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Background and Objective: Compliance with weight reducing programs can be improved by intensive care and control. We tested a telemetrically-guided weight reduction program in overweight and obese persons., Patients and Methods: 200 outpatients (62 males) with a mean body mass index of 34 kg/m (2) and a mean age of 47 years participated in a prospective study for one year. During the first six months, telemetrical support (weight-transmission via Bluetooth (short range)-technology, 20-minutes telephone consultation with a nutritionist) was given weekly. After six months, participants were randomly assigned either to a group with further telemonitoring support (telemetric group) or to a group without contact to our clinic (control group). At baseline, and after six and twelve months, body weight, body composition (bioelectrical impedance analysis), and parameters of the metabolic syndrome were assessed at our clinic., Results: 16 participants terminated the study prematurely during the first 6 months and 19 participants (10 from the telemetric group and 9 from the control group) during the second 6 months. According to the intention-to-treat principle, mean weight loss was 6.7 kg (p < 0,001), mean loss of body fat was 5.1 kg (p < 0,001), and mean loss of fat-free mass was 1.6 kg (p < 0,001) within the first six months. Moreover, metabolic and cardiovascular risk markers such as waist circumference, blood pressure, serum triglycerides and blood glucose declined significantly (p < 0,001). Prevalence of the metabolic syndrome fell from 49.5% to 42.0 % (p < 0,05). During the second six months body fat content, waist circumference, and blood glucose increased again in the control group but not in the telemetric group (p < 0,05-0,001)., Conclusion: The telemetrically-guided weight loss program was a more efficacious measure than the less intensive support without telemonitoring.

Background: The formation of abdominal aortic aneurysm (AAA) is caused by a destructive remodeling of the extracellular matrix in the vascular wall. Proteoglycan content and biosynthesis have been shown to be altered in AAA. Xylosyltransferase I (XT-I) is the initial and rate-limiting enzyme in the biosynthesis of the proteoglycan-linked glycosaminoglycan chains. A familial predisposition to AAA is well recognized. Thus, variations in the XT-I coding gene XYLT1 might be risk factors for AAA formation., Methods: We performed genotyping of two genetic variations in the XYLT1 gene which, have been already linked to proteoglycan-associated diseases, in 129 AAA patients and 129 age- and sex-matched healthy controls., Results: The T-allele of the polymorphism c.343G>T (p.A115S) was found to be significantly more frequent in AAA patients compared to the healthy control group, demonstrating that carriers of the T-allele have a 5-fold increased risk of developing AAA (odds ratio 4.87, 95%-CI 1.38-17.19; p=0.011)., Conclusions: Our results show that XT-I polymorphisms potentially confer to the genetic susceptibility of AAA.

Objectives: Pseudoxanthoma elasticum (PXE) is a hereditary disorder predominantly affecting the skin, retina and vascular system. The aim of this study was to measure cell adhesion molecules in PXE patients., Design and Methods: Soluble P-, E- and L-selectins were measured in 61 non-consanguineous PXE patients. The distribution of the variants E-selectin S128R and P-selectin T715R were determined., Results: P-selectin concentrations were significantly increased in male and female PXE patients. Furthermore, P-selectin levels correlated with the ABCC6 gene status of the PXE patients. Patients harboring two mutant ABCC6 alleles had 1.5-fold increased P-selectin concentrations in comparison to patients with at least one wild-type allele. E- and L-selectin levels were within normal range and the allelic frequencies of the investigated polymorphisms did not differ between patients and age- and sex-matched controls., Conclusions: Our data show elevated P-selectin levels in PXE patients potentially due to oxidative stress and elevated protease activity in PXE.

Objectives: Recent studies have revealed the involvement of calcification inhibitory proteins in the pathogenesis of pseudoxanthoma elasticum (PXE)., Design and Methods: We analyzed serum concentrations of the calcification inhibitor matrix Gla protein (MGP) in a large cohort of patients suffering from PXE (n=101), 34 first-degree relatives and 67 healthy controls. Moreover, we determined the distribution of the two MGP promoter polymorphisms c.-7G>A and c.-138T>C in the three cohorts., Results: We found significantly lower total MGP concentrations in the sera of PXE patients compared to healthy controls (p=0.0002). Furthermore, higher serum MGP concentrations could be correlated with a later PXE onset. Analysis of MGP promoter polymorphism frequencies revealed one MGP haplotype to be a potential protective co-factor in PXE., Conclusions: Our findings point to a role of the local calcification inhibitor MGP in PXE manifestation.

Advanced glycation endproducts (AGEs) and oxidative stress (OS) contribute to the development and progression of diabetic complications. We have reported that dietary AGEs and OS induce acute endothelial dysfunction in vivo, but little is known about their effects on adipokines. Twenty inpatients with type 2 diabetes mellitus (mean age: 55.9; range: 32-71 years), received a standard diabetes diet for 6 days. On days 4 and 6, the acute effects of a high-AGE (HAGE) or a low-AGE (LAGE) meal (15.100 vs. 2.750 kU AGE) were studied in a randomized, cross-over, investigator-blinded design. Measurements were performed after an overnight fast, at baseline (B) and at 2, 4, and 6 h after the HAGE or LAGE meals. Both meals had the same ingredients and differed only by the cooking method. Two h following HAGE, a significant decrease from baseline occurred in adiponectin (-10%*double dagger vs. +0%) and leptin (-22%*double dagger vs. -13%*), and a significant increase occurred in vascular cell adhesion molecule 1 (+19%*double dagger vs. -5%) and thiobarbituric acid reactive substances (+23%*double dagger vs. +6%). These changes did not occur, or occurred to a lesser extent, following LAGE. At 4 h following HAGE, an increase in methylglyoxal (+20%double dagger vs. -5%) and E-selectin (+54%*double dagger vs. -3%) occurred. Urinary AGEs increased only after HAGE (+51%*double dagger vs. -2%; values presented as HAGE vs. LAGE; *P < 0.05 vs. baseline, double daggerP < 0.05 vs. LAGE). The postprandial excursions in glucose, insulin, and triglycerides were similar between both meals. A meal rich in AGEs induces acute endothelial and adipocyte dysfunction. These effects were prevented by changing the cooking method.

Background: Vitamin D receptor knockout mice develop typical signs of congestive heart failure (CHF). In approximately 20% of stable CHF patients, frankly low concentrations of the vitamin D hormone calcitriol are found., Aims: We investigated whether serum calcitriol concentrations predict clinical outcome in end-stage CHF., Methods and Results: We collected blood samples in 383 end-stage CHF patients who were on a waiting list for cardiac transplantation. We assessed associations of calcitriol with disease severity and freedom from event (death or cardiac transplantation) during 1-year follow-up. In electively listed patients (n=325), 31% had deficient calcitriol levels (<43 pmol/l) compared to 47% in urgently/high urgently listed patients (n=58; P<0.001). As determined by multivariable logistic regression, calcitriol was an independent predictor of the listing status 'urgent/high urgent' (P<0.001). Calcitriol concentrations were also significantly lower in patients with an event (n=233) compared to those who survived on the waiting list (P<0.001). Cox regression analysis revealed that patients in the highest calcitriol tertile had a hazard ratio (95% CI) for an event of 0.506 (0.334-0.767) compared with patients in the lowest calcitriol tertile (P=0.005), after adjustment for potential confounders., Conclusion: Data indicate that low serum calcitriol concentrations are independently associated with poor clinical outcome in end-stage CHF.

