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Biomedical subjects

A J Day

Publications and source records attributed to A J Day.

At least 37 records · Page 2Linked to original sources
PubMed2000

Dietary flavonoid and isoflavone glycosides are hydrolysed by the lactase site of lactase phlorizin hydrolase.

Lactase phlorizin hydrolase (LPH; EC 3.2.1.62) is a membrane-bound, family 1 beta-glycosidase found on the brush border of the mammalian small intestine. LPH, purified from sheep small intestine, was capable of hydrolysing a range of flavonol and isoflavone glycosides. The catalytic efficiency (k(cat)/K(m)) for the hydrolysis of quercetin-4'-glucoside, quercetin-3-glucoside, genistein-7-glucoside and daidzein-7-glucoside was 170, 137, 77 and 14 (mM(-1) s(-1)) respectively. The majority of the activity occurred at the lactase and not phlorizin hydrolase site. The ability of LPH to deglycosylate dietary (iso)flavonoid glycosides suggests a possible role for this enzyme in the metabolism of these biologically active compounds.

Animals↗
PubMed2000

Separate effects of triploidy, parentage and genomic diversity upon feeding behaviour, metabolic efficiency and net energy balance in the Pacific oyster Crassostrea gigas.

Triploid oysters were induced using cytochalasin B upon retention of either the first (meiosis I triploids) or the second (meiosis II triploids) polar body in embryos from a single cohort derived from mixed parentage. Allozyme and microsatellite assays enabled the confirmation of both parentage and triploidy status in each oyster. Comparison of meiosis I triploids, meiosis II triploids and diploid siblings established that improved physiological performance in triploids was associated with increased allelic variation, rather than with the quantitative dosage effects of ploidy status. An unidentified maternal influence also interacted with genotype. Among full sibs, allelic variation measured as multi-locus enzyme heterozygosity accounted for up to 42% of the variance in physiological performance; significant positive influences were identified upon feeding rate, absorption efficiency, net energy balance and growth efficiency (= net energy balance divided by energy absorbed). Whilst allelic variation was greater in both meiosis I and meiosis II triploids than in diploid siblings, both allelic variation and net energy balance were highest in triploids induced at meiosis I. This suggests that it may be preferable to induce triploidy by blocking meiosis I, rather than meiosis II as has traditionally been undertaken during commercial breeding programmes.

Animals↗
PubMed2000

Human metabolic pathways of dietary flavonoids and cinnamates.

Flavonoids and cinnamates are widespread phenolic secondary metabolites synthesized by plants for defensive purposes. Many foods and beverages contain high levels of phenolic compounds. Certain phenolics in the diet are particularly bioactive and have pronounced effects on mammalian cells. These effects, together with epidemiological studies and animal models, have led to the hypothesis that dietary phenolics contribute to the health benefits of a diet rich in fruit and vegetables. This paper examines the biochemistry of the uptake and metabolic route of two groups of plant phenolics, the flavonols and hydroxycinnamates.

Biological Transport↗
PubMed2000

Intestinal transport of quercetin glycosides in rats involves both deglycosylation and interaction with the hexose transport pathway.

Flavonoids are polyphenolic plant secondary metabolites with antioxidant and other biological activities potentially beneficial to health. Food-borne flavonoids occur mainly as glycosides, some of which can be absorbed in the human small intestine; however, the mechanism of uptake is uncertain. We used isolated preparations of rat small intestine to compare the uptake of the quercetin aglycone with that of some quercetin glucosides commonly found in foods, and investigated interactions between quercetin-3-glucoside and the intestinal hexose transport pathway. The nature of any metabolism of quercetin and its glucosides during small intestinal transport in vitro was determined by HPLC. The presence of quercetin-3-glucoside in the mucosal medium suppressed the uptake of labeled galactose by competitive inhibition and stimulated the efflux of preloaded galactose. Quercetin-3-glucoside and quercetin-4'-glucoside, but not quercetin-3,4'-diglucoside, were transported into everted sacs significantly more quickly than quercetin aglycone. Intact quercetin glucosides were not detected in mucosal tissue or within the serosal compartment, but both free quercetin and its metabolites were present, mainly as quercetin-3-glucuronide and quercetin-7-glucuronide. Evidently, quercetin derived from quercetin-3-glucoside passes across the small intestinal epithelium more rapidly than free quercetin aglycone. Monoglucosides of quercetin interact with the sodium-dependent glucose transporter. During passage across the epithelium, quercetin-3-glucoside is rapidly deglycosylated and then glucuronidated.

