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1.
Glycoconj J ; 30(3): 237-45, 2013 Apr.
Article in English | MEDLINE | ID: mdl-22886070

ABSTRACT

Recently, we demonstrated that the human xylosyltransferase II (XT-II) has enzymatic activity and is able to catalyze the initial and rate-limiting step in the biosynthesis of glycosaminoglycans (GAGs) like chondroitin and dermatan sulfate, as well as heparan sulfate and heparin. Therefore, this enzyme also very likely assumes a crucial regulatory role in the biosynthesis of proteoglycans (PGs). In this study, we identified and characterized for the first time the XYLT2 gene promoter region and transcription factors involved in its regulation. Several binding sites for members of the Sp1 family of transcription factors were identified as being necessary for transcriptional regulation of the XYLT2 gene. This was determined by mithramycin A treatment, electrophoretic mobility shift and supershift assays, as well as numerous site-directed mutagenesis experiments. Different 5' and 3' deletion constructs of the predicted GC rich promoter region, which lacks a canonical TATA and CAAT box, revealed that a 177 nts proximal promoter element is sufficient and indispensable to drive the constitutive transcription in full strength in HepG2 hepatoma cells. In addition, we also detected the transcriptional start site using 5'-RACE (rapid amplification of cDNA ends). Our results provide an insight into transcriptional regulation of the XYLT2 gene and may contribute to understanding the manifold GAG-involving processes in health and disease.


Subject(s)
Pentosyltransferases/genetics , Promoter Regions, Genetic , Transcription, Genetic , 3' Flanking Region , 5' Flanking Region , Base Sequence , GC Rich Sequence , Gene Expression Regulation, Neoplastic , Hep G2 Cells , Humans , Molecular Sequence Data , Mutagenesis , Pentosyltransferases/metabolism , Sp1 Transcription Factor/metabolism , UDP Xylose-Protein Xylosyltransferase
2.
Methods Mol Biol ; 943: 91-103, 2013.
Article in English | MEDLINE | ID: mdl-23104283

ABSTRACT

Despite considerable advances in the safety of blood components based on the application of highly sensitive and specific screening methods to minimize the viral infection risk, the prevention of transfusion-associated bacterial infection remains a major challenge in transfusion medicine. In particular, platelet concentrates represent the greatest infectious risk of transfusion-transmitted bacterial sepsis. The detection of bacterial contamination in platelet concentrates has been implemented in several blood services as a routine quality control testing. Although culture is likely to remain the gold standard method of detecting bacterial contamination, the use of rapid methods is likely to increase and play an important role in transfusion medicine in the future. In particular, flow cytometric methods and nucleic acid amplification techniques are powerful tools in bacterial screening assays. Compared to culture-based methods, the combination of high sensitivity and specificity, low contamination risk, ease of performance, and speed has made those technologies appealing alternatives to conventional culture-based testing methods.


Subject(s)
Bacteria/isolation & purification , Blood Platelets/microbiology , Flow Cytometry/methods , Real-Time Polymerase Chain Reaction/methods , Bacteria/genetics , Humans , Nucleic Acids/isolation & purification , Specimen Handling/methods
3.
J Clin Microbiol ; 48(10): 3475-81, 2010 Oct.
Article in English | MEDLINE | ID: mdl-20702673

ABSTRACT

Bacterial contamination of platelet concentrates (PCs) still represents an ongoing risk in transfusion-transmitted sepsis. Recently the Pan Genera Detection (PGD) system was developed and FDA licensed for screening of bacterial contamination of PCs directly prior to transfusion. The test principle is based on the immunological detection of lipopolysaccharide (for Gram-negative bacteria) or lipoteichoic acid (for Gram-positive bacteria). In the present study we analyzed the applicability of this method with regard to detection limit, practicability, implementation, and performance. PCs were spiked with Staphylococcus aureus, Bacillus subtilis, and five different Klebsiella pneumoniae strains, as well as eight different Escherichia coli strains. The presence of bacteria was assessed by the PGD immunoassay, and bacteria were enumerated by plating cultures. Application of the PGD immunoassay showed that it is a rapid test with a short hands-on time for sample processing and no demand for special technical equipment and instrument operation. The lower detection limits of the assay for Gram-positive bacteria showed a good agreement with the manufacturer's specifications (8.2 × 10(3) to 5.5 × 10(4) CFU/ml). For some strains of K. pneumoniae and E. coli, the PGD test showed analytical sensitivities (>10(6) CFU/ml) that were divergent from the designated values (K. pneumoniae, 2.0 × 10(4) CFU/ml; E. coli, 2.8 × 10(4) CFU/ml). Result interpretation is sometimes difficult due to very faint bands. In conclusion, our study demonstrates that the PGD immunoassay is an easy-to-perform bedside test for the detection of bacterial contamination in PCs. However, to date there are some shortcomings in the interpretation of results and in the detection limits for some strains of Gram-negative bacteria.