Mesenchymal stem cells (MSCs) are multipotent adult stem cells capable to differentiate into osteoblasts. Therefore, they represent attractive cell sources for tissue engineering applications, especially for bone replacement. Proteoglycans (PGs) exhibit a crucial role for matrix assembly and remodeling. Nevertheless, since bone development is a highly dynamic and complex process, the regulation of the extracellular matrix (ECM) formation remains elusive. Consequently, the aim of this study was to investigate the mRNA expression levels of genes involved in PG assembly in different stages of osteogenesis. For the rate-limiting enzyme in glycosaminoglycan (GAG) biosynthesis xylosyltransferase I (XT-I), maximal mRNA expression levels (3.89 +/- 0.83-fold increase) and elevated enzyme activities (285 +/- 17 dpm/mug DNA) were observed 10 days after osteogenic induction, simultaneously to the beginning mineralization of the ECM, whereas the highly homologous protein XT-II showed no specific alterations. The differential expression of chondroitin sulfate, dermatan sulfate and heparan sulfate chains was determined by analyzing the mRNA expression of EXTL2 (alpha-1,4-N-acetylhexosaminyltransferase), GalNAcT (beta-1,4-N-acetylgalactosaminyltransferase), and GlcAC5E (glucuronyl C5-epimerase) as they represent crucial enzymes in GAG biosynthesis. Besides GlcAC5E, all key enzymes showed upregulated mRNA contents (up to 3.6-fold) around day 10. Except for decorin, which exhibited heightened mRNA levels even in the early stages of osteogenesis, we found similar upregulated mRNA contents (up to 14.6-fold) for all investigated PG core proteins. The synchronized expression profiles demonstrate the coordinated biosynthesis of the PGs during bone formation and osteogenic stem cell differentiation occurring in parallel to the mineralization of the extracellular matrix.

Human xylosyltransferase II (EC 2.4.2.26, XT-II) represents an isoform of xylosyltransferase I (XT-I). Recently, we and others provided first evidence that XT-II is capable of initiating the biosynthesis of glycosaminoglycan chains in proteoglycans. Here, a soluble form of human XT-II was expressed in the yeast Pichia pastoris and the substrate specificity for various acceptors was investigated, pointing to a modified bikunin peptide to be the optimal XT-II acceptor (K(M)=1.9 microM). Furthermore, biochemical characterization of XT-II showed that this enzyme was strongly inhibited by nucleotides and glycosaminoglycans. Its temperature optimum, stability, and ion dependency were further examined, demonstrating necessity for Mg(2+) or Mn(2+) ions for its enzymatic activity. Our data show for the first time that XT-I and XT-II are xylosyltransferases with similar but not identical properties, pointing to their potential role in modulating the cellular proteoglycan pool.

Background: Pseudoxanthoma elasticum (PXE) is an inherited disorder leading to characteristic calcified elastic fibers in skin, eyes and vasculature. PXE-like fibers have not been described in inflammatory skin disease in the absence of other signs of PXE., Methods: The histopathology of inflamed skin from 13 patients that contained PXE-like fibers but lacked clinical evidence of PXE were studied. Six of these and six comparison specimens from known patients with PXE were subjected to polymerase chain reaction amplification and sequencing of exons 24 and 28 of the PXE-associated gene ABCC6. This genetic analysis employed a novel assay utilizing paraffin-embedded tissue., Results: Incidental PXE-like fibers were found in patients without clinical suspicion of PXE in lesional tissue showing lipodermatosclerosis, granuloma annulare, lichen sclerosus, morphea profunda, erythema nodosum, septal panniculitis, basal cell carcinoma and fibrosing dermatitis. Two patients with PXE-like fibers but without clinical findings of PXE were heterozygous for a PXE-associated ABCC6 sequence alteration., Conclusions: This pilot study shows elastic fibers similar to those of PXE in the lesional skin of patients with a variety of inflammatory skin diseases in the absence of clinical evidence of PXE; and some of these patients harbor changes in ABCC6.

Background: Pseudoxanthoma elasticum (PXE) is a rare hereditary disorder characterized by progressive calcification and fragmentation of elastic fibers in connective tissues. PXE is caused by mutations in the ABCC6 gene, which encodes the membrane transporter multidrug resistance-associated protein 6. Chronic oxidative stress was recently suggested to play a crucial role in the pathogenesis of the disease. Our aim was to investigate the association of PXE with genetic variation in genes coding for antioxidant enzymes., Methods: We used restriction fragment length polymorphism and allele-specific PCR analyses to evaluate the distribution of single-nucleotide polymorphisms in the genes encoding catalase (CAT), superoxide dismutase 2 (SOD2), and glutathione peroxidase 1 (GPX1) in DNA samples from 117 German PXE patients and 117 healthy age- and sex-matched control individuals., Results: The investigated genetic variants had previously been shown to affect the activities of these antioxidant enzymes. We found a correlation between genotype and age of disease onset for polymorphisms in CAT (c.-262C>T), SOD2 (c.47C>T), and GPX1 (c.593C>T). Furthermore, the age of disease onset was inversely correlated with the number of mutated alleles, indicating a cumulative effect on the time of disease onset [mean (SD) age of 40.9 (13.6) years, 32.4 (16.3) years, and 25.7 (15.9) years for carriers of 0, 1-2, and >2 mutated alleles, respectively; P = 0.03]., Conclusion: Our findings demonstrate that increased oxidative stress due to activity-affecting polymorphisms in genes encoding antioxidant enzymes leads to earlier PXE onset.

In cardiac fibrosis remodeling of the failing myocardium is associated with a complex reorganization of the extracellular matrix (ECM). Xylosyltransferase I and Xylosyltransferase II (XT-I and XT-II) are the key enzymes in proteoglycan biosynthesis, which are an important fraction of the ECM. XT-I was shown to be a measure for the proteoglycan biosynthesis rate and a biochemical fibrosis marker. Here, we investigated the XT-I and XT-II expression in cardiac fibroblasts and in patients with dilated cardiomyopathy and compared our findings with nonfailing donor hearts. We analyzed XT-I and XT-II expression and the glycosaminoglycan (GAG) content in human cardiac fibroblasts incubated with transforming growth factor (TGF)-beta(1) or exposed to cyclic mechanical stretch. In vitro and in vivo no significant changes in the XT-II expression were detected. For XT-I we found an increased expression in parallel with an elevated chondroitin sulfate-GAG content after incubation with TGF-beta(1) and after mechanical stretch. XT-I expression and subsequently increased levels of GAGs could be reduced with neutralizing anti-TGF-beta(1) antibodies or by specific inhibition of the activin receptor-like kinase 5 or the p38 mitogen-activated protein kinase pathway. Usage of XT-I small interfering RNA could specifically block the increased XT-I expression under mechanical stress and resulted in a significantly reduced chondroitin sulfate-GAG content. In the left and right ventricular samples of dilated cardiomyopathy patients, our data show increased amounts of XT-I mRNA compared with nonfailing controls. Patients had raised levels of XT-I enzyme activity and an elevated proteoglycan content. Myocardial remodeling is characterized by increased XT-I expression and enhanced proteoglycan deposition. TGF-beta(1) and mechanical stress induce XT-I expression in cardiac fibroblasts and have impact for ECM remodeling in the dilated heart. Specific blocking of XT-I expression confirmed that XT-I catalyzes a rate-limiting step during fibrotic GAG biosynthesis.

Mesenchymal stem cells are multipotent progenitor cells that can differentiate into the chondrogenic lineage. To date, only limited knowledge about the formation and remodeling of the cartilaginous extracellular matrix is available. We recently analyzed the coordinated expression of proteins involved in the biosynthesis of proteoglycans and collagens, the two major components of cartilage matrix, to understand matrix formation and to provide potential tools to improve the quality of tissue-engineered cartilage.

