Behaviour change, weight loss and remission of Type 2 diabetes: a community-based prospective cohort study.

AIM: To quantify the association between behaviour change and weight loss after diagnosis of Type 2 diabetes, and the likelihood of remission of diabetes at 5-year follow-up. METHOD: We conducted a prospective cohort study in 867 people with newly diagnosed diabetes aged 40-69 years from the ADDITION-Cambridge trial. Participants were identified via stepwise screening between 2002 and 2006, and underwent assessment of weight change, physical activity (EPAQ2 questionnaire), diet (plasma vitamin C and self-report), and alcohol consumption (self-report) at baseline and 1 year after diagnosis. Remission was examined at 5 years after diabetes diagnosis via HbA1c level. We constructed log binomial regression models to quantify the association between change in behaviour and weight over both the first year after diagnosis and the subsequent 1-5 years, as well as remission at 5-year follow-up. RESULTS: Diabetes remission was achieved in 257 participants (30%) at 5-year follow-up. Compared with people who maintained the same weight, those who achieved ≥ 10% weight loss in the first year after diagnosis had a significantly higher likelihood of remission [risk ratio 1.77 (95% CI 1.32 to 2.38; p<0.01)]. In the subsequent 1-5 years, achieving ≥10% weight loss was also associated with remission [risk ratio 2.43 (95% CI 1.78 to 3.31); p<0.01]. CONCLUSION: In a population-based sample of adults with screen-detected Type 2 diabetes, weight loss of ≥10% early in the disease trajectory was associated with a doubling of the likelihood of remission at 5 years. This was achieved without intensive lifestyle interventions or extreme calorie restrictions. Greater attention should be paid to enabling people to achieve weight loss following diagnosis of Type 2 diabetes.

Cutaneous targets for topical pain medications in patients with neuropathic pain: individual differential expression of biomarkers supports the need for personalized medicine.

Introduction: Numerous potential cutaneous targets exist for treating chronic pain with topically applied active pharmaceutical ingredients. This preliminary human skin tissue investigation was undertaken to characterize several key biomarkers in keratinocytes and provide proof-of-principle data to support clinical development of topical compounded formulations for peripheral neuropathic pain syndromes, such as postherpetic neuralgia (PHN). Objectives: The study intended to identify objective biomarkers in PHN skin on a patient-by-patient personalized medicine platform. The totality of biopsy biomarker data can provide a tissue basis for directing individualized compounded topical preparations to optimize treatment efficacy. Methods: Referencing 5 of the most common actives used in topical pain relief formulations (ketamine, gabapentin, clonidine, baclofen, and lidocaine), and 3 well-established cutaneous mediators (ie, neuropeptides, cannabinoids, and vanilloids), comprehensive immunolabeling was used to quantify receptor biomarkers in skin biopsy samples taken from ipsilateral (pain) and contralateral (nonpain) dermatomes of patients with PHN. Results: Epidermal keratinocyte labeling patterns were significantly different among the cohort for each biomarker, consistent with potential mechanisms of action among keratinocytes. Importantly, the total biomarker panel indicates that the enriched PHN cohort contains distinct subgroups. Conclusion: The heterogeneity of the cohort differences may explain studies that have not shown statistical group benefit from topically administered compounded therapies. Rather, the essential need for individual tissue biomarker evaluations is evident, particularly as a means to direct a more accurately targeted topical personalized medicine approach and generate positive clinical results.

Two year follow-up study of stressors and occupational stress in submariners.

