Copper transport during lactation in transgenic mice expressing the human ATP7A protein

Both copper transporting ATPases, ATP7A and ATP7B, are expressed in mammary epithelial cells but their role in copper delivery to milk has not been clarified. We investigated the role of ATP7A in delivery of copper to milk using transgenic mice that over-express human ATP7A. In mammary gland of transgenic mice, human ATP7A protein was 10- to 20-fold higher than in control mice, and was localized to the basolateral membrane of mammary epithelial cells in lactating mice. The copper concentration in the mammary gland of transgenic dams and stomach contents of transgenic pups was significantly reduced compared to non-transgenic mice. The mRNA levels of endogenous Atp7a, Atp7b, and Ctr1 copper transporters in the mammary gland were not altered by the expression of the ATP7A transgene, and the protein levels of Atp7b and ceruloplasmin were similar in transgenic and non-transgenic mice. These data suggest that ATP7A plays a role in removing excess copper from the mammary epithelial cells rather than supplying copper to milk.

Structural and biochemical characterisation of the oxidoreductase NmDsbA3 from Neisseria meningitidis

DsbA is an enzyme found in the periplasm of Gram-negative bacteria that catalyses the formation of disulfide bonds in a diverse array of protein substrates, many of which are involved in bacterial pathogenesis. Whilst most bacteria possess only a single essential DsbA, Neisseria meningitidis is unusual in that it possesses three DsbAs, although the reason for this additional redundancy is unclear. Two of these N. meningitidis enzymes (NmDsbA1 and NmDsbA2) play an important role in meningococcal attachment to human epithelial cells, whilst NmDsbA3 is considered to have a narrow substrate repertoire. To begin to address the role of DsbAs in the pathogenesis of N. meningitidis, we have determined the structure of NmDsbA3 to 2.3 Å resolution. Although the sequence identity between NmDsbA3 and other DsbAs is low, the NmDsbA3 structure adopted a DsbA-like fold. Consistent with this finding, we demonstrated that NmDsbA3 acts as a thiol-disulfide oxidoreductase in vitro and is reoxidised by Escherichia coli DsbB (EcDsbB). However, pronounced differences in the structures between DsbA3 and EcDsbA, which are clustered around the active site of the enzyme, suggested a structural basis for the unusual substrate specificity that is observed for NmDsbA3.

Examination of the role of intestinal Fatty acid-binding protein in drug absorption using a parallel artificial membrane permeability assay

Transcellular diffusion across the absorptive epithelial cells (enterocytes) of the small intestine is the main route of absorption for most orally administered drugs. The process by which lipophilic compounds transverse the aqueous environment of the cytoplasm, however, remains poorly defined. In the present study, we have identified a structurally diverse group of lipophilic drugs that display low micromolar binding affinities for a cytosolic lipid-binding protein—intestinal fatty acid-binding protein (I-FABP). Binding to I-FABP significantly enhanced the transport of lipophilic drug molecules across a model membrane, and the degree of transport enhancement was related to both drug lipophilicity and I-FABP binding affinity. These data suggest that intracellular lipid-binding proteins such as I-FABP may enhance the membrane transport of lipophilic xenobiotics and facilitate drug access to the enterocyte cytoplasm and cytoplasmic organelles.

The interaction of lipophilic drugs with intestinal fatty acid-binding protein

Intestinal fatty acid-binding protein (I-FABP) is a small protein that binds long-chain dietary fatty acids in the cytosol of the columnar absorptive epithelial cells (enterocytes) of the intestine. The binding cavity of I-FABP is much larger than is necessary to bind a fatty acid molecule, which suggests that the protein may be able to bind other hydrophobic and amphipathic ligands such as lipophilic drugs. Herein we describe the binding of three structurally diverse lipophilic drugs, bezafibrate, ibuprofen (both R- and S-isomers) and nitrazepam to I-FABP. The rank order of affinity for I-FABP determined for these compounds was found to be R-ibuprofen {approx} bezafibrate > S-ibuprofen >> nitrazepam. The binding affinities were not directly related to aqueous solubility or partition coefficient of the compounds; however, the freely water-soluble drug diltiazem showed no affinity for I-FABP. Drug-I-FABP interaction interfaces were defined by analysis of chemical shift perturbations in NMR spectra, which revealed that the drugs bound within the central fatty acid binding cavity. Each drug participated in a different set of interactions within the cavity; however, a number of common contacts were observed with residues also involved in fatty acid binding. These data suggest that the binding of non-fatty acid lipophilic drugs to I-FABP may increase the cytosolic solubility of these compounds and thereby facilitate drug transport from the intestinal lumen across the enterocyte to sites of distribution and metabolism.

Bovine Muc1 is a highly polymorphic gene encoding an extensively glycosylated mucin that binds bacteria

The bovine Muc1 protein is synthesized by mammary epithelial cells and shed into milk as an integral component of the milk fat globule membrane; however, the structure and functions of this mucin, particularly in relation to lactation, are poorly defined. The objectives of this investigation were to investigate the Muc1 gene and protein structures in the context of lactation and to test the hypothesis that Muc1 has a role in innate immune defense. Polymerase chain reaction analysis of genomic DNA from 630 cattle revealed extensive polymorphism in the variable number of tandem repeats (VNTR) in the bovine Muc1 gene. Nine allelic
variants spanning 7 to 23 VNTR units, each encoding 20 AA, were identified. Three alleles, containing 11, 14, and 16 VNTR units, respectively, were predominant. In addition, a polymorphism in one of the VNTR units has the potential to introduce a unique site for N-linked glycosylation. Statistical analysis indicated weak associations between the VNTR alleles and milk protein and fat percentages in a progeny-tested population of Holstein-Friesian dairy cattle. No association with somatic cell count could be demonstrated. Bovine Muc1 was purified from milk fat globule membranes and characterized. The protein was highly glycosylated, primarily with O-linked sialylated T-antigen [Neu5Ac(α2–3)-Gal(β1–3)-GalNAcα1] and, to a lesser extent, with N-linked oligosaccharides, which together accounted for approximately 60% of the apparent mass of Muc1. Purified bovine Muc1 directly bound fluorescently labeled Escherichia coli BioParticles (Invitrogen, Mount Waverley, Australia) and inhibited their binding to bovine mammary epithelial cells grown in vitro.

Gut health immunomodulatory and anti-inflammatory functions of gut enzyme digested high protein micro-nutrient dietary supplement-Enprocal

Background: Enprocal is a high-protein micro-nutrient rich formulated supplementary food designed to meet the nutritional needs of the frail elderly and be delivered to them in every day foods. We studied the potential of Enprocal to improve gut and immune health using simple and robust bioassays for gut cell proliferation, intestinal integrity/permeability, immunomodulatory, anti-inflammatory and anti-oxidative activities. Effects of Enprocal were compared with whey protein concentrate 80 (WPC), heat treated skim milk powder, and other commercially available milk derived products.

Results: Enprocal (undigested) and digested (Enprocal D) selectively enhanced cell proliferation in normal human intestinal epithelial cells (FHs74-Int) and showed no cytotoxicity. In a dose dependent manner Enprocal induced cell death in Caco-2 cells (human colon adencarcinoma epithelial cells). Digested Enprocal (Enprocal D: gut enzyme cocktail treated) maintained the intestinal integrity in transepithelial resistance (TEER) assay, increased the permeability of horseradish peroxidase (HRP) and did not induce oxidative stress to the gut epithelial cells. Enprocal D upregulated the surface expression of co-stimulatory (CD40, CD86, CD80), MHC I and MHC II molecules on PMA differentiated THP-1 macrophages in coculture transwell model, and inhibited the monocyte/lymphocyte (THP-1/Jurkat E6-1 cells)-epithelial cell adhesion. In cytokine secretion analyses, Enprocal D down-regulated the secretion of proinflammatory cytokines (IL-1β and TNF-α) and up-regulated IFN-γ, IL-2 and IL-10.

Conclusion: Our results indicate that Enprocal creates neither oxidative injury nor cytotoxicity, stimulates normal gut cell proliferation, up regulates immune cell activation markers and may aid in the production of antibodies. Furthermore, through downregulation of proinflammatory cytokines, Enprocal appears to be beneficial in reducing the effects of chronic gut inflammatory diseases such as inflammatory bowel disease (IBD). Stimulation of normal human fetal intestinal cell proliferation without cell cytotoxicity indicates it may also be given as infant food particularly for premature babies.

Diversity and clonotypic composition of influenza-specific CD8+ TCR repertoires remain unaltered in the absence of Aire

TCR repertoire diversity is important for the protective efficacy of CD8⁺ T cells, limiting viral escape and cross-reactivity between unrelated epitopes. The exact mechanism for selection of restricted versus diverse TCR repertoires is far from clear, although one thought is that the epitopes resembling self-peptides might select a limited array of TCR due to the deletion of autoreactive TCR. The molecule Aire promotes the expression of tissue-specific Ag on thymic medullary epithelial cells and the deletion of autoreactive cells, and in the absence of Aire autoreactive cells persist. However, the contribution of Aire-dependent peptides to the selection of the Ag-specific TCR repertoire remains unknown. In this study, we dissect restricted (D^bNP₃₆₆%⁺CD8⁺) and diverse (D^bPA₂₂₄%⁺CD8⁺, K^dNP₁₄₇%⁺CD8⁺) TCR repertoires responding to three influenza-derived peptides in Aire-deficient mice on both B6 and BALB/c backgrounds. Our study shows that the number, qualitative characteristics and TCR repertoires of all influenza-specific, D^bNP₃₆₆%⁺CD8⁺, D^bPA₂₂₄%⁺CD8⁺ and K^dNP₁₄₇%⁺CD8⁺ T cells are not significantly altered in the absence of Aire. This provides the first demonstration that the selection of an Ag-specific T-cell repertoire is not significantly perturbed in the absence of Aire.

Molecular characterization and analysis of a truncated serotonin receptor gene expressed in neural and reproductive tissues of abalone

In molluscs, the neurotransmitter serotonin (5-HT) has been linked to a variety of biological roles including gamete maturation and spawning. The possible involvement of 5-HT in abalone gamete release was demonstrated by a dose-dependent increase in Haliotis rubra gonad contractile bioactivity following 5-HT stimulation. Physiological functions associated with 5-HT, are mediated through binding to 5-HT receptors. A cDNA encoding a putative 5-HT receptor consisting of 359 amino acids was isolated from the tropical abalone H. asinina, termed 5-HT_{1 ha}. The 5-HT_{1 ha} shares G-protein-coupled receptor motifs with metazoan 5-HT receptors, including predicted transmembrane domains, active sites for protein kinase action, and N-linked glycosylation sites. However, the third intracellular loop of 5-HT_{1 ha} is relatively short, and only six transmembrane domains are predicted, implying a truncated receptor. Phylogenetic analysis with known 5-HT receptor genes suggests that 5-HT_{1 ha} belongs to the type 1 5-HT receptor family. Expression analysis by RT-PCR showed that 5-HT_{1 ha}  mRNA was present in all tissues examined, including the neural ganglia and gonad tissues. Immunocytochemistry revealed the presence of 5-HT_{1 ha} specifically within the soma of neuronal cells located in the outer cortex of both cerebral and pleuropedal ganglia. In ovarian and testicular tissues, 5-HT_{1 ha} immunoreactivity was observed in epithelial cells of the outer capsule and connective tissue of the trabeculae to which the gamete follicles adhere. Whether this receptor transcript is translated to a functional protein needs to be verified, but if so, it could play a role in reproduction.

A novel approach identified the FOLR1 gene, a putative regulator of milk protein synthesis

This study has utilised comparative functional genomics to exploit animal models with extreme adaptation to lactation to identify candidate genes that specifically regulate protein synthesis in the cow mammary gland. Increasing milk protein production is valuable to the dairy industry. The lactation strategies of both the Cape fur seal (Artocephalus pusillus pusillus) and the tammar wallaby (Macropus eugenii) include periods of high rates of milk protein synthesis during an established lactation and therefore offer unique models to target genes that specifically regulate milk protein synthesis. Global changes in mammary gene expression in the Cape fur seal, tammar wallaby, and the cow (Bos taurus) were assessed using microarray analysis. The folate receptor α (FOLR1) showed the greatest change in gene expression in all three species [cow 12.7-fold (n = 3), fur seal 15.4-fold (n = 1), tammar 2.4-fold (n = 4)] at periods of increased milk protein production. This compliments previous reports that folate is important for milk protein synthesis and suggests FOLR1 may be a key regulatory point of folate metabolism for milk protein synthesis within mammary epithelial cells (lactocytes). These data may have important implications for the dairy industry to develop strategies to increase milk protein production in cows. This study illustrates the potential of comparative genomics to target genes of interest to the scientific community.

Bovine lactoferrin : potential antioxidant effector molecule in colon carcinoma

This research generated important insight into the antioxidant capacity and effector molecule capability of bovine lactoferrin in both its Apo- (iron free) and metal saturated forms within the gastrointestinal system, giving rise to bovine lactoferrin's potential at regulating oxidative stress associated signalling molecules within colon carcinoma.

A Pseudomonas aeruginosa toxin that hijacks the host ubiquitin proteolytic system

Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen chronically infecting the lungs of patients with chronic obstructive pulmonary disease (COPD), pneumonia, cystic fibrosis (CF), and bronchiectasis. Cif (PA2934), a bacterial toxin secreted in outer membrane vesicles (OMV) by P. aeruginosa, reduces CFTR-mediated chloride secretion by human airway epithelial cells, a key driving force for mucociliary clearance. The aim of this study was to investigate the mechanism whereby Cif reduces CFTR-mediated chloride secretion. Cif redirected endocytosed CFTR from recycling endosomes to lysosomes by stabilizing an inhibitory effect of G3BP1 on the deubiquitinating enzyme (DUB), USP10, thereby reducing USP10-mediated deubiquitination of CFTR and increasing the degradation of CFTR in lysosomes. This is the first example of a bacterial toxin that regulates the activity of a host DUB. These data suggest that the ability of P. aeruginosa to chronically infect the lungs of patients with COPD, pneumonia, CF, and bronchiectasis is due in part to the secretion of OMV containing Cif, which inhibits CFTR-mediated chloride secretion and thereby reduces the mucociliary clearance of pathogens.

The ΔF508-CFTR mutation results in increased biofilm formation by Pseodomonas aeruginosa by increasing iron availability

Enhanced antibiotic resistance of Pseudomonas aeruginosa in the cystic fibrosis (CF) lung is thought to be due to the formation of biofilms. However, there is no information on the antibiotic resistance of P. aeruginosa biofilms grown on human airway epithelial cells or on the effects of airway cells on biofilm formation by P. aeruginosa. Thus we developed a coculture model and report that airway cells increase the resistance of P. aeruginosa to tobramycin (Tb) by >25-fold compared with P. aeruginosa grown on abiotic surfaces. Therefore, the concentration of Tb required to kill P. aeruginosa biofilms on airway cells is 10-fold higher than the concentration achievable in the lungs of CF patients. In addition, CF airway cells expressing ΔF508-CFTR significantly enhanced P. aeruginosa biofilm formation, and ΔF508 rescue with wild-type CFTR reduced biofilm formation. Iron (Fe) content of the airway in CF is elevated, and Fe is known to enhance P. aeruginosa growth. Thus we investigated whether enhanced biofilm formation on ΔF508-CFTR cells was due to increased Fe release by airway cells. We found that airway cells expressing ΔF508-CFTR released more Fe than cells rescued with WT-CFTR. Moreover, Fe chelation reduced biofilm formation on airway cells, whereas Fe supplementation enhanced biofilm formation on airway cells expressing WT-CFTR. These data demonstrate that human airway epithelial cells promote the formation of P. aeruginosa biofilms with a dramatically increased antibiotic resistance. The ΔF508-CFTR mutation enhances biofilm formation, in part, by increasing Fe release into the apical medium.

Chemotoxicity of doxorubicin and surface expression of P-glycoprotein (MDR1) is regulated by the Pseudomonas aeruginosa toxin Cif

P-glycoprotein (Pgp), a member of the adenosine triphosphate-binding cassette (ABC) transporter superfamily, is a major drug efflux pump expressed in normal tissues, and is overexpressed in many human cancers. Overexpression of Pgp results in reduced intracellular drug concentration and cytotoxicity of chemotherapeutic drugs and is thought to contribute to multidrug resistance of cancer cells. The involvement of Pgp in clinical drug resistance has led to a search for molecules that block Pgp transporter activity to improve the efficacy and pharmacokinetics of therapeutic agents. We have recently identified and characterized a secreted toxin from Pseudomonas aeruginosa, designated cystic fibrosis transmembrane conductance regulator (CFTR) inhibitory factor (Cif). Cif reduces the apical membrane abundance of CFTR, also an ABC transporter, and inhibits the CFTR-mediated chloride ion secretion by human airway and kidney epithelial cells. We report presently that Cif also inhibits the apical membrane abundance of Pgp in kidney, airway, and intestinal epithelial cells but has no effect on plasma membrane abundance of multidrug resistance protein 1 or 2. Cif increased the drug sensitivity to doxorubicin in kidney cells expressing Pgp by 10-fold and increased the cellular accumulation of daunorubicin by 2-fold. Thus our studies show that Cif increases the sensitivity of Pgp-overexpressing cells to doxorubicin, consistent with the hypothesis that Cif affects Pgp functional expression. These results suggest that Cif may be useful to develop a new class of specific inhibitors of Pgp aimed at increasing the sensitivity of tumors to chemotherapeutic drugs, and at improving the bioavailability of Pgp transport substrates.