The xylosyltransferases I and II (XT-I, XT-II, EC 2.4.2.26) catalyze the transfer of xylose from UDP-xylose to selected serine residues in the proteoglycan core protein, which is the initial and ratelimiting step in glycosaminoglycan biosynthesis. Both xylosyltransferases are Golgi-resident enzymes and transfer xylose to similar core proteins acceptors. XT-I and XT-II are differentially expressed in cell types and tissues, although the reason for the existence of two xylosyltransferase isoforms in all higher organisms remains elusive. Serum xylosyltransferase activity was found to be a biochemical marker for the assessment of disease activity in systemic sclerosis and for the diagnosis of fibrotic remodeling processes. Furthermore, sequence variations in the XT-I and XT-II coding genes were identified as risk factors for diabetic nephropathy, osteoarthritis or pseudoxanthoma elasticum. These findings point to the important role of the xylosyltransferases as disease modifiers in pathologies which are characterized by an altered proteoglycan metabolism. The present review discusses recent advances in mammalian xylosyltransferases and the impact of xylosyltransferases in proteoglycan-associated diseases.

Background: Progressive calcification and fragmentation of elastic fibers are characteristic hallmarks of pseudoxanthoma elasticum (PXE), which is caused by mutations in ABCC6 encoding multidrug resistance-associated protein 6 (MRP6). Because of the great clinical variability of PXE, secondary genetic risk factors are suspected to exist. We investigated whether SPP1 (secreted phosphoprotein 1; previously OPN, osteopontin) promoter polymorphisms are associated with PXE., Methods: We screened an approximately 2-kb region spanning the theoretical promoter of the SPP1 gene for sequence variations by denaturing HPLC and direct sequencing in 93 PXE patients. Sequence variations with a prevalence >5% were genotyped in 93 age- and sex-matched healthy controls. Statistical and haplotype association analyses were performed using Fisher exact test, PHASE v2.1.1, and Haploview 3.2., Results: Mutational screening revealed 9 different sequence variations. Three SPP1 promoter polymorphisms (c.-1748A>G, c.-155&#95;156insG, and c.244&#95;245insTG) were significantly more frequent in PXE patients than in 93 age- and sex-matched healthy controls (P(corrected) < 0.05 each). The odds ratios (95% CI) for PXE among carriers of the 3 alleles were, respectively, 2.16 (1.34-3.48), 2.41 (1.51-3.82), and 1.97 (1.23-3.15). Haplotype analysis of 6 SPP1 promoter polymorphisms revealed 1 haplotype to be significantly reduced among PXE patients (P(corrected) = 0.035, odds ratio 1.80, 95% CI 1.19-2.71)., Conclusions: Polymorphisms in the SPP1 promoter are secondary genetic risk factors contributing to PXE susceptibility.

Background: An advanced glycation endproducts (AGEs)-rich diet induces significant increases in inflammatory and endothelial dysfunction markers in type 2 diabetes mellitus (T2DM)., Objective: The aim was to investigate the acute effects of dietary AGEs on vascular function in T2DM patients., Design: Twenty inpatients with T2DM [x (+/-SEM) age: 55.4 +/- 2.2 y; glycated hemoglobin: 8.8 +/- 0.5%] were investigated. In a randomized crossover design, the effects of a low-AGE (LAGE) and high-AGE (HAGE) meal on macrovascular [by flow-mediated dilatation (FMD)] and microvascular (by Laser-Doppler flowmetry) function, serum markers of endothelial dysfunction (E-selectin, intracellular adhesion molecule 1, and vascular cell adhesion molecule 1), oxidative stress, and serum AGE were assessed. The meals had identical ingredients but different AGE amounts (15.100 compared with 2.750 kU AGE for the HAGE and LAGE meals, respectively), which were obtained by varying the cooking temperature and time. The measurements were performed at baseline and 2, 4, and 6 h after each meal., Results: After the HAGE meal, FMD decreased by 36.2%, from 5.77 +/- 0.65% (baseline) to 3.93 +/- 0.48 (2 h), 3.70 +/- 0.42 (4 h), and 4.42 +/- 0.54% (6 h) (P<0.01 for all compared with baseline). After the LAGE meal, FMD decreased by 20.9%, from 6.04 +/- 0.68% (baseline) to 4.75 +/- 0.48% (2 h), 4.69 +/- 0.51% (4 h), and 5.62 +/- 0.63% (6 h), respectively (P<0.01 for all compared with baseline; P<0.001 for all compared with the HAGE meal). This impairment of macrovascular function after the HAGE meal was paralleled by an impairment of microvascular function (-67.2%) and increased concentrations of serum AGE and markers of endothelial dysfunction and oxidative stress., Conclusions: In patients with T2DM, a HAGE meal induces a more pronounced acute impairment of vascular function than does an otherwise identical LAGE meal. Therefore, chemical modifications of food by means of cooking play a major role in influencing the extent of postprandial vascular dysfunction.

Human xylosyltransferase I (XT-I) initiates the biosynthesis of the glycosaminoglycan (GAG) linkage tetrasaccharide in proteoglycans. Xylosyltransferase II (XT-II) is a protein homologous to XT-I but with hitherto unknown activity or physiological function. Here, we report the enzymatic activity of XT-II and provide evidence that XT-II initiates the biosynthesis of both heparan sulfate and chondroitin sulfate GAGs. Transfection of the xylosyltransferase-deficient Chinese hamster ovary mutant pgsA-745 with XT-I or XT-II coding cDNA completely restored GAG biosynthesis. GAG disaccharide analysis revealed that XT-I- and XT-II-transfected pgsA-745 cells produced similar amounts of chondroitin sulfate and heparan sulfate. Furthermore, a high xylosyltransferase activity was measured after transfection with cDNAs encoding either isozyme. Analysis of the enzyme activity revealed that XT-II catalyzes the transfer of xylose to similar peptide acceptors as XT-I but with different efficiency. The optimal XT-II acceptor was observed using a bikunin-related peptide (K(m) 5.2 microM). Analysis of XT-I and XT-II mRNA expression in murine tissues showed a differential expression pattern for both enzymes. In particular, XT-II is highly expressed in liver tissue, where XT-I transcripts were not detected. This is the first report on the enzyme activity of XT-II and its involvement in chondroitin sulfate and heparan sulfate biosynthesis.

Objective: Tissue factor pathway inhibitor (TFPI) is a multivalent Kunitz-type serine proteinase inhibitor that plays a central role in the extrinsic pathway of blood coagulation and is mainly expressed by endothelial cells. In this study we examined the in vitro effects of heparin and other glycosaminoglycans on TFPI mRNA-expression in cultivated human endothelial (Ea.hy 926) and in chondrosarcoma (SW 1353) cells., Methods: We used a LightCycler-based method for relative quantification of the TFPI-mRNA expression before and after stimulation. The cells were stimulated with different concentrations of heparin (with and without addition of protamin), heparan sulfate (HS) and chondroitin-6-sulfate (CS). Cells were harvested after incubation times of 4, 8 and 24h, total RNA was isolated, and cDNA was synthesized and quantified relatively to a constantly expressed housekeeping gene., Results: Stimulation of Ea.hy 926 cells with unfractionated heparin (UFH) or low-molecular-weight heparin (LMWH) caused a time- and dose-dependent upregulation of TFPI-mRNA expression with LMWH showing the stronger effect. In contrast to this, HS led to a strongly and CS to a slightly decreased TFPI-mRNA expression. SW 1353 cells which were stimulated with LMWH/UFH and HS/CS did not show a significant up- or downregulative effect., Conclusion: Our results show that we have developed a versatile method for the relative quantification of TFPI-mRNA expression. As a conclusion, the determined heparin-induced upregulation of TFPI-mRNA expression can be considered a major component of the modulation of the anticoagulant properties of the endothelium.