Analysis of Variance↗
PubMed1999

Purification of cytosolic beta-glucosidase from pig liver and its reactivity towards flavonoid glycosides.

Flavonoid glycosides are common dietary components which may have health-promoting activities. The metabolism of these compounds is thought to influence their bioactivity and uptake from the small intestine. It has been suggested that the enzyme cytosolic beta-glucosidase could deglycosylate certain flavonoid glycosides. To test this hypothesis, the enzyme was purified to homogeneity from pig liver for the first time. It was found to have a molecular weight (55 kDa) and specific activity (with p-nitrophenol glucoside) consistent with other mammalian cytosolic beta-glucosidases. The pure enzyme was indeed found to deglycosylate various flavonoid glycosides. Genistein 7-glucoside, daidzein 7-glucoside, apigenin 7-glucoside and naringenin 7-glucoside all acted as substrates, but we were unable to detect activity with naringenin 7-rhamnoglucoside. Quercetin 4'-glucoside was a substrate, but neither quercetin 3, 4'-diglucoside, quercetin 3-glucoside nor quercetin 3-rhamnoglucoside were deglycosylated. Estimates of K(m) ranged from 25 to 90 microM while those for V(max) were about 10% of that found with the standard artificial substrate p-nitrophenol glucoside. The non-substrate quercetin 3-glucoside was found to partially inhibit deglycosylation of quercetin 4'-glucoside, but it had no effect upon activity with p-nitrophenol glucoside. This study confirms that mammalian cytosolic beta-glucosidase can deglycosylate some, but not all, common dietary flavonoid glycosides. This enzyme may, therefore, be important in the metabolism of these compounds.

Animals↗
PubMed1998

Deglycosylation of flavonoid and isoflavonoid glycosides by human small intestine and liver beta-glucosidase activity.

Flavonoid and isoflavonoid glycosides are common dietary phenolics which may be absorbed from the small intestine of humans. The ability of cell-free extracts from human small intestine and liver to deglycosylate various (iso)flavonoid glycosides was investigated. Quercetin 4'-glucoside, naringenin 7-glucoside, apigenin 7-glucoside, genistein 7-glucoside and daidzein 7-glucoside were rapidly deglycosylated by both tissue extracts, whereas quercetin 3,4'-diglucoside, quercetin 3-glucoside, kaempferol 3-glucoside, quercetin 3-rhamnoglucoside and naringenin 7-rhamnoglucoside remained unchanged. The Km for hydrolysis of quercetin 4'-glucoside and genistein 7-glucoside was approximately 32+/-12 and approximately 14+/-3 microM in both tissues respectively. The enzymatic activity of the cell-free extracts exhibits similar properties to the cytosolic broad-specificity -glucosidase previously described in mammals.

Cell Extracts↗
PubMed1998

TSG-6 interacts with hyaluronan and aggrecan in a pH-dependent manner via a common functional element: implications for its regulation in inflamed cartilage.

Cartilage matrix is stabilised by the interactions of proteins with hyaluronan (HA). We compare the pH dependences of HA binding by aggrecan, link protein and TSG-6. Aggrecan and link protein exhibit maximal binding across a wide pH range (6.0-8.0). TSG-6, a protein that is only produced during inflammation, binds maximally at about pH 6.0 but shows a dramatic loss of function with increasing pH. TSG-6 also interacts with aggrecan, with a similar pH dependence, and this can be inhibited by HA. Thus, a common binding surface on TSG-6 may be involved in HA and aggrecan binding. We propose that TSG-6 is involved in matrix dissociation and that this is regulated by pH gradients in cartilage.

