following exposure to urea or thermal denaturation, and by studying iron loss from holo transferrins following exposure to urea or thermal denaturation. Other than expected differences in isoelectric points, and slightly faster rates of iron loss from variant holo transferrins titrated at physiological pH, all variant transferrins were found to show similar characteristics to Tf Cl, with identical molecular weights and sialic acid content, similar metal-binding properties, and similar stabilities to urea or thermal denaturation. The results indicate that the structure and function of the variant transferrins are not adversely affected by their differences in primary structure. The second study examined the relationship between plasma transferrin concentration and transferrin phenotypes representing five of the six most common Tf C phenotypes (i. e. Tf Cl, Tf C2, Tf C2-1, Tf C3-1 and Tf C3-2), to determine whether plasma concentrations were dependent on transferrin phenotype as suggested in literature. Transferrin phenotypes of 931 unrelated individuals were determined by electrophoresis of plasma on polyacrylamide isoelectric focusing gels. Plasma transferrin concentrations were determined by single radial immunodiffusion using rabbit anti-human transferrin IgG. A significant difference was found between the plasma concentrations of the five transferrin phenotypes (p < 0.01) indicating that transferrin concentration was dependent on phenotype. The results suggest that the two Tf C alleles, Tf C2 and Tf C3 are associated with low plasma concentrations. The third study was initiated to investigate a report in literature that a nondegenerate nucleotide difference of adenine for guanine at base 1086 in exon 8 of the human transferrin gene, may indicate the presence of a hitherto unrecognised transferrin variant with Asn rather than a wild-type Asp at position 310 of the amino acid sequence. The nucleotide sequenceo f exon 8 from 220 unrelatedi ndividuals showing transferrin phenotypes associated with the three most common Tf C alleles, Tf Cl, Tf C2 and Tf C3, and from 5 individuals showing abnormal transferrin phenotypes in the first study, were amplified by polymerase chain reaction. Amplified products were digested with the restriction endonuclease, Fok I which has a single recognition site in exon 8 containing the proposed wild-type guanine at base 1086, or with Bsm I which also shows a single recognition site containing the proposed adenine nucleotide. The study failed to detect the presence of the proposed transferrin variant, confirming that the wild-type guanine was present in all 225 individuals.Biotechnology and Biological Sciences Research Counci

Extensive variation in the intelectin gene family in laboratory and wild mouse strains

Intelectins are a family of multimeric secreted proteins that bind microbe-specific glycans. Both genetic and functional studies have suggested that intelectins have an important role in innate immunity and are involved in the etiology of various human diseases, including inflammatory bowel disease. Experiments investigating the role of intelectins in human disease using mouse models are limited by the fact that there is not a clear one-to-one relationship between intelectin genes in humans and mice, and that the number of intelectin genes varies between different mouse strains. In this study we show by gene sequence and gene expression analysis that human intelectin-1 (ITLN1) has multiple orthologues in mice, including a functional homologue Itln1; however, human intelectin-2 has no such orthologue or homologue. We confirm that all sub-strains of the C57 mouse strain have a large deletion resulting in retention of only one intelectin gene, Itln1. The majority of laboratory strains have a full complement of six intelectin genes, except CAST, SPRET, SKIVE, MOLF and PANCEVO strains, which are derived from different mouse species/subspecies and encode different complements of intelectin genes. In wild mice, intelectin deletions are polymorphic in Mus musculus castaneus and Mus musculus domesticus. Further sequence analysis shows that Itln3 and Itln5 are polymorphic pseudogenes due to premature truncating mutations, and that mouse Itln1 has undergone recent adaptive evolution. Taken together, our study shows extensive diversity in intelectin genes in both laboratory and wild-mice, suggesting a pattern of birth-and-death evolution. In addition, our data provide a foundation for further experimental investigation of the role of intelectins in disease

SGP28, a novel matrix glycoprotein in specific granules of human neutrophils with similarity to a human testis-specific gene product and to a rodent sperm-coating glycoprotein

AbstractA novel 28 kDa glycoprotein was purified from exocytosed material from human neutrophils and its primary structure partially determined. Degenerate oligonucleotide primers were used to amplify cDNA clones from a human bone marrow cDNA library. The deduced 245 amino acid sequence of the 2124 bp full-length cDNA showed high degrees of similarity to the deduced sequences of the human gene TPX-1 and of sperm coating glycoprotein from rat and mouse. Subcellular fractionation of human neutrophils indicated that the protein is localized in specific granules. The protein was named SGP28 (specific granule protein of 28 kDa)

Amylase α-2A Autoantibodies: Novel Marker of Autoimmune Pancreatitis and Fulminant Type 1 Diabetes

OBJECTIVE— The pathogenesis of autoimmune pancreatitis (AIP) and fulminant type 1 diabetes remains unclear, although it is known that immune-mediated processes severely compromise the endocrine and exocrine functions in both diseases