Ex vivo expansion is being used to increase the number of stem and progenitor cells for autologous cell therapy. Initiation of pivotal clinical trials testing the efficacy of these cells for tissue repair has been hampered by the challenge of assuring safe and high-quality cell production. A strategy is described here for clinical-scale expansion of bone marrow (BM)-derived stem cells within a mixed cell population in a completely closed process from cell collection through postculture processing using sterile connectable devices. Human BM mononuclear cells (BMMNC) were isolated, cultured for 12 days, and washed postharvest using either standard open procedures in laminar flow hoods or using automated closed systems. Conditions for these studies were similar to long-term BM cultures in which hematopoietic and stromal components are cultured together. Expansion of marrow-derived stem and progenitor cells was then assessed. Cell yield, number of colony forming units (CFU), phenotype, stability, and multilineage differentiation capacity were compared from the single pass perfusion bioreactor and standard flask cultures. Purification of BMMNC using a closed Ficoll gradient process led to depletion of 98% erythrocytes and 87% granulocytes, compared to 100% and 70%, respectively, for manual processing. After closed system culture, mesenchymal progenitors, measured as CD105+CD166+CD14-CD45- and fibroblastic CFU, expanded 317- and 364-fold, respectively, while CD34+ hematopoietic progenitors were depleted 10-fold compared to starting BMMNC. Cultured cells exhibited multilineage differentiation by displaying adipogenic, osteogenic, and endothelial characteristics in vitro. No significant difference was observed between manual and bioreactor cultures. Automated culture and washing of the cell product resulted in 181 x 10(6) total cells that were viable and contained fibroblastic CFU for at least 24 h of storage. A combination of closed, automated technologies enabled production of good manufacturing practice (GMP)-compliant cell therapeutics, ready for use within a clinical setting, with minimal risk of microbial contamination.

Background: Xylosyltransferase I (XT-I), the key enzyme in the biosynthesis of glycosaminoglycan chains in proteoglycans, has increased activity in the blood serum of patients with connective tissue diseases. Therefore, the measurement of serum XT-I activity is useful to monitor disease activity in these patients., Methods: We developed an HPLC electrospray ionization tandem mass spectrometry method to assay XT-I activity in serum by use of a synthetic peptide (Bio-BIK-F) as the XT-I substrate. On the basis of XT-I-mediated transfer of D-xylose from UDP-D-xylose to the synthetic peptide to form Bio-BIK-F-Xyl, we determined XT-I activity in human serum samples., Results: Multiple calibration curves for the analysis of Bio-BIK-F-Xyl exhibited consistent linearity and reproducibility in the range of 0.20-20 mg/L, corresponding to XT-I activity of 1.14-114 mU/L under assay conditions. The mean (SD, range) XT-I activity values in 30 blood donor sera were 18.4 (3.0, 8.7-24.8) mU/L. The limit of detection and lower limit of quantification were 8.5 microg/L (0.05 mU/L) and 163 microg/L Bio-BIK-F-Xyl (0.93 mU/L XT-I activity), respectively. Interassay imprecision (CV) was 5.4%-26.1% in the range of 0.64 to 129 mU/L, and mean recovery was 107% (range, 96%-129%). Method comparison with the radiochemical assay showed a moderate correlation (r = 0.79). The Passing-Bablok regression line was: radiochemical assay = 0.045 LC-MS/MS + 0.061 mU/L, S(y/x) = 0.186., Conclusions: This simple and robust LC-MS/MS assay permits the rapid and accurate determination of XT-I activity in human serum.

In vitro differentiation of mesenchymal stem cells (MSCs) into chondrogenic cells and their transplantation is promising as a technique for the treatment of cartilaginous defects. But the regulation of extracellular matrix (ECM) formation remains elusive. Therefore, the objective of this study was to analyze the regulation of proteoglycan (PG) biosynthesis during the chondrogenic differentiation of MSCs. In different stages of chondrogenic differentiation, we analyzed mRNA and protein expression of key enzymes and PG core proteins involved in ECM development. For xylosyltransferase I (XT-I), we found maximum mRNA levels 48 hours after chondrogenic induction with a 5.04 +/- 0.58 (mean +/- SD)-fold increase. This result correlates with significantly elevated levels of enzymatic XT-I activity (0.49 +/- 0.03 muU/1 x 10(6) cells) at this time point. Immunohistochemical staining of XT-I revealed a predominant upregulation in early chondrogenic stages. The highly homologous protein XT-II showed 4.7-fold (SD 0.6) increased mRNA levels on day 7. To determine the differential expression of heparan sulfate (HS), chondroitin sulfate (CS), and dermatan sulfate (DS) chains, we analyzed the mRNA expression of EXTL2 (alpha-4-N-acetylhexosaminyltransferase), GalNAcT (beta-1,4-N-acetylgalactosaminyltransferase), and GlcAC5E (glucuronyl C5 epimerase). All key enzymes showed a similar regulation with temporarily downregulated mRNA levels (up to -87-fold) after chondrogenic induction. In accordance to previous studies, we observed a similar increase in the expression of PG core proteins. In conclusion, we could show that key enzymes for CS, DS, and HS synthesis, especially XT-I, are useful markers for the developmental stages of chondrogenic differentiation.

Objective: Xylosyltransferase I (XT-I) is the chain-initiating enzyme in the biosynthesis of proteoglycans in basement membranes. It catalyzes the transfer of xylose to selected serine residues in the core protein. The XYLT-II gene codes for a protein highly homologous to XT-I. Proteoglycans are important components of basement membranes and are responsible for their permeability properties. Type 1 diabetic patients have an altered proteoglycan metabolism, which results in microvascular complications. Thus, genetic variations in the xylosyltransferase genes might be implicated in the initiation and progression of these complications., Research Design and Methods: Genotyping of four genetic variations in the genes XYLT-I and XYLT-II was performed in 912 type 1 diabetic patients (453 with and 459 without diabetic nephropathy) using restriction fragment-length polymorphism., Results: The distribution of the c.343G>T polymorphism in XYLT-I is significantly different between patients with and without diabetic nephropathy (P = 0.03). T-alleles were more frequent in patients with diabetic nephropathy (odds ratio 2.47 [95% CI 1.04-5.83]). The allelic frequencies of the other investigated XYLT-I and XYLT-II variations (XYLT-I: c.1989T>C in exon 9; XYLT-II: IVS6-9T>C and IVS6-14&#95;IVS6-13insG in intron 5; and c.2402C>G: p.T801R in exon 11) were not different between patients with and without diabetic nephropathy., Conclusions: The XYLT-I c.343G>T polymorphism contributes to the genetic susceptibility to development of diabetic nephropathy in type 1 diabetic patients.

Background: Pseudoxanthoma elasticum (PXE) is a heritable connective tissue disorder caused by mutations in the ABCC6 gene. Fragmentation of elastic fibres and deposition of proteoglycans result in a highly variable clinical picture. The altered proteoglycan metabolism suggests that enzymes from this pathway function as genetic co-factors in the severity of PXE. Therefore, we propose the XYLT genes encoding xylosyltransferase I (XT-I) as the chain-initiating enzyme in the biosynthesis of proteoglycans and the highly homologous XT-II as potential candidate genes., Methods: We screened all XYLT exons in 65 German PXE patients using denaturing high performance liquid chromatography and analysed the influence of the variations on clinical characteristics., Results: We identified 22 variations in the XYLT genes. The missense variation p.A115S (XT-I) is associated with higher serum XT activity (p = 0.005). The amino acid substitution p.T801R (XT-II; c.2402C>G) occurs with significantly higher frequency in patients under 30 years of age at diagnosis (43% v 26%; p = 0.04); all PXE patients with this variation suffer from skin lesions compared to only 75% of the wild type patients (p = 0.002). c.166G>A, c.1569C>T, and c.2402C>G in the XYLT-II gene were found to be more frequent in patients with higher organ involvement (p = 0.04, p = 0.01, and p = 0.02, respectively)., Conclusions: Here we show for the first time that variations in the XYLT-II gene are genetic co-factors in the severity of PXE. Furthermore, the higher XT activity in patients with the exchange p.A115S (XT-I) indicates that this polymorphism is a potential marker for increased remodelling of the extracellular matrix.