Aggrecans↗
PubMed1998

Identification of CD44 residues important for hyaluronan binding and delineation of the binding site.

CD44 is a widely distributed cell surface protein that plays a role in cell adhesion and migration. As a proteoglycan, CD44 is also implicated in growth factor and chemokine binding and presentation. The extracellular region of CD44 is variably spliced, giving rise to multiple CD44 isoforms. All isoforms contain an amino-terminal domain, which is homologous to cartilage link proteins. The cartilage link protein-like domain of CD44 is important for hyaluronan binding. The structure of the link protein domain of TSG-6 has been determined by NMR. Based on this structure, a molecular model of the link-homologous region of CD44 was constructed. This model was used to select residues for site-specific mutagenesis in an effort to identify residues important for ligand binding and to outline the hyaluronan binding site. Twenty-four point mutants were generated and characterized, and eight residues were identified as critical for binding or to support the interaction. In the model, these residues form a coherent surface the location of which approximately corresponds to the carbohydrate binding sites in two functionally unrelated calcium-dependent lectins, mannose-binding protein and E-selectin (CD62E).

Amino Acid Sequence↗
PubMed1998

Characterization of a functional hyaluronan-binding domain from the human CD44 molecule expressed in Escherichia coli.

The CD44 molecule is a widely distributed cell surface receptor for the extracellular matrix glycosaminoglycan hyaluronan. The ligand-binding site which is located in the membrane distal portion of the molecule encompasses a region of approximately 100 amino acids termed the Link domain, a structural unit that is conserved among members of the Hyaladherin superfamily which includes cartilage link protein, aggrecan, and tumor necrosis factor-stimulated gene-6 (TSG-6). In contrast to these other Hyaladherins, however, the ligand-binding domain of CD44 appears to extend beyond the Link domain to involve additional basic residues located toward the membrane proximal region. Furthermore, recent molecular modeling studies indicate that within the CD44 Link domain itself, the spatial arrangement of critical residues involved in HA binding is likely to differ significantly from the prototypic TSG-6 Link module. In order to obtain material to solve the CD44 solution structure we have developed an optimized method for the expression and purification of functionally active CD44 ectodomains encompassing both the Link module and the additional downstream HA-binding residues in Escherichia coli. Here we describe the details of the method which involves solubilization of recombinant CD44 from inclusion bodies in 8 M urea, followed by refolding and purification of intact monomers using size-exclusion and reverse-phase chromatography. We show the method yields CD44 molecules that (1) retain reactivity with a panel of conformation-sensitive antibodies, (2) possess similar hyaluronan-binding characteristics to authentically folded CD44 molecules expressed in eukaryotic cells, and (3) display one-dimensional NMR spectra that indicate the presence of a single conformational species. This method should enable sufficient amounts of functional CD44 Link module to be produced for comprehensive structural analyses by multidimensional NMR spectroscopy.

Binding Sites↗
PubMed1998

Characterization of flavonoids as monofunctional or bifunctional inducers of quinone reductase in murine hepatoma cell lines.