BACKGROUND: Submariners are an occupational group within the Royal Navy (RN) who work in isolated and extreme conditions. This preliminary study forms part of a longitudinal study of occupational stress in the RN. AIMS: To compare stress prevalence in submariners with matched controls and to identify predictors of occupational stress in submariners over a 2 year follow-up period. METHODS: Participants completed a Work and Well-Being Questionnaire to measure occupational stressors and the General Health Questionnaire-12 (GHQ-12) to measure stress at time point 1, and a follow-up GHQ-12 2 years later. Demographically matched controls from the surface fleet of the RN were identified for each submariner. Regression models were developed for submariners and their controls to predict future stress at time point 2 using psychosocial predictors from time point 1. RESULTS: Participants comprised 144 submariners and 144 general service controls. There were no differences between submariners and their surface fleet counterparts in the prevalence of occupational stress. Nevertheless, different predictors for the development of stress were found between the two groups. For submariners, over-commitment and rank were the main predictors; whereas for controls, the predictors were length of service, body mass index and physical work. CONCLUSIONS: Submariners were not more likely to suffer from occupational stress than surface fleet controls in the RN. However, the psychosocial predictors of stress were significantly different for this RN specialist group, demonstrating the importance of developing individual models of stress for different occupational groups.

Safety and efficacy of compounded bioidentical hormone therapy (cBHT) in perimenopausal and postmenopausal women: a systematic review and meta-analysis of randomized controlled trials.

IMPORTANCE: More information is needed about the efficacy and safety of compounded bioidentical hormone therapy (cBHT) in the published literature. A thorough synthesis of existing data is not currently available. OBJECTIVE: To provide a systematic review and meta-analysis of the existing evidence related to the safety and efficacy of commonly prescribed cBHT preparations in perimenopausal and postmenopausal women. EVIDENCE REVIEW: PubMed, ClinicalTrials.gov, and The Cochrane Central Register of Controlled Trials were searched. Randomized controlled trials (RCTs) comparing cBHT with a placebo or FDA-approved products in perimenopausal or postmenopausal women were eligible. The risk of bias was assessed by the Cochrane risk of bias tool. The primary safety outcome was changes in lipid profile and glucose metabolism, and the primary efficacy outcome was the change of vaginal atrophy symptoms. The secondary outcomes included the change of endometrial thickness, risk of adverse events, vasomotor symptoms, change of serum hormone levels, and change of bone mineral density. FINDINGS: A total of 29 RCTs reported in 40 articles containing 1,808 perimenopausal and postmenopausal women were included. Two risk factors of cardiovascular disease, lipid profile, and glucose metabolism, were evaluated with cBHT. The results showed that compounded androgen was not associated with change of lipid profile or glucose metabolism. There was no change in endometrial thickness or serious adverse events. There were more androgenic side effects with compounded dehydroepiandrosterone compared with placebo as expected. Other safety measures including clinical cardiovascular events, endometrial biopsy, and risk of breast cancer were not studied. cBHT in the form of compounded vaginal androgen was found to significantly improve vaginal atrophy symptoms (SMD -0.66 [95% CI, -1.28 to -0.04]; I2 = 86.70%). This finding was supported by the association between compounded vaginal androgen and improved female sexual function scores. The changes of serum hormone levels were also evaluated. Despite the variations in absorption from different types of compounded hormones, routes, and strengths, the trends were consistent with published data from FDA-approved products. CONCLUSIONS AND RELEVANCE: This review found that cBHT used in primarily short-term RCTs is not associated with adverse changes in lipid profile or glucose metabolism. cBHT in the form of vaginal androgens appears beneficial for vaginal atrophy symptoms. There are insufficient RCTs of cBHT to assess clinical risk of breast cancer, endometrial cancer, or cardiovascular disease. Long-term studies with clinical endpoints are needed.

Characterization of hyaluronan and TSG-6 in skin scarring: differential distribution in keloid scars, normal scars and unscarred skin.