Objective: Diabetes is characterized by marked postprandial endothelial dysfunction induced by hyperglycemia, hypertriglyceridemia, advanced glycation end products (AGEs), and dicarbonyls (e.g., methylglyoxal [MG]). In vitro hyperglycemia-induced MG formation and endothelial dysfunction could be blocked by benfotiamine, but in vivo effects of benfotiamine on postprandial endothelial dysfunction and MG synthesis have not been investigated in humans until now., Research Design and Methods: Thirteen people with type 2 diabetes were given a heat-processed test meal with a high AGE content (HAGE; 15.100 AGE kU, 580 kcal, 54 g protein, 17 g lipids, and 48 g carbohydrates) before and after a 3-day therapy with benfotiamine (1,050 mg/day). Macrovascular flow-mediated dilatation (FMD) and microvascular reactive hyperemia, along with serum markers of endothelial disfunction (E-selectin, vascular cell adhesion molecule-1, and intracellular adhesion molecule-1), oxidative stress, AGE, and MG were measured during both test meal days after an overnight fast and then at 2, 4, and 6 h postprandially., Results: The HAGE induced a maximum reactive hyperemia decrease of -60.0% after 2 h and a maximum FMD impairment of -35.1% after 4 h, without affecting endothelium-independent vasodilatation. The effects of HAGE on both FMD and reactive hyperemia were completely prevented by benfotiamine. Serum markers of endothelial dysfunction and oxidative stress, as well as AGE, increased after HAGE. These effects were significantly reduced by benfotiamine., Conclusions: Our study confirms micro- and macrovascular endothelial dysfunction accompanied by increased oxidative stress following a real-life, heat-processed, AGE-rich meal in individuals with type 2 diabetes and suggests benfotiamine as a potential treatment.

Pseudoxanthoma elasticum (PXE) is a genetic disorder characterized by calcification of elastic fibers in dermal, ocular, and cardiovascular tissues. Recently, ABCC6 mutations were identified as causing PXE. In this follow-up study we report the investigation of 61 German PXE patients from 53 families, hitherto the largest cohort of German PXE patients screened for the complete ABCC6 gene. In addition, we characterized the proximal ABCC6 promoter of PXE patients according to mutation. In this study we identified 32 disease-causing ABCC6 variants, which had been described previously by us and others, and 10 novel mutations (eight missense mutations and two splice site alterations). The mutation detection rate among index patients was 87.7%. Frequent alterations were the PXE-mutations p.R1141X, Ex23,&#95;Ex29del, and c.2787+1G > T. In the ABCC6 promoter we found the polymorphisms c.-127C > T, c.-132C > T, and c.-219A > C. The difference in the c.-219A > C frequencies between PXE patients and controls were determined as statistically significant. Interestingly, c.-219A > C is located in a transcriptional activator sequence of the ABCC6 promoter and occurred in a binding site for a transcriptional repressor, predominantly found in genes that participate in lipid metabolism. Obtaining these genetic data signifies our contribution to elucidating the pathogenetics of PXE.

Aims: Decreased content of heparan sulphate proteoglycans (HSPGs) is a characteristic of the glomerular basement membrane (GBM) in diabetes and contributes to the development of diabetic nephropathy (DN). Xylosyltransferase I (XT-I) is the chain-initiating enzyme involved in the biosynthesis of HSPGs. This study investigated a possible association between XYLT-I sequence variants and susceptibility to DN., Methods: Screening of all XYLT-I exons was performed in 74 caucasians with Type 1 diabetes (48 with and 26 without DN) and in 13 non-diabetic control subjects using denaturing high-performance liquid chromatography., Results: Fifteen XYLT-I sequence variants were identified. Of these, six were previously unknown. There were significant differences in the allele frequencies of the three polymorphisms (c.343G-->T (p.A115S), IVS3+10C-->T, IVS3+30G-->C) in Type 1 diabetic patients and healthy controls., Conclusions: The occurrence of DN is independent of the XYLT-I variants detected in our study. However, three XYLT-I polymorphisms may be linked to Type 1 diabetes. Since we have previously proposed that one of these polymorphisms was not associated with Type 1 diabetes (Schön S et al. Kidney Int 2005; 68: 1483-1490), larger-scale analysis is clearly necessary to pinpoint the significance of this mutation.

Pseudoxanthoma elasticum (PXE) is an inherited disorder that is associated with accumulation of mineralized and fragmented elastic fibers in the skin, vessel walls, and Bruch's membrane. Clinically, patients exhibit characteristic lesions of the skin (soft, ivory-colored papules in a reticular pattern that predominantly affect the neck), the posterior segment of the eye (peau d'orange, angioid streaks, choroidal neovascularizations), and the cardiovascular system (peripheral arterial occlusive disease, coronary occlusion, gastrointestinal bleeding). There is no causal therapy. Recent studies suggest that PXE is inherited almost exclusively as an autosomal recessive trait. Its prevalence has been estimated to be 1:25,000-100,000. The ABCC6 gene on chromosome 16p13.1 is associated with the disease. Mutations within the ABCC6 gene cause reduced or absent transmembraneous transport that leads to accumulation of substrate and calcification of elastic fibers. Although based on clinical features the diagnosis appears readily possible, variability in phenotypic expressions and the low prevalence may be responsible that the disease is underdiagnosed. This review covers current knowledge of PXE and presents therapeutic approaches.

Xylosyltransferase I (XT-I) catalyzes the transfer of xylose from UDP-xylose to serine residues in proteoglycan core proteins. This is the first and apparently rate-limiting step in the biosynthesis of the tetrasaccharide linkage region in glycosaminoglycan-containing proteoglycans. The XYLT-II gene codes for a highly homologous protein, but its physiological function is not yet known. Here we present for the first time the construction of a vector encoding the full-length GFP-tagged human XT-I and the recombinant expression of the active enzyme in mammalian cells. We expressed XT-I-GFP and various GFP-tagged XT-I and XT-II mutants with C-terminal truncations and deletions in HEK-293 and SaOS-2 cells in order to investigate the intracellular localization of XT-I and XT-II. Immunofluorescence analysis showed a distinct perinuclear pattern of XT-I-GFP and XT-II-GFP similar to that of alpha-mannosidase II, which is a known enzyme of the Golgi cisternae. Furthermore, a co-localization of native human XT-I and alpha-mannosidase II could also be demonstrated in untransfected cells. Using brefeldin A, we could also show that both xylosyltransferases are resident in the early cisternae of the Golgi apparatus. For its complete Golgi retention, XT-I requires the N-terminal 214 amino acids. Unlike XT-I, for XT-II, the first 45 amino acids are sufficient to target and retain the GFP reporter in the Golgi compartment. Here we show evidence that the stem regions were indispensable for Golgi localization of XT-I and XT-II.