The ability of flavonoid compounds to induce the activity of the phase II anticarcinogenic marker enzyme, quinone reductase (QR), has been studied in a wild-type murine hepatoma cell line (Hepalclc7) and in an Ah-receptor-defective mutant of the same cell line (Hepalclc7 bp(r)cl). The results showed that 10 (beta-naphthoflavone, kaempferide, tamarixetin, rhamnetin, quercetin, kaempferol, quercetin-4'-glucoside, isorhamnetin, daidzein and genistein) of the 13 flavonoids tested induced QR activity in the wild-type cells. Only the latter six also showed such activity in the bp(r)cl mutant, which indicates that they induce phase II enzymes directly (monofunctional inducers), whereas the others induce phase 11 enzymes only in cells with an operative Ah receptor system (bifunctional inducers). The metabolism of representatives of monofunctional (quercetin) and bifunctional (tamarixetin and rhamnetin) flavonol inducers were studied in both wild-type and bp(r)cl cells. In all cases, the major metabolites were glucuronides. Quercetin produced identical metabolites in both cell types, whereas one glucuronide of tamarixetin and two glucuronides of rhamnetin were not formed in the mutant cells. This shows that flavonoids can be mono- or bifunctional inducers depending on their chemical structure, and that the glucuronidation pattern of bifunctional inducers is altered by the presence of a functional Ah receptor system.

Animals↗
PubMed1997

The SH2 domain from the tyrosine kinase Fyn in complex with a phosphotyrosyl peptide reveals insights into domain stability and binding specificity.

BACKGROUND: SH2 domains are found in a variety of signal transduction proteins; they bind phosphotyrosine-containing sequences, allowing them to both recognize target molecules and regulate intramolecular kinase activity. Fyn is a member of the Src family of tyrosine kinases that are involved in signal transduction by association with a number of membrane receptors. The kinase activity of these signalling proteins is modulated by switching the binding mode of their SH2 and SH3 domains from intramolecular to intermolecular. The molecular basis of the signalling roles observed for different Src family members is still not well understood; although structures have been determined for the SH2 domains of other Src family molecules, this is the first structure of the Fyn SH2 domain. RESULTS: The structure of the Fyn SH2 domain in complex with a phosphotyrosyl peptide (EPQpYEEIPIYL) was determined by high resolution NMR spectroscopy. The overall structure of the complex is analogous to that of other SH2-peptide complexes. Noteworthy aspects of the structure are: the BG loop, which contacts the bound peptide, contains a type-I' turn; a capping-box-like interaction is present at the N-terminal end of helix alpha A; cis-trans isomerization of the Val beta G1-Pro beta G2 peptide bond causes conformational heterogeneity of residues near the N and C termini of the domain. CONCLUSIONS: Comparison of the Fyn SH2 domain structure with other structures of SH2 domains highlights several interesting features. Conservation of helix capping interactions among various SH2 domains is suggestive of a role in protein stabilisation. The presence of a type-I' turn in the BG loop, which is dependent on the presence of a glycine residue at position BG3, is indicative of a binding pocket, characteristic of the Src family, SykC and Abl, rather than a binding groove found in PLC-gamma 1C, p85 alpha N and Shc, for example.

Amino Acid Sequence↗
PubMed1997

Overlapping sites on the Link module of human TSG-6 mediate binding to hyaluronan and chrondroitin-4-sulphate.

Link modules are hyaluronan-binding domains that are involved in the formation and stability of extracellular matrix and cell migration. We have examined the glycosaminoglycan specificity of the Link module from the arthritis-associated protein, human TSG-6, by microtitre plate-based assays employing biotinylated-hyaluronan or mono-biotinylated Link module. This domain was found to interact specifically with chondroitin-4-sulphate (C4S), with similar affinity to hyaluronan, but not with chondroitin-6-sulphate or heparin. Competition experiments indicate that C4S and hyaluronan have overlapping binding surfaces on the TSG-6 Link module. Disease-associated changes in C4S expression may influence the localisation and biological role of TSG-6.

Aggrecans↗
PubMed1997

Method for quantitative refolding of the link module from human TSG-6.

We have developed a procedure for the quantitative refolding of the Link module from human tumor necrosis factor-stimulated gene 6. This significantly simplifies the previously described method of production of this protein domain (Day et al., Protein Expression Purif. 8, 1-16, 1996). The refolding is carried out under nondenaturing conditions at pH 6.0 in the presence of a 100-fold molar excess of beta-mercaptoethanol. After 2 days the starting material, which consists of three species that differ only with respect to their disulfide bond organization, has rearranged to give a single homogeneous species with the correct disulfide bridges. This method allows the production of about 20 mg of folded protein per liter of Escherichia coli culture.