Subject(s)
Bacteria/classification , Bacteria/isolation & purification , Bacteriological Techniques/methods , Biological Products , Blood Platelets/microbiology , Quality Assurance, Health Care/methods , Humans , Immunoassay/methods , Lipopolysaccharides/analysis , Lipopolysaccharides/immunology , Point-of-Care Systems , Sensitivity and Specificity , Teichoic Acids/analysis , Teichoic Acids/immunology , Time Factors
4.
Clin Chim Acta ; 411(19-20): 1487-90, 2010 Oct 09.
Article in English | MEDLINE | ID: mdl-20541540

ABSTRACT

BACKGROUND: Pseudoxanthoma elasticum (PXE) is a rare hereditary disorder predominantly affecting the skin, the eyes and the cardiovascular system. The disease is caused by mutations in the ABCC6 gene and characterized by ectopic calcification and extracellular matrix (ECM) alterations. Matrix metalloproteinases (MMPs) play a pivotal role in the process of ECM remodeling and are likely implied in PXE pathology. The aim of the present study was to investigate the association of single nucleotide polymorphisms (SNPs) in the promoter of the MMP2 gene, and PXE. METHODS: We evaluated the allelic distribution of five SNPs in the MMP2 promoter in DNA samples from 168 German patients affected by PXE and in 168 healthy, age- and sex-matched control subjects using restriction fragment length polymorphism analysis. RESULTS: The alleles c.-1575G, c.-1306C, and c.-790T were more abundant in the PXE patients' group. Furthermore, the haplotype GCTCG was significantly associated with PXE (OR 1.56, 95% CI 1.14-2.12, P(corrected)=0.026). CONCLUSIONS: Our results may indicate an involvement of MMP2 in the pathology of PXE. The promoter polymorphisms associated with PXE may lead to increased MMP2 expression and thereby contribute to the elevated proteolytic activity observed in PXE in vitro and in vivo.


Subject(s)
Matrix Metalloproteinase 2/genetics , Polymorphism, Single Nucleotide , Promoter Regions, Genetic/genetics , Pseudoxanthoma Elasticum/genetics , Adult , Aged , Alleles , Calcinosis , Case-Control Studies , Extracellular Matrix Proteins , Female , Germany , Haplotypes , Humans , Male , Middle Aged , Multidrug Resistance-Associated Proteins/genetics , Pseudoxanthoma Elasticum/pathology
5.
Glycoconj J ; 27(3): 359-66, 2010 Apr.
Article in English | MEDLINE | ID: mdl-20198421

ABSTRACT

Xylosylation of core proteins takes place in the Golgi-apparatus as the transfer of xylose from UDP-xylose to specific serine residues in proteoglycan core proteins. This initial and rate-limiting step in glycosaminoglycan biosynthesis is catalyzed by human xylosyltransferase I (XT-I). XT-I is proteolytically cleaved from the Golgi surface and shed in its active form into the extracellular space. The secreted, circulating glycosyltransferase represents a serum biomarker for various diseases with an altered proteoglycan metabolism, whereas a physiological function of secreted XT-I is still unknown. To shed light on the secretion process of XT-I and on its biological function, the cleavage site was examined and the group of proteases involved in the cleavage was identified in this study. The peptide mass fingerprint from partly purified secreted XT-I revealed the cleavage site to be localized in the aminoterminal 231 amino acids. The addition of a cysteine protease inhibitor cocktail to cells recombinantly expressing XT-I led to a concentration-dependent shift of enzyme activity towards the cell lysates attended by consistent total intracellular and extracellular XT-I activities. In conclusion, our findings provide a first insight into the XT-I secretion process regulated by a cysteine protease and may contribute to understanding the biological and pathological role of this process.