Objective: The hallmark in osteoarthritis (OA) is the loss of proteoglycans (PGs) in articular cartilage (AC). Xylosyltransferase I (XT-I) catalyzes the transfer of xylose to serine residues in the core protein and initiates the biosynthesis of PGs in AC. The XYLT-II gene encodes a highly homologous protein but its biological function is not yet known. Here we investigate for the first time genetic variations in the XYLT-genes and serum XT-I activities and their implication in OA., Methods: Denaturing high-performance liquid chromatography (DHPLC) was used for the screening of the XYLT-genes in 49 OA patients. For a detailed characterization of XT-I amino acid exchanges we performed recombinant expression of XT-I mutants in insect cells. Furthermore, the XT activity was measured in the patients' serum., Results: The variation c.1569C>T (XYLT-II) occurs with a significantly higher frequency in younger OA patients in comparison with the older ones (P<0.001) and the controls (P<0.02). Furthermore, significantly higher serum XT activities were found in patients with a long disease duration of OA (P<0.04). The recombinant XT-I mutants p.P385L and p.I552S had reduced enzymatic activity (85% and 74%) compared with the wildtype (wt)., Conclusions: Our findings indicate a correlation of the c.1569 T-allele in XYLT-II with an earlier manifestation of OA and that the serum XT activity is a potential biochemical marker for staging and monitoring the progression of AC damage in OA. Comparison of XT-I activity in mutant enzymes in vivo and in vitro revealed that heterozygous mutations are not involved in OA.

Analysis of glycosaminoglycans (GAGs) is of increasing importance concerning alterations in extracellular matrix composition and selectivity of glomerular basement membrane. In this report we describe the analysis of chondroitin sulfate disaccharides as an example of GAG delta disaccharide analysis using standard DNA sequencing equipment (DNA sequencer-assisted GAG disaccharide separation, DSA-GAGS). The presented methodology allows nanomolar quantification of 8-aminopyrene-1,3,6-trisulfonic acid (APTS)-derived GAG disaccharides. In comparison to RP-HPLC the established method is much more sensitive, showing detection limits of 38 fmol/microL. Variation coefficients were approximately 10%, enabling exact quantifications after run times of 17 min at 30 degrees C and an electrophoresis voltage of 15 kV; using a capillary DNA sequencer, available in many molecular laboratories, presented advantages like automated sample injection, opportunity of high-throughput analyses, separation of even sulfated disaccharide epimers, and the possibility of using APTS-derived fucose as an internal standard. Furthermore, highly reproducible retention times rendered easy identification of specific signals (SD 0.02). With regard to these results, the described method is a useful tool for the quantification of GAG disaccharides in low amounts, indicating advantages of obverse RP-HPLC and slab gel polyacrylamide electrophoresis in sensitivity, error-proneness, automation, and handling.

Human XT-I (xylosyltransferase I; EC 2.4.2.26) initiates the biosynthesis of the glycosaminoglycan linkage region and is a diagnostic marker of an enhanced proteoglycan biosynthesis. In the present study, we have investigated mutant enzymes of human XT-I and assessed the impact of the N-terminal region on the enzymatic activity. Soluble mutant enzymes of human XT-I with deletions at the N-terminal domain were expressed in insect cells and analysed for catalytic activity. As many as 260 amino acids could be truncated at the N-terminal region of the enzyme without affecting its catalytic activity. However, truncation of 266, 272 and 273 amino acids resulted in a 70, 90 and >98% loss in catalytic activity. Interestingly, deletion of the single 12 amino acid motif G261KEAISALSRAK272 leads to a loss-of-function XT-I mutant. This is in agreement with our findings analysing the importance of the Cys residues where we have shown that C276A mutation resulted in a nearly inactive XT-I enzyme. Moreover, we investigated the location of the heparin-binding site of human XT-I using the truncated mutants. Heparin binding was observed to be slightly altered in mutants lacking 289 or 568 amino acids, but deletion of the potential heparin-binding motif P721KKVFKI727 did not lead to a loss of heparin binding capacity. The effect of heparin or UDP on the XT-I activity of all mutants was not significantly different from that of the wild-type. Our study demonstrates that over 80% of the nucleotide sequence of the XT-I-cDNA is necessary for expressing a recombinant enzyme with full catalytic activity.

Tissue factor pathway inhibitor (TFPI) is a multivalent Kunitz-type serine proteinase inhibitor that plays a central role in the extrinsic pathway of blood coagulation. In earlier studies we could identify the [P151L]TFPI mutant, and we could also demonstrate that heterozygous carriers of this mutant show a nine-fold increased risk for deep venous thrombosis (DVT). To express greater amounts of both proteins and to enable their characterization, we expressed wild-type TFPI as well as [P151L]TFPI in High Five insect cells with expression rates of up to 215 ng/ml for wild-type TFPI and 214 ng/ml for [P151L]TFPI. The specific inhibitory activities for the recombinant proteins were determined as 11.3 and 11.5 mU/ng, respectively. Both proteins were detected via Western blot analysis and ELISA. The recombinant proteins' inhibitory activities were characterized by a chromogenic assay and by the determination of a modified activated thromboplastin time (aPTT) in which both of them proved to be inhibitorily active. We also examined both recombinant proteins' binding properties to glycoproteins, glycosaminoglycans, lipoproteins and tissue factor. Our results show that we have developed an efficient model system for the recombinant expression of inhibitorily active wild-type TFPI as well as [P151L]TFPI in insect cells, and we were able to characterize both proteins' inhibitory properties by determination of their influence on the aPTT and also their binding properties. Although both recombinant proteins did not show a significant difference in their effect on the aPTT, their binding properties differed significantly between the wild type and mutant protein.

Background: Pseudoxanthoma elasticum (PXE) is a hereditary disorder of the connective tissue affecting the skin, retina, and cardiovascular system and characterized by progressive calcification of abnormal and fragmented elastic fibers in the extracellular matrix. The aim of the present study was to investigate the association of fetuin-A, a major systemic inhibitor of calcification, with PXE., Methods: Fetuin-A was measured by quantitative sandwich enzyme immunoassay in sera from 110 German patients with PXE, 53 unaffected first-degree family members, and 80 healthy blood donors. We determined the distribution of the fetuin-A polymorphisms c.742C>T (p.T248M) and c.766C>G (p.T256S) in these same 3 groups. The occurrences of the frequent ABCC6 gene mutations c.3421C>T (p.R1141X) and c.EX23&#95;EX29del were also assessed., Results: Serum fetuin-A concentrations in male and female PXE patients were lower than in unaffected first-degree relatives and controls [mean (SD) concentrations, 0.55 (0.11) g/L in patients; 0.70 (0.23) g/L in relatives; and 0.80 (0.23) g/L in controls (P <0.0001)]. Serum fetuin-A was higher in female PXE patients with cardiovascular involvement than in the corresponding male group (P <0.05). The fetuin-A polymorphism frequencies did not differ among PXE patients, family members, and blood donors., Conclusion: A deficiency of multidrug resistance-associated protein 6 leads to alteration of circulating substrates, e.g., inhibitors of calcification as fetuin-A, leading to progressive mineralization of elastic fibers in PXE.

Objectives: Increased total homocysteine (tHcy) level is an independent risk factor for atherosclerosis and cardiovascular disease. Here, we describe a rapid tHcy micro-scale assay., Methods: We developed an easy sample preparation and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the measurement of tHcy in human serum and plasma, respectively., Results: The tHcy assay was linear up to 61.6 micromol/L, the detection limit was 1.0 micromol/L, the lower limit of quantification was 1.8 micromol/L, the imprecision in the range of 9.30-25.9 micromol/L was less than 5.9% and the analytical recovery was 94.7 +/- 6.3%., Conclusions: Our novel tHcy LC-MS/MS assay is a quick, precise and robust method for tHcy determination in routine diagnostics.