BACKGROUND: Hyaluronan (HA) is a major component of the extracellular matrix (ECM) with increased synthesis during tissue repair. Tumour necrosis factor-stimulated gene-6 (TSG-6) is known to catalyze the covalent transfer of heavy chains (HC1 and HC2) from inter-α-inhibitor (IαI) onto HA, and resultant HC•HA complexes have been implicated in physiological and pathological processes related to remodelling and inflammation. OBJECTIVE: The aims of this study were to determine the expression of HA, TSG-6 and the IαI polypeptides in unscarred skin, normal scars and keloid scars. METHODS: Formalin-fixed paraffin-embedded sections of unscarred skin, normal scars and keloid scars were prepared from patient samples collected during scar revision surgery. Haematoxylin and eosin, as well as immunofluorescent staining for HA, TSG-6 and the three polypeptide chains of IαI (i.e. HC1, HC2 and bikunin) were performed. RESULTS: All skin types stained positive for TSG-6, HC1, HC2 and bikunin, associated with keratinocytes, fibroblasts and skin appendages all in close proximity to HA. Keloid lesions showed altered HA organization patterns compared with unscarred skin and normal scars. TSG-6 staining was significantly more intense in the epidermis compared with the dermis of all sample types. There was a significant reduction in TSG-6 levels within keloid lesions compared with the dermis of unscarred skin (P=0.017). CONCLUSION: TSG-6 is expressed in unscarred skin, where its close association with HA and IαI could give rise to TSG-6-mediated HC•HA formation within this tissue. A reduction in the beneficial effects of TSG-6, caused by diminished protein levels in keloid lesions, could contribute to this abnormal scarring process.

Evaluation of Containment Ventilated Enclosure Performance in Absence of Negative Pressure Containment Secondary Engineering Control.

The purpose of this study was to evaluate the efficacy of a containment ventilated enclosure on preventing powder exposure to surrounding areas during a high-particle generating compounding procedure in absence of a negative pressure compounding secondary engineering control. Air samples were collected under actual compounding conditions to assess compounder and room exposure to estradiol powder. Samples were collected from several locations. The entire compounding process took place in a containment ventilated enclosure after assessing performance characteristics via velocity and smoke sampling. The particle generating compounding process consisted of weighing, multiple transfers, trituration, and capsule filling. Compounding procedures were performed by three different compounders. The laboratory detection limit for estradiol was determined, and collection parameters were targeted to ensure adequate samples were obtained to prevent false negative results. Sampling time for each compounder ranged from 24 minutes to 38 minutes. All samples collected during compounding procedures showed less than detectable amounts of estradiol. Observation of the compounding procedure showed notable potential particle generation during the compounding procedure, including spills and vigorous jostling of compounding equipment prior to encapsulation. Despite a significant potential for particulate generation inside the containment ventilated enclosure, no estradiol was detected outside of the enclosure. These findings suggest that powder chemical exposure to the compounder is minimal when using a properly certified containment ventilated enclosure. The findings also show a lack of exposure of the room to aerosolized estradiol even when using a neutral containment secondary engineering control.

Pediatric Compounding Pharmacy: Taking on the Responsibility of Providing Quality Customized Prescriptions.

Compounding pharmacy has an important role to play in the field of pediatric medicine. These specialized pharmacies can offer solutions to the unique patient needs that arise in the pediatric population. Medication can be tailored to the child to allow better compliance in cases when the commercial product is unable to meet the needs of the patient. For example, a suspension, suppository, or lozenge formulation is sometimes needed when the manufactured products are only offered as solid oral dosage forms. Sensory processing disorder (SPD), patients with food allergies, and specific dietary needs can also be a big challenge for caregivers and practitioners who need alternatives to the commercially available forms. Three example cases are presented to help describe the process of collaboration between the pharmacist, patient, and doctor to solve the patient's needs.

Superficial zone chondrocytes in normal and osteoarthritic human articular cartilages synthesize novel truncated forms of inter-alpha-trypsin inhibitor heavy chains which are attached to a chondroitin sulfate proteoglycan other than bikunin.