Subject(s)
Cysteine Proteases/metabolism , Pentosyltransferases/metabolism , Amino Acid Sequence , Animals , CHO Cells , Cricetinae , Cricetulus , Cysteine Proteases/genetics , Cysteine Proteinase Inhibitors/pharmacology , Enzyme Activation/drug effects , Extracellular Matrix/enzymology , Extracellular Matrix/metabolism , Glycosaminoglycans/metabolism , Humans , Molecular Sequence Data , Mutagenesis, Site-Directed , Pentosyltransferases/antagonists & inhibitors , Pentosyltransferases/genetics , Proteoglycans/metabolism , Sequence Homology, Amino Acid , UDP Xylose-Protein Xylosyltransferase
6.
BMC Microbiol ; 10: 78, 2010 Mar 16.
Article in English | MEDLINE | ID: mdl-20233397

ABSTRACT

BACKGROUND: Streptococcus gallolyticus subsp. gallolyticus is an important causative agent of infective endocarditis (IE) but the knowledge on virulence factors is limited and the pathogenesis of the infection is poorly understood. In the present study, we established an experimental in vitro IE cell culture model using EA.hy926 and HUVEC cells to investigate the adhesion and invasion characteristics of 23 Streptococcus gallolyticus subsp. gallolyticus strains from different origins (human IE-derived isolates, other human clinical isolates, animal isolates). Adhesion to eight components of the extracellular matrix (ECM) and the ability to form biofilms in vitro was examined in order to reveal features of S. gallolyticus subsp. gallolyticus endothelial infection. In addition, the strains were analyzed for the presence of the three virulence factors gtf, pilB, and fimB by PCR. RESULTS: The adherence to and invasion characteristics of the examined S. gallolyticus subsp. gallolyticus strains to the endothelial cell line EA.hy926 differ significantly among themselves. In contrast, the usage of three different in vitro models (EA.hy926 cells, primary endothelial cells (HUVECs), mechanical stretched cells) revealed no differences regarding the adherence to and invasion characteristics of different strains. Adherence to the ECM proteins collagen I, II and IV revealed the highest values, followed by fibrinogen, tenascin and laminin. Moreover, a strong correlation was observed in binding to these proteins by the analyzed strains. All strains show the capability to adhere to polystyrole surfaces and form biofilms. We further confirmed the presence of the genes of two known virulence factors (fimB: all strains, gtf: 19 of 23 strains) and demonstrated the presence of the gene of one new putative virulence factor (pilB: 9 of 23 strains) by PCR. CONCLUSION: Our study provides the first description of S. gallolyticus subsp. gallolyticus adhesion and invasion of human endothelial cells, revealing important initial information of strain variability, behaviour and characteristics of this as yet barely analyzed pathogen.


Subject(s)
Endocarditis, Bacterial/microbiology , Endothelial Cells/microbiology , Streptococcal Infections/microbiology , Streptococcus/physiology , Analysis of Variance , Bacterial Adhesion/physiology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Biofilms/growth & development , Cell Line, Transformed , Colony Count, Microbial , Extracellular Matrix Proteins/genetics , Extracellular Matrix Proteins/metabolism , Host-Pathogen Interactions/physiology , Humans , Statistics, Nonparametric , Streptococcus/genetics , Streptococcus/pathogenicity , Virulence
7.
Talanta ; 80(5): 1894-8, 2010 Mar 15.
Article in English | MEDLINE | ID: mdl-20152429