Tissue factor pathway inhibitor 2 (TFPI-2) is a Kunitz-type serine protease inhibitor with homology to TFPI-1, an important regulator of the extrinsic pathway of blood coagulation. Recent studies have focused on TFPI-2 and its implications for atherosclerosis. The promoter region and the exons of the human TFPI-2 gene were screened for sequence variations in 41 apoplectic patients and 140 blood donors with no history of ischemic stroke. The sequence variations -567T>C, -546T>C, -353A>G, -161G>C, -167G>A, -47C>A, and -18C>A, which are located in the TFPI-2 promoter, were discovered in both cohorts with allelic frequencies ranging from 0.3 to 2.4%. The influence of these sequence variations on the transcriptional activity of the TFPI-2 gene was investigated in HEK-293 cells using a promoter test system. A wild-type TFPI-2 promoter fragment 716 bp upstream of the translation start site was cloned into a secreted alkaline phosphatase expression vector, and the sequence variations were introduced by site-directed mutagenesis. Interestingly, the promoter activity of the tested mutants was reduced by 1.3- to 2.8-fold compared to that of wild-type control. The variation -18C>A, where a putative binding site of the transcription factor Sp-1 is located, had the strongest effect on transcriptional activity. In conclusion, our present study shows that the transcription of TFPI-2 is changed by single nucleotide polymorphisms and that the sequence variations in transcription factor binding sites of the TFPI-2 promoter may influence the regulation of this gene.

Pseudoxanthoma elasticum (PXE) is a hereditary disorder of the connective tissue characterized by extracellular matrix alterations with elastin fragmentation and excessive proteoglycan deposition. Xylosyltransferase I (XT-I, E.C. 2.4.2.26) is the initial enzyme in the biosynthesis of the glycosaminoglycan chains in proteoglycans and has been shown to be a marker of tissue remodeling processes. Here, we investigated for the first time serum XT-I activities in a large cohort of German PXE patients and their unaffected relatives. XT-I activities were measured in serum samples from 113 Caucasian patients with PXE and 103 unaffected first-degree family members. The occurrence of the frequent ABCC6 gene mutation c.3421C>T (R1141X) and the hypertension-associated genetic variants T174M and M235T in the angiotensinogen (AGT) gene were determined. Serum XT-I activities in male and female PXE patients were significantly increased compared to unaffected family members (male patients, mean value 0.96 mU/l, SD 0.37; male relatives, 0.78 mU/l, SD 0.29; female patients, 0.91 mU/l, SD 0.31; female relatives, 0.76 mU/l, SD 0.34; p<0.05). The mean XT-I activities in PXE patients with hypertension were 24% higher than in patients without increased blood pressure (p<0.05). The AGT T174M and M235T frequencies were not different in hypertensive PXE patients, normotensive PXE patients, family members or blood donors. Our data show that the altered proteoglycan biosynthesis in PXE patients is closely related to an increased XT-I activity in blood. Serum XT-I, the novel fibrosis marker, may be useful for the assessment of extracellular matrix alterations and disease activity in PXE.

Background: Tissue factor pathway inhibitor (TFPI) is a multivalent Kunitz-type serine proteinase inhibitor which plays a central role in the extrinsic pathway of blood coagulation. A significant role of TFPI for the follicular development has been discussed in recent studies, and thrombotic complications during IVF procedure are a common problem. To elucidate the pathophysiological mechanisms underlying these problems, we have measured TFPI levels in human follicular fluid (hFF) of women undergoing in vitro fertilisation (IVF)., Methods: Total TFPI concentrations were determined in hFF of 28 women undergoing IVF treatment, 6 of whom developed an ovarian hyperstimulation syndrome (OHSS)., Results and Conclusions: This is the first study to demonstrate an age-dependance of TFPI concentrations in hFF. Additionally, TFPI levels in hFF of women developing OHSS were determined as 323+/-66.8 ng/mL (mean+/-SD) in comparison with 279+/-137 ng/mL for non-OHSS patients. Our findings demonstrate that, unlike the decreased TFPI levels found in OHSS patients' blood, there is no statistically significant difference in hFF TFPI levels between OHSS and non-OHSS patients. Furthermore, we could show that the outcome of the IVF procedure is not correlated with TFPI levels in hFF.

Objectives: Hyperhomocysteinemia is a risk factor for cardiovascular diseases, while tissue factor pathway inhibitor (TFPI) regulates the extrinsic pathway of blood coagulation. We investigated the in vivo influence of elevated homocysteine concentrations on TFPI levels., Methods: Total TFPI levels of 82 hyperhomocysteinemic patients were determined., Results: The patients' TFPI levels were significantly elevated compared to blood donors, with a more pronounced effect in females than in males., Conclusions: We have shown a gender-dependent TFPI elevation in hyperhomocysteinemic patients.

Background: Xylosyltransferase I (XT-I) is the chain-initiating enzyme in the biosynthesis of heparan sulfate proteoglycans (HSPGs). It catalyses the transfer of xylose to specific serine residues in the core protein. The XYLT-II gene codes for a protein highly homologous to the XT-I but its biologic function is not yet known. HSPGs are thought to play an important role in the permeability properties of the glomerular basement membrane (GBM) and thus the xylotransferase genes might be potential candidate genes predisposing to diabetic nephropathy in type 1 diabetic patients., Methods: We screened all XYLT-I and XYLT-II exons and flanking intron regions in 96 Caucasians with type 1 diabetes (48 with and 48 without diabetic nephropathy) using denaturing high-performance liquid chromatography (DHPLC). We also studied a nondiabetic control group., Results: Applying this technique we identified 13 variations in XYLT-I and 20 in XYLT-II. The variations IVS6-9T>C and IVS6-14&#95;IVS6-13insG in XYLT-II were found with a frequency of 5.2% (5/96) in nondiabetic nephropathy patients, while all nephropathy patients were negative (P= 0.06). Nondiabetic controls also showed the single nucleotide polymorphisms (SNP) at a frequency of 1.1% (5/440). The investigation of the SNPs' influence on clinical characteristics revealed significant associations for c.1989T>C (XYLT-I) and c.2402C>G (XYLT-II) with patients' blood pressure., Conclusion: We detected in our cohort associations between DNA sequence variations of genes encoding xylosyltransferases and the occurrence of altered clinical characteristics.

Human basic fibroblast growth factor (bFGF) is a heparin-binding growth factor containing a G-S-G-motif which is a potential recognition sequence of xylosyltransferase I (XT-I). Here, we show that the recombinant human bFGF was xylosylated in vitro by human XT-I and that the fragment bFGF (1-24) is a good XT-I acceptor (K(m) = 20.8 microM for native XT-I and K(m) = 22.3 microM for recombinant XT-I). MALDI and MALDI-PSD time-of-flight mass spectrometric analyses of the xylosylated bFGF protein demonstrate the transfer of xylose to the serine residue of the G-S-G-motif in the amino terminal end of bFGF. The peptide bFGF (1-24) is well suitable as an acceptor substrate for XT-I and can be used in a radiochemical assay to measure the XT-I activity in cell culture supernatant and human body fluids, respectively. Furthermore, we could demonstrate that the XT-I interacts strongly with heparin and that this glycosaminoglycan is a predominantly non-competitive inhibitor of the enzyme using the fragment bFGF (1-24) as xylose acceptor.

Pseudoxanthoma elasticum (PXE) is a heritable disorder of the connective tissue affecting the skin, eyes, and cardiovascular system. Recently, the PXE candidate gene ABCC6 was identified and a limited number of ABCC6 mutations were observed in different PXE cohorts. To identify novel PXE-causing ABCC6 mutations in German patients with PXE, we investigated a cohort of 54 German PXE patients and 23 family members from 49 apparently nonconsanguineous families. From the mutational analysis we found 27 different ABCC6 sequence variations. Among these, 11 were polymorphisms or neutral alterations and 16 were PXE-causing mutations. The most common mutation in our PXE cohort was the nonsense mutation p.R1141X, which occurred with an allele frequency of 25.9%. Furthermore, we found nine missense, one additional nonsense, and two putative splice site mutations as well as three single-nucleotide deletions. Most of these mutations were unique and occurred in cytoplasmic regions of the MRP6 protein; these mutations are proposed to be critical for the physiological function of the MRP6 protein. In these regions we also found the three novel PXE-causing mutations p.R1114C, p.Y1239H, and p.G1311E, which were identified in three alleles from patients with PXE and were absent in 200 healthy control subjects. In addition, the first genotype-phenotype correlation was observed. By obtaining these genetic mutation data, we are contributing to an overview of all ABCC6 mutations leading to PXE and the pathogenetics of this disease.