OBJECTIVE: We have examined the occurrence of the inflammation-associated inter-alpha-trypsin inhibitor (IalphaI) components, bikunin, heavy chain (HC)1 and HC2 in normal cartilage and osteoarthritis (OA) cartilage and synovial fluids. DESIGN/METHODS: Cartilage extracts from normal donors and late-stage OA patients, and synovial fluids from OA patients were studied by Western blot with multiple antibodies to bikunin, HC1 and HC2. Cell and matrix localization was determined by immunohistochemistry and mRNA by RT-PCR. RESULTS: Bikunin.chondroitin sulfate (CS) and IalphaI were abundant in OA cartilages, but virtually undetectable in normal. In both OA and normal cartilages, HCs were largely present in a novel C-terminally truncated 50-kDa form, with most, if not all of these being attached to CS on a proteoglycan other than bikunin. Synovial fluids from OA patients contained bikunin.CS and full-length (approximately 90 kDa) HCs linked to hyaluronan (HA) as HC.HA (SHAP.HA). Immunohistochemistry showed intracellular and cell-associated staining for bikunin and HCs, consistent with their synthesis by superficial zone chondrocytes. PCR on multiple human normal and OA cartilage samples detected transcripts for HC1 and HC2 but not for bikunin. In OA cartilages, immunostaining was predominantly matrix-associated, being most intense in regions with a pannus-like fibrotic overgrowth. CONCLUSION: The truncated structure of HCs, their attachment to a proteoglycan other than bikunin, PCR data and intracellular staining are all consistent with synthesis of HC1 and HC2 by human articular chondrocytes. The presence of bikunin.CS and IalphaI in OA cartilage, but not in normal, appears to be due to diffusional uptake and retention through fibrillated (but not deeply fissured) cartilage surfaces.

The role of transdermal compounding in opioid safety.

Since the number of prescriptions for opioid medications have continued to rise, there have been questions about the safety of using opioids in pain management. Traditionally, opioid analgesics were reserved for a few select conditions, such as terminal illness and surgery, but currently opioids have been readily prescribed for multiple conditions. The objective of this manuscript is to clarify the current state of opioid use and to discuss alternative transdermal analgesic therapies in pain management. Transdermal compounded medications are patient-specific and customizable to include different types of drugs, in various dosage strengths, that are to be delivered simultaneously in one application. Due to the different origins and types of pain, treatments may be most beneficial with multiple classes of drugs with various mechanisms of action. In addition, combination drug therapy may include nontraditional pain management options, and has the ability to maximize therapeutic effects of medications through additive or synergistic properties, without increasing the dosage strengths of the drugs. Many of the challenges faced when using oral opioid therapy may be overcome by using transdermal drug delivery since this route of administration reduces adverse effects, increases patient compliance, and limits exposure to potentially abusive drugs. Although prescribing practices surrounding opioids remains to be a controversial topic, the use of compounded pain medications may help healthcare providers effectively treat their patients while avoiding the use of addictive drugs.

Hyaluronan: polysaccharide chaos to protein organisation.

Molecular dynamics simulations of hyaluronan have revealed the inherent flexibility of this glycosaminoglycan in solution. Crystal structures of hyaluronan-digesting enzymes have provided the first direct insights into the molecular basis of hyaluronan-protein interactions. Various studies on hyaluronan-binding proteins suggest there is considerable diversity in their mode of interaction with hyaluronan, which might result in many different bound conformations of the polysaccharide.

Bone mineral density, polyphenols and caffeine: a reassessment.

Several studies have shown beneficial associations between tea consumption and bone mineral density (BMD) and fracture risk. Current investigations into potential mechanisms of benefit are focused upon the F and polyphenol components of tea. However, previous studies have pointed towards caffeine consumption as a potential risk factor for low BMD and high fracture risk. Tea, therefore, represents an interesting paradox as a mildly caffeinated beverage that may enhance bone health. Fruit and vegetable intake has also been associated with BMD, and it is now apparent that several fruit and vegetable components, including polyphenols, may contribute positively to bone health. Evidence surrounding the function(s) of polyphenol-rich foods in bone health is examined, along with more recent studies challenging the relevance of caffeine consumption to in vivo Ca balance. Plant foods rich in polyphenols such as tea, fruit and vegetables, as significant factors in a healthy diet and lifestyle, may have positive roles in bone health, and the negative role of caffeine may have been overestimated. The present review covers evidence of dietary mediation in positive and negative aspects of bone health, in particular the roles of tea, fruit and vegetables, and of caffeine, flavonoids and polyphenols as components of these foods. Since the deleterious effects of caffeine appear to have been overstated, especially in respect of the positive effects of flavonoids, it is concluded that a reassessment of the role of caffeinated beverages may be necessary.