ABSTRACT

Mycophenolic acid (MPA) is an immunosuppressant drug which powerfully inhibits lymphocyte proliferation. Since the early 1990s it has been used to prevent rejection in organ transplantation. The requirement of therapeutic drug monitoring shown in previous studies raises the necessity of acquiring accurate and sensitive methods to measure MPA and its major metabolite mycophenolic acid glucuronide (MPAG). The authors developed a sample cleanup-free, rapid, and highly specific method for simultaneous measurement of MPA and MPAG in human plasma and serum using the novel technology of ultra-performance liquid chromatography-electrospray ionization tandem mass spectrometry. MPA- and MPAG-determinations were performed during a 2.0-min run time. Multiple calibration curves for the analysis of MPA and MPAG exhibited consistent linearity and reproducibility in the range of 0.05-100 (r>0.999) mg L(-1) and 4-4000 mg L(-1) (r>0.999), respectively. Limits of Detection were 0.014 mg L(-1) for MPA and 1.85 mg L(-1) for MPAG. Lower Limits of Quantification were 0.05 mg L(-1) for MPA and 2.30 mg L(-1) for MPAG. Interassay imprecision was <10% for both substances. Mean recovery was 103.6% (range 78.1-129.7%) for MPA and 111.1% (range 73.0-139.6%) for MPAG. Agreement was good for MPA and MPAG between the presented method and a validated HPLC-MS/MS method. The Passing-Bablok regression line for MPA and MPAG was HPLC-MS/MS=1.14 UPLC-MS/MS-0.14 [ mg L(-1)], r=0.96, and HPLC-MS/MS=0.77 UPLC-MS/MS+0.50 [ mg L(-1)], r=0.97, respectively. This sample cleanup-free and robust LC-MS/MS assay facilitates the rapid, accurate and simultaneous determination of MPA and MPAG in human body fluids.


Subject(s)
Chromatography, High Pressure Liquid/methods , Glucuronides/blood , Mycophenolic Acid/analogs & derivatives , Mycophenolic Acid/blood , Spectrometry, Mass, Electrospray Ionization/methods , Calibration , Chromatography, High Pressure Liquid/economics , Humans , Limit of Detection , Spectrometry, Mass, Electrospray Ionization/economics
8.
Eur Heart J ; 31(9): 1105-13, 2010 May.
Article in English | MEDLINE | ID: mdl-20093256

ABSTRACT

AIMS: A new diagnostic strategy to improve the detection of pathogens in heart valves (HVs) from patients with infective endocarditis (IE) was evaluated. METHODS AND RESULTS: Three hundred and fifty seven HVs surgically removed from 326 patients with proven IE or suspicious intra-operative findings, examined by 16S rDNA polymerase chain reaction (PCR) and culture were retrospectively analysed according to the predictive value of various PCR methods. Patients were classified into four categories: active IE, IE with ambiguous infective status, healed IE, and valve diseases but no IE. Retained samples of 200 HVs were analysed by real-time PCR targeting bacterial 23S rDNA, fungal 28S rDNA, and mycoplasmal tuf gene. 16S rDNA PCR revealed 80.6% sensitivity, 100% specificity, 100% positive predictive value, and 71% negative predictive value (NPV), compared with cultivation with 33.4, 96.6, 95.5, and 40.9%, respectively. The use of real-time PCR increased diagnostic sensitivity to 96.4%, and NPV to 92.5%. Bacterial load, C-reactive protein, and white blood cell counts (WBCs) decreased during antibiotic treatment. Bacterial load showed no correlation to C-reactive protein or WBCs, whereas C-reactive protein and WBCs were significantly correlated. CONCLUSION: 23S rDNA real-time PCR of surgically removed HVs improves the diagnosis of IE. Polymerase chain reaction analysis of explanted HVs allow the optimization of the antimicrobial therapy, especially in patients with culture-negative IE.


Subject(s)
DNA, Ribosomal/metabolism , Endocarditis/diagnosis , Heart Valve Diseases/diagnosis , Polymerase Chain Reaction/methods , Adolescent , Adult , Aged , Biomarkers/metabolism , Female , Heart Valves/metabolism , Humans , Male , Middle Aged , Young Adult
9.
Biochem Biophys Res Commun ; 391(1): 685-91, 2010 Jan 01.
Article in English | MEDLINE | ID: mdl-19944077