Objectives: Serum xylosyltransferase I (XT-I) is a marker for the determination of tissue remodeling in systemic sclerosis. Here, we investigated whether renal insufficiency affects XT-I levels in blood., Methods: We measured serum XT-I activity in 236 patients with different serum creatinine levels., Results: XT-I activities in cohorts with increased creatinine levels were not significantly altered compared to controls., Conclusions: Serum XT-I activity is applicable as a fibrosis marker independent from renal function.

XT-I (xylosyltransferase I) is the initial enzyme in the post-translational biosynthesis of glycosaminoglycan chains in proteoglycans. To gain insight into the structure-function relationship of the enzyme, a soluble active form of human XT-I was expressed in High Five insect cells with an apparent molecular mass of 90 kDa. Analysis of the electrophoretic mobility of the protein under non-reducing and reducing conditions indicated that soluble XT-I does not form homodimers through disulphide bridges. In addition, the role of the cysteine residues was investigated by site-directed mutagenesis combined with chemical modifications of XT-I by N-phenylmaleimide. Replacement of Cys471 or Cys574 with alanine led to a complete loss of catalytic activity, indicating the necessity of these residues for maintaining an active conformation of soluble recombinant XT-I by forming disulphide bonds. On the other hand, N-phenylmaleimide treatment showed no effect on wild-type XT-I but strongly inactivated the cysteine mutants in a dose-dependant manner, indicating that seven intramolecular disulphide bridges are formed in wild-type XT-I. The inhibitory effect of UDP on the XT-I activity of C561A (Cys561-->Ala) mutant enzyme was significantly reduced compared with all other tested cysteine mutants. In addition, we tested for binding to UDP-agarose beads. The inactive mutants revealed no significantly different nucleotide-binding properties. Our study demonstrates that recombinant XT-I is organized as a monomer with no free thiol groups and strongly suggests that the catalytic activity does not depend on the presence of free thiol groups, furthermore, we identified five cysteine residues which are critical for enzyme activity.

Pseudoxanthoma elasticum (PXE; OMIM 177850 and 264800) is a rare heritable disorder of the connective tissue affecting the extracellular matrix of the skin, eyes, gastrointestinal system, and cardiovascular system. It has recently been found that mutations in the ABCC6 gene encoding the multidrug resistance-associated protein (MRP) 6 cause PXE. This study examined novel mutations in the ABCC6 gene in our cohort of 76 German PXE patients and 54 unaffected or not yet affected relatives with a view to expanding the known mutational spectrum of the gene. Mutational analysis was performed using denaturing high-performance liquid chromatography and direct sequencing. The mutational screening revealed a total of 22 different ABCC6 sequence variations. We identified seven novel and four previously described PXE-associated mutations as well as eight novel neutral ABCC6 sequence variants. The new PXE-associated mutations included five missense mutations, one single base pair deletion, and one larger out-of-frame deletion. We suspect that the novel missense mutations lead to an impaired function of MRP6. Both deletions are predicted to result in a dysfunctional MRP6 protein. The seven new ABCC6 mutations were not present in 200 alleles from healthy blood donors which served as a control cohort. Most of the PXE patients who were found to carry PXE-causing ABCC6 mutations were assumed to manifest the PXE phenotype because of a compound heterozygous genotype. However, a genotype-phenotype correlation could not be established for the detected ABCC6 mutations. In summary, our data give a further insight into the spectrum of ABCC6 mutations in PXE patients.

Human xylosyltransferase I (XT-I) is the initial enzyme involved in the biosynthesis of the glycosaminoglycan linker region in proteoglycans. Here, we tested the importance of the DXD motifs at positions 314-316 and 745-747 for enzyme activity and the nucleotide binding capacity of human XT-I. Mutations of the 314DED316 motif did not have any effect on enzyme activity, whereas alterations of the 745DWD747 motif resulted in reduced XT-I activity. Loss of function was observed after exchange of the highly conserved aspartic acid at position 745 with glycine. However, mutation of Asp745 to glutamic acid retained full enzyme activity, indicating the importance of an acidic amino acid at this position. Reduced substrate affinity was observed for mutants D747G (Km=6.9 microm) and D747E (Km=4.4 microm) in comparison with the wild-type enzyme (Km=0.9 microm). Changing the central tryptophan to a neutral, basic, or acidic amino acid resulted in a 6-fold lower Vmax, with Km values comparable with those of the wild-type enzyme. Despite the major effect of the DWD motif on XT-I activity, nucleotide binding was not abolished in the D745G and D747G mutants, as revealed by UDP-bead binding assays. Ki values for inhibition by UDP were determined to be 1.9-24.6 microm for the XT-I mutants. The properties of binding of XT-I to heparin-beads, the Ki constants for noncompetitive inhibition by heparin, and the activation by protamine were not altered by the generated mutations.

summary In many countries, screening of hepatitis B virus (HBV) in blood donors is limited to HBsAg testing. However, if anti-HBc testing and sensitive HBV nucleic acid amplification testing (NAT) for routine screening are not prescribed, HBV viraemia might remain unrecognized. A clinically inconspicuous HBsAg-negative 35-year-old female blood donor was detected with anti-HBc antibodies following the introduction of anti-HBc screening of donors. Based on her history, she had seroconverted to anti-HBs positive (titre >7000 IU/L) after vaccination. Blood donations were routinely tested HBV-DNA negative by minipool NAT. The individual donor samples were reinvestigated by an ultrasensitive NAT with a lower detection limit of 3.8 IU/mL. Intermittent HBV viraemia was detected over a 7-year period from this donor, with a concentration ranging from 8 to 260 IU/mL. In the subsequent donor-directed lookback study, no post-transfusion hepatitis was detected. Low-level HBV viraemia in simultaneous anti-HBc- and anti-HBs-positive blood donors could only be identified with enhanced sensitivity individual polymerase chain reaction assays and is not detectable by pool HBV NAT.

Pseudoxanthoma elasticum (PXE) is a heritable disorder of the connective tissue. Mutations in the ABCC6 gene could be linked to this disease and, just recently, the c.3421C>T mutation was also associated with a high risk of coronary artery disease. We have now developed new real-time PCR assays for the accurate and rapid determination of the c.3421C>T genotype. Using our new assay, we analyzed the presence of the c.3421C>T mutation in the largest collection of DNA samples from unrelated German PXE patients (n=64) and in a control cohort (n=910). For assay setup, two sets of samples with known genotype for the c.3421C>T mutation were analyzed over a period of 14 days. Results were confirmed by restriction endonuclease mapping, sequence-specific PCR and DNA sequencing. In order to ensure that no further mutations or deletions interfered with the c.3421C>T genotyping, we scanned the exon 24 of the ABCC6 gene by DHPLC and investigated the presence of the ABCC6del23-29 deletion in all patients. The assay has been set up on a group of patients with known genotype and validated on 64 PXE patients. In this group four PXE patients (6.3%) were found to be homozygous and 25 (39.0%) to be heterozygous carriers of the c.3421C>T mutation. The common ABCC6del23-29 deletion, possibly interfering with genotype determination, was searched and excluded. Furthermore, two novel mutations in the ABCC6 gene could be identified in two patients. The novel mutations c.3389C>T and c.3341G>A did not interfere with our new assay. Our new c.3421C>T genotyping assays can be used for the rapid identification of this frequent mutation in PXE patients and of the recently newly proposed cardiac risk factor in young patients with myocardial infarcts of unknown origin.