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.

Localization and characterization of the hyaluronan-binding site on the link module from human TSG-6.

BACKGROUND: The interactions of hyaluronan (HA) with proteins are important in extracellular matrix integrity and leukocyte migration and are usually mediated by a domain termed a Link module. Although the tertiary structure of a Link module has been determined, the molecular basis of HA-protein interactions remains poorly understood. RESULTS: Isothermal titration calorimetry was used to characterize the interaction of the Link module from human TSG-6 (Link_TSG6) with HA oligosaccharides of defined length (HA(4)-HA(16)). All oligomers bound (except HA(4)) with K(d) values ranging from 0.2-0.5 microM at 25 degrees C. The reaction is exothermic with a favourable entropy and the thermodynamic profile is similar to those of other glycosaminoglycan-protein interactions. The HA(8) recognition site on Link_TSG6 was localized by comparing nuclear magnetic resonance (NMR) spectra from a 1:1 complex with free protein. Residues perturbed on HA binding include both amino acids that are likely to be directly involved in the interaction (i.e., Lys11, Tyr59, Asn67, Phe70, Lys72 and Tyr78) and those affected by a ligand-induced conformational change in the beta4/beta5 loop. The sidechain of Asn67 becomes more rigid in the complex suggesting that it is in close proximity to the binding site. CONCLUSIONS: In TSG-6 a single Link module is sufficient for a high-affinity interaction with HA. The HA-binding surface on Link_TSG6 is found in a similar position to that suggested previously for CD44, indicating that its location might be conserved across the Link module superfamily. Here we find no evidence for the involvement of linear sequence motifs in HA binding.

Amylin and the amylin gene: structure, function and relationship to islet amyloid and to diabetes mellitus.

Amylin, the major peptide component of the islet amyloid commonly found in the pancreases of patients with type 2 (non-insulin-dependent) diabetes mellitus (NIDDM), is a recently discovered islet polypeptide. This peptide has many structural and functional features suggesting that it is a novel hormone, which may control carbohydrate metabolism in partnership with insulin and other glucoregulatory factors. Amylin is synthesised in, and probably secreted from, the beta-cells of the islets of Langerhans, where it has recently been immunolocalised to secretory granules. DNA cloning studies indicate that in the human and the rat, amylin is generated from a precursor, preproamylin, which displays a typical signal peptide followed by a small prohormone-like sequence containing the amylin sequence. The presence of the signal peptide suggests that amylin is secreted and plays a physiological role. Amylin is probably generated by proteolytic processing similar to that for proinsulin and other islet prohormones. The human amylin gene encodes the complete polypeptide precursor in two exons which are separated by an intron of approx. 5 kb, and is located on chromosome 12. Amylin is a potent modulator of glycogen synthesis and glucose uptake in skeletal muscle, and is capable of inducing an insulin-resistant state in this tissue in vitro, and perhaps also in the liver in vivo. In normal metabolism, amylin could act in concert with insulin as a signal for the body to switch the site of carbohydrate disposal from glycogen to longer-term stores in adipose tissue, by making skeletal muscle relatively insulin-resistant, whilst at the same time leaving rates of insulin-stimulated carbohydrate metabolism in adipose tissue unaltered. Several lines of evidence now implicate elevated amylin levels in the pathogenic mechanisms underlying NIDDM, and suggest to us that the obesity which frequently accompanies this syndrome is a result of, rather than a risk factor for, NIDDM. Following the beta-cell destruction which occurs in type 1 (insulin-dependent) diabetes mellitus (IDDM), it is probable that amylin secretion disappears in addition to that of insulin. As patients with insulin-treated IDDM frequently experience problems with hypoglycaemia, and as amylin acts to modulate the action of insulin in various tissues, it is possible that amylin deficiency may contribute to morbidity in insulin-treated IDDM, perhaps through the loss of a natural damping mechanism which guards against hypoglycaemia under conditions of normal physiology.