Cell Adhesion Molecules↗
PubMed1997

The C1q and collectin binding site within C1q receptor (cell surface calreticulin).

C1q receptor (C1qR/collectin receptor/cC1qR) has an almost complete amino acid sequence identity with calreticulin (CRT). C1qR/CRT is located on the surface of many cell types. Binding of C1q to C1q receptor elicits a range of immunological responses. C1qR also interacts with the collectins SP-A, MBL, CL43 and conglutinin via a cluster of charged residues on the collagen tails of the ligands. In order to localise C1q and collectin binding activity within C1qR/CRT, recombinant C1qR/CRT domains [N (residues 18-196), P (197-308) and C (309-417)] were produced. Both the N- and P-domains bound to C1q, demonstrating that the binding site spans the intersection of these domains. Amino acid alignment analysis identified a putative CUB module within this region. This S-domain (residues 160-283) was expressed and showed concentration-dependent binding to immobilised C1q, demonstrating that it contains the C1q binding site. Competitive inhibition studies of the S-domain-C1q interaction revealed that the S-domain binds to C1q collagen tails and to the collectin proteins, SP-A, MBL, CL43 and conglutinin. The C1q and collection binding site on C1qR/CRT has therefore been localised to the S-domain.

Amino Acid Sequence↗
PubMed1996

Solution structure of the link module: a hyaluronan-binding domain involved in extracellular matrix stability and cell migration.

Link modules are hyaluronan-binding domains found in proteins involved in the assembly of extracellular matrix, cell adhesion, and migration. The solution structure of the Link module from human TSG-6 was determined and found to consist of two alpha helices and two antiparallel beta sheets arranged around a large hydrophobic core. This defines the consensus fold for the Link module superfamily, which includes CD44, cartilage link protein, and aggrecan. The TSG-6 Link module was shown to interact with hyaluronan, and a putative binding surface was identified on the structure. A structural database search revealed close similarity between the Link module and the C-type lectin domain, with the predicted hyaluronan-binding site at an analogous position to the carbohydrate-binding pocket in E-selectin.

Amino Acid Sequence↗
PubMed1996

Prediction from sequence comparisons of residues of factor H involved in the interaction with complement component C3b.

The amino acid sequence of the region of bovine factor H containing the C3b binding site has been derived from sequencing overlapping cDNA clones. A cDNA sequence encoding 669 amino acids was obtained. Like human and mouse factor H the sequence can be arranged into a number of internally homologous units (CPs), each of which is about 60 amino acids long and is based on a framework of four conserved cysteine residues. Bovine factor H is of the same molecular mass as human and mouse factor H, and is therefore likely to be composed of 20 contiguous CPs. Comparisons with human and mouse factor H indicate that the partial bovine sequence encodes CPs 2-12 inclusive of bovine factor H. Bovine factor H binds to human ammonia-treated C3 (causing thiolester cleavage) [C3(NH3)] and promotes the cleavage of human C3(NH3) in the presence of bovine factor I. Other studies indicate that CPs 2-5 of human factor H encompass the C3b binding and factor I cofactor activity site. Multiple sequence alignments of human factor H, mouse factor H (which also interacts with human C3b) and bovine factor H with CP modules whose structures have been determined experimentally, have been used to predict residues in the hypervariable loops of CPs 2-5 and to identify residues of potential importance in human C3 binding and factor I cofactor activity. Leu-17 and Gly-20 of CP 2, Ser-17, Ala-19, Glu-21, Asp-23 and Glu-25 of CP 3 and Lys-18 of CP 4 are all conserved between the three species. It may be that CPs 3 and 4 interact with C3(NH3) directly, whilst CPs 2 and 5 maintain the correct orientation for CPs 3 and 4 to interact.

Amino Acid Sequence↗