ABSTRACT

The xylosyltransferase (XT) isoforms XT-I and XT-II initiate the posttranslational glycosaminoglycan (GAG) synthesis. Here, we determined the relative expression of both isoforms in 33 human cell lines. The majority of tested cell lines showed dominant XYLT2 gene expression, while only in 23132/87, JAR, NCI-H510A and THP-1 was the XT-I mRNA expression higher. Nearly equal expression levels were detected in six cell lines. Additionally, to shed light on putative differences in acceptor specificities the acceptor properties of potential acceptor sequences were determined. Peptides were expressed as glutathione-S-transferase fusion proteins containing putative or known GAG attachment sites of in vivo proteoglycans. Kinetic analysis showed that K(m) and V(max) values for XT-I mediated xylosylation were slightly higher than those for XT-II, and that XT-I showed a lesser stringency concerning the acceptor sequence. Mutagenesis of the bikunin peptide sequence in the G-S-G attachment site and flanking regions generated potential acceptor molecules. Here, mutations on the N-terminal side and the attachment site were found to be more susceptible to a loss of acceptor function than mutations in the C-terminus. Altogether the known consensus sequence a-a-a-a-G-S-G-a-a/G-a ('a' representing Asp or Glu) for XT-I mediated xylosylation could be approved and additionally extended to apply to XT-II as well.


Subject(s)
Gene Expression Regulation, Enzymologic , Pentosyltransferases/chemistry , Pentosyltransferases/genetics , Proteoglycans/chemistry , Alpha-Globulins/chemistry , Alpha-Globulins/genetics , Amino Acid Motifs , Amino Acid Sequence , Cell Line , Glutathione Transferase/chemistry , Glutathione Transferase/genetics , Glycosaminoglycans/chemistry , Glycosaminoglycans/genetics , Humans , Isoenzymes/genetics , Molecular Sequence Data , Mutagenesis , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Xylose/chemistry , UDP Xylose-Protein Xylosyltransferase
10.
J Pharm Biomed Anal ; 51(1): 210-6, 2010 Jan 05.
Article in English | MEDLINE | ID: mdl-19720490

ABSTRACT

A stable isotope dilution liquid chromatography-electrospray ionization tandem mass spectrometry (LC-MS/MS) assay to measure amiodarone, the most frequently used agent for maintaining sinus rhythm in patients with atrial fibrillation, and its major metabolite desethylamiodarone in human plasma and serum was developed. Measurement of amiodarone and desethylamiodarone was performed during a 4.0-min run-time using amiodarone-D(4) and desethylamiodarone-D(4) as internal standards. Calibration curves covering 12 calibrators measured in four replicates each for the analysis of both amiodarone and desethylamiodarone were linear and reproducible in the range of 0.01-40.0 mg/L (r>0.999). Limits of detection in plasma matrix were 2.7 microg/L for amiodarone and 1.9 microg/L for desethylamiodarone, and lower limits of quantification in plasma matrix were 7.5 microg/L for amiodarone and 2.5 microg/L for desethylamiodarone. Interassay imprecision and inaccuracy were <8% and <9% for both substances. Mean extraction yield was 99.6% (range 92.6-107.7%) for amiodarone and 90.2% (range 80.0-94.7%) for desethylamiodarone. Agreement was moderate for amiodarone (n=162) and desethylamiodarone (n=117), respectively, between the present method and a HPLC method with UV detection using a commercially available reagent set for the HPLC analysis of these drugs. The Passing-Bablok regression line was HPLC=0.98 (LC-MS/MS)+0.10 [mg/L]; r=0.94 for amiodarone and HPLC=1.05 (LC-MS/MS)+0.02 [mg/L]; r=0.90 for desethylamiodarone. This sensitive and interference-free LC-MS/MS assay permits rapid and accurate determination of amiodarone and desethylamiodarone in human plasma and other body fluids.


Subject(s)
Amiodarone/analogs & derivatives , Chromatography, Liquid/methods , Spectrometry, Mass, Electrospray Ionization/methods , Tandem Mass Spectrometry/methods , Amiodarone/blood , Anti-Arrhythmia Agents/blood , Calibration , Chromatography, High Pressure Liquid/methods , Drug Monitoring/methods , Humans , Reproducibility of Results
11.
J Biol Chem ; 284(45): 30775-82, 2009 Nov 06.
Article in English | MEDLINE | ID: mdl-19762916

ABSTRACT

Human xylosyltransferase I catalyzes the initial and rate-limiting step in the biosynthesis of glycosaminoglