Natural Polyphenols May Normalize Hypochlorous Acid-Evoked Hemostatic Abnormalities in Human Blood.

During pathogen invasion, activated neutrophils secrete myeloperoxidase (MPO), which generates high local concentrations of hypochlorous acid (HOCl), a strong antimicrobial agent. Prolonged or uncontrolled HOCl production may, however, affect hemostasis, manifesting in inhibition of platelet aggregation and thrombus formation and in elevated fibrin density and attenuated fibrinolysis. In this report, we investigated whether three plant-derived polyphenols with well-known antioxidant properties, i.e., quercetin (Que), epigallocatechin gallate (EGCG), and resveratrol (Resv), at concentrations not affecting platelet responses per se, may normalize particular aspects of hemostasis disturbed by HOCl. Specifically, Que (5-25 µM) and EGCG (10-25 µM) abolished HOCl-evoked inhibition of platelet aggregation (assessed by an optical method), while the simultaneous incubation of platelet-rich plasma with Resv (10-25 µM) enhanced the inhibitory effect of HOCl. A similar effect was observed in the case of thrombus formation under flow conditions, evaluated in whole blood by confocal microscope. When plasma samples were incubated with HOCl, a notably higher density of fibrin (recorded by confocal microscope) was detected, an effect that was efficiently normalized by Que (5-25 µM), EGCG (10-25 µM), and Resv (5-25 µM) and which corresponded with the normalization of the HOCl-evoked prolongation of fibrinolysis, measured in plasma by a turbidimetric method. In conclusion, this report indicates that supplementation with Que and EGCG may be helpful in the normalization of hemostatic abnormalities during inflammatory states associated with elevated HOCl production, while the presence of Resv enhances the inhibitory action of HOCl towards platelets.

IGFBP5 induces cell adhesion, increases cell survival and inhibits cell migration in MCF-7 human breast cancer cells.

Maintenance of tissue boundaries is crucial for control of metastasis. We describe a new signalling pathway in which epithelial cell disruption can be minimised and thereby restricts epithelial-mesenchymal transgressions. This involves the release of insulin-like growth factor (IGF)-binding protein 5 (IGFBP5) from apoptotic cells, which increases the adhesion of epithelial cells on mesenchymal but not epithelial extracellular matrix (ECM), and involves the direct interaction of IGFBP5 and α2ß1 integrins. IGFBP5 also induced cell adhesion to vitronectin in the absence of αVß3 integrin, the vitronectin receptor, again through an α2ß1-integrin-dependent action, suggesting that IGFBP5 can induce spreading on matrices, even in the absence of the integrins normally used in this process. Using IGFBP5 mutants we demonstrate that the effect is IGF-independent but requires the heparin-binding domain in the C-terminus of IGFBP5. A truncated mutant containing only the C-terminal of IGFBP5 also induced adhesion. Adhesion induced by IGFBP5 was dependent on Cdc42 and resulted in activation of integrin-linked kinase (ILK) and Akt. Consistent with these changes, IGFBP5 facilitated prolonged cell survival in nutrient-poor conditions and decreased phosphorylation of the stress-activated kinase p38 MAPK (MAPK14). Whereas IGFBP5 enhanced adhesion, it inhibited cell migration, although this was not evident using the truncated C-terminal mutant, suggesting that effects of IGFBP5 on adhesion and migration involve different mechanisms. We anticipate that these responses to IGFBP5 would reduce the metastatic potential of cells.

IGFBP-5 induces epithelial and fibroblast responses consistent with the fibrotic response.

Fibrosis involves activation of fibroblasts, increased production of collagen and fibronectin and transdifferentiation into contractile myofibroblasts. The process resembles aspects of wound-healing but remains unresolved and can be life-threatening when manifest in the kidneys, lungs and liver, in particular. The causes are largely unknown, but recent suggestions that repetitive micro-injury results in the eventual failure of epithelial cell repair due to replicative senescence are gaining favour. This is consistent with the onset of fibrotic diseases in middle age. Because epithelial injury often involves blood loss, inflammatory responses associated with the fibrotic response have been considered as therapeutic targets. However, this has proved largely unsuccessful and focus is now switching to earlier events in the process. These include EMT (epithelial-mesenchymal transition) and fibroblast activation in the absence of inflammation. TGFbeta1 (transforming growth factor-beta1) induces both EMT and fibroblast activation and is considered to be a major pro-fibrotic factor. Recently, IGFBP-5 [IGF (insulin-like growth factor)-binding protein-5] has also been shown to induce similar effects on TGFbeta1, and is strongly implicated in the process of senescence. It also stimulates migration of peripheral blood mononuclear cells, implicating it in the inflammatory response. In this paper, we examine the evidence for a role of IGFBP-5 in fibrosis and highlight its structural relationship with other matrix proteins and growth factors also implicated in tissue remodelling.

EMMPRIN (basigin/CD147) expression is not correlated with MMP activity during adult mouse mammary gland development.

Extracellular matrix metalloproteinase inducer (EMMPRIN/basigin/CD147) is a cell surface protein, which has been associated with the induction of matrix metalloproteinase (MMP) genes during cancer metastasis. EMMPRIN plays a role in a variety of physiological processes as is evident by the diverse deficiencies detectable in EMMPRIN knockout mice. We have analysed the role of EMMPRIN in the induction of MMP genes during mammary gland differentiation and involution. Co-transfection studies showed that EMMPRIN has diverse effects on MMP promoter activity in different mammary and non-mammary cell lines. Expression of EMMPRIN mRNA is enhanced markedly by insulin in a mammary gland cell line but appears to have no direct effect on MMP gene expression in these cells. Microarray analysis and quantitative PCR show that EMMPRIN is expressed throughout mammary gland differentiation in the mouse. Its expression decreases during early pregnancy and briefly after induction of mammary gland involution by litter removal. Immunohistochemical analysis shows that EMMPRIN expression is limited to the stromal compartment during pregnancy, whereas it is strongly expressed in the epithelium during lactation. In summary the data argue against a causal role for EMMPRIN for the induction of MMP gene expression during adult mammary gland development. These data therefore support a physiological role for EMMPRIN other than MMP induction in mammary gland biology.

Molecular interactions in the insulin-like growth factor (IGF) axis: a surface plasmon resonance (SPR) based biosensor study.

This review describes a comprehensive analysis of a surface plasmon resonance (SPR)-based biosensor study of molecular interactions in the insulin-like growth factor (IGF) molecular axis. In this study, we focus on the interaction between the polypeptide growth factors IGF-I and IGF-II with six soluble IGF binding proteins (IGFBP 1-6), which occur naturally in various biological fluids. We have describe the conditions required for the accurate determination of kinetic rate constants for these interactions and highlight the experimental and theoretical pitfalls, which may be encountered in the early stages of such a study. We focus on IGFBP-5 and describe a site-directed mutagenesis study, which examines the contribution of various residues in the protein to high affinity interaction with IGF-I and -II. We analyse the interaction of IGFBP-5 (and IGFBP-3) with heparin and other biomolecules and describe experiments, which were designed to monitor multi-protein complex formation in this molecular axis.

Cumulative mutagenesis of the basic residues in the 201-218 region of insulin-like growth factor (IGF)-binding protein-5 results in progressive loss of both IGF-I binding and inhibition of IGF-I biological action.

We have reported previously that mutation of two conserved nonbasic amino acids (G203 and Q209) within the highly basic 201-218 region in the C-terminal domain of IGF-binding protein-5 (IGFBP-5) decreases binding to IGFs. This study reveals that cumulative mutagenesis of the 10 basic residues in this region, to create the C-Term series of mutants, ultimately results in a 15-fold decrease in the affinity for IGF-I and a major loss in heparin binding. We examined the ability of mutants to inhibit IGF-mediated survival of MCF-7 cells and were able to demonstrate that this depended not only upon the affinity for IGF-I, but also the kinetics of this interaction, because IGFBP-5 mutants with similar affinity constants (K(D)) values, but with different association (Ka) and dissociation (Kd) rate values, had markedly different inhibitory properties. In contrast, the affinity for IGF-I provided no predictive value in terms of the ability of these mutants to enhance IGF action when bound to the substratum. Instead, these C-Term mutants appeared to enhance the actions of IGF-I by a combination of increased dissociation of IGF-IGFBP complexes from the substratum, together with dissociation of IGF-I from IGFBP-5 bound to the substratum. These effects of the IGFBPs were dependent upon binding to IGF-I, because a non-IGF binding mutant (N-Term) was unable to inhibit or enhance the actions of IGF-I. These results emphasize the importance of the kinetics of association/dissociation in determining the enhancing or inhibiting effects of IGFBP-5 and demonstrate the ability to generate an IGFBP-5 mutant with exclusively IGF-enhancing activity.

Regulation of genes encoding proteolytic enzymes during mammary gland development.

The mammary gland undergoes extensive tissue remodelling during each lactation cycle. During pregnancy, the epithelial compartment of the gland is vastly expanded (Benaud et al. 1998). At the end of lactation the epithelial cells undergo apoptosis and adipocyte differentiation is induced (Lilla et al. 2002). Ductal and alveolar growth during puberty and pregnancy, and the involution process require the action of proteolytic enzymes (including matrix metalloproteinases, plasminogen and membrane-peptidases) and the corresponding genes are activated during these periods (Benaud et al. 1998; Alexander et al. 2001). Matrix metalloproteinases (MMP) are expressed in several cell types of the mammary gland including stromal fibroblasts (e.g., MMP3, MMP2), epithelial cells (e.g., MMP7 or MMP9), adipocytes (e.g., MMP2) and lymphoid cells (e.g., MMP9) (Crawford et al. 1996; Lund et al. 1996; Wiseman et al. 2003). A number of knock-out mice, which are deficient for individual MMP genes (e.g., MMP2, MMP3) or plasminogen, display alterations to mammary gland structure and impairment of lactation (Lund et al. 1999; Wiseman et al. 2003).

Molecular recognition characteristics in the insulin-like growth factor (IGF)-insulin-like growth factor binding protein -3/5 (IGFBP-3/5) heparin axis.

Insulin-like growth factor binding proteins (IGFBPs) -3 and -5 are known to interact with various components of the extracellular matrix (ECM; e.g. heparin and heparan sulphate) and this interaction is believed to affect the affinity of both IGFBP species for their cognate ligands--IGF-I and -II. There is little detail on the nature of the molecular complex formed between ECM components, IGFBPs and IGFs although the glycosaminoglycan (GAG) heparin has been reported to reduce the affinity of IGFBP-5 for IGF-I. In order to investigate this phenomenon further, we have undertaken an extensive surface plasmon resonance based biosensor study to report the affinity of IGFBP-3 and -5 for binding heparin (22 and 7 nM respectively). We have also shown that pre-complexation of IGFBP with IGF-I and -II inhibits the subsequent association of IGFBP with heparin and conversely that heparin complexation of IGFBP-3 and -5 inhibits IGFBP binding to biosensor surfaces containing immobilised IGF-I. In addition we have used both IGF-I and heparin coated biosensor surfaces in an attempt to build ternary IGF-IGFBP-heparin complexes in order to gain some insight into the nature of inhibition by heparin of IGFI-IGFBP complex formation. Our data lead us to conclude that the inhibition by heparin is partly competitive in nature, and that ternary complexes of IGF-IGFBP-heparin are either unable to form, or only form unstable transient complexes. The potential biological significance of our data is highlighted by the demonstration that IGF-I and IGF-II can displace endogenous IGFBP-5 from monolayer cultures of the mouse mammary epithelial cell line HC11.

The DNA-binding capacity of genetic variants of the bovine STAT5A transcription factor.

The STATs are a family of transcription factors. STAT5A, previously known as MGF, transduces prolactin signals to the milk protein genes. Here, we describe the detection of nucleotide sequence polymorphism in exon 16 of the bovine STAT5A gene, coding for the SH2 domain. SSCP was found in a 281-bp PCR amplified gene fragment, lying between positions 12,525 and 12,806, and encompassing parts of intron 15 and exon 16 of the bovine STAT5A gene (GenBank AJ 237937). Three SSCP patterns (genotypes) were identified in a group of 108 animals of different cattle breeds. The DNA sequencing showed that they differed by a CCT deletion at position from 12,549 in intron 15, and a T-->C substitution at position 12,743 in exon 16. The latter mutation changes an amino acid sequence in the STAT5A protein - a Val/Ala substitution at position 686. Since T-->C substitution creates a new MslI site, genetic variants in the bovine STAT5A gene can be distinguished with RFLP analysis. The frequency of alleles T and C varied between the different cattle breeds studied; the CC genotype was the least frequent and the frequency of alleles T and C was 0.842 and 0.158, respectively. Proteins were extracted from the cell nuclei of liver tissues derived from bulls of different STAT5A genotypes and subjected to EMSA in order to study if the amino acid substitution might change the DNA-binding capacity of STAT5A transcription factor. Statistically significant (p<0.05) differences in nuclear protein binding to DNA were observed between genotypes TT and CC; nuclear proteins derived from CC animals always showed less DNA protein complexing than those of TT animals. EMSA competition experiments confirmed that STAT5 transcription factors take part in the formation of the DNA-protein complexes.

The impact of genetic polymorphisms on the protein composition of ruminant milks.

The purpose of this review is to give an overview of our current knowledge on the polymorphisms occurring in genes coding for milk proteins and responsible for quantitative variability in their expression, thus influencing the protein composition of livestock ruminant milk. The overall genomic organisation of the 6 main ruminant milk protein genes: alpha-lactalbumin, beta-lactoglobulin and the four caseins (alpha(s1), alpha(s2), beta and kappa), their chromosomal location and their expression pattern are first summarised before presenting general mechanisms controlling gene expression both at the transcriptional and the post-transcriptional levels. Polymorphisms found in cis-regulatory elements, mainly within the 5'-flanking region of the genes encoding beta-lactoglobulin and alpha(s1)- and alpha(s2)-caseins, have been found, in cattle, to influence their transcription rate. In addition, polymorphisms found in the transcription unit, within intron as well as exon sequences, have been shown to be responsible for defects in the processing of primary transcripts and/or the export of messenger RNA to the cytoplasm. Mutations responsible for the occurrence of premature stop codons in alpha(s1)- and beta-casein mRNAs have been shown to be associated both with a decrease in the level of the relevant transcripts and the existence of multiple forms of messengers due to alternative splicing (exon skipping, usage of cryptic splice sites). Such a situation, well-exemplified by the gene encoding alpha(s1)-casein in the goat, may have dramatic biological consequences (secretion pathway, casein micelle structure, fat content, etc.) by modifying the message and accordingly the primary structure of the protein as well as its expression. Since some of these polymorphisms dramatically affect technological properties of milk, including cheese yields and organoleptic characteristics, methods mainly based on the PCR technique have been designed and applied in selection and breeding programmes to improve milk protein quality.