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.

Three-dimensional structure of a complement control protein module in solution.

The complement control protein (CCP) modules (also known as short consensus repeats) are defined by a consensus sequence within a stretch of about 60 amino acid residues. These modules have been identified more than 140 times in over 20 proteins, including 12 proteins of the complement system. The solution structure of the 16th CCP module from human complement factor H has been determined by a combination of 2-dimensional nuclear magnetic resonance spectroscopy and restrained simulated annealing. In all, 548 structurally important nuclear Overhauser enhancement cross-peaks were quantified as distance restraints and, together with 41 experimentally measured angle restraints, were incorporated into a simulated annealing protocol to determine a family of closely related structures that satisfied the experimental observations. The CCP structure is shown to be based on a beta-sandwich arrangement; one face made up of three beta-strands hydrogen-bonded to form a triple-stranded region at its centre and the other face formed from two separate beta-strands. Both faces of the molecule contribute highly conserved hydrophobic side-chains to a compact core. The regions between the beta-strands are composed of both well-defined turns and less well-defined loops. Analysis of CCP sequence alignments, in light of the determined structure, reveals a high degree of conservation amongst residues of obvious structural importance, while almost all insertions, deletions or replacements observed in the known sequences are found in the less well-defined loop regions. On the basis of these observations it is postulated that models of other CCP modules that are based on the structure presented here will be accurate. Certain families of CCP modules differ from the consensus in that they contain extra cysteine residues. As a test of structural consensus, the extra disulphide bridges are shown to be easily accommodated within the determined CCP model.

Characterization of Xenopus laevis complement factor I structure--conservation of modular structure except for an unusual insert not present in human factor I.

Factor I (C3b/C4b inactivator) is a regulatory protein of the classical and alternative complement pathways. In this paper, we report the sequence of Xenopus factor I cDNA and the deduced protein structure. The basic structure of human preprofactor I, NH2-heavy chain-cleavage peptide-light chain-COOH, is conserved in the frog. However, the frog heavy chain contains a highly charged segment of 29 amino acids, encoded by a poly dA-rich mRNA insert, which is not found in human factor I. The modular structure of the frog heavy chain was analyzed, and found to differ vis-à-vis previously published analyses of human factor I. We also evaluate the timing of factor I transcription during frog embryogenesis.

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.

Conjugation position of quercetin glucuronides and effect on biological activity.

Quercetin glycosides are common dietary antioxidants. In general, however, potential biological effects of the circulating plasma metabolites (e.g., glucuronide conjugates) have not been measured. We have determined the rate of glucuronidation of quercetin at each position on the polyphenol ring by human liver cell-free extracts containing UDP-glucuronosyltransferases. The apparent affinity of UDP-glucuronosyltransferase followed the order 4'- > 3'- > 7- > 3, although the apparent maximum rate of formation was for the 7-position. The 5-position did not appear to be a site for conjugation. After isolation of individual glucuronides, the inhibition of xanthine oxidase and lipoxygenase were assessed. The K(i) for the inhibition of xanthine oxidase by quercetin glucuronides followed the order 4'- > 3'- > 7- > 3-, with quercetin-4'-glucuronide a particularly potent inhibitor (K(i) = 0. 25 microM). The glucuronides, with the exception of quercetin-3-glucuronide, were also inhibitors of lipoxygenase. Quercetin glucuronides are metabolites of quercetin in humans, and these compounds can retain some biological activity depending on conjugation position at expected plasma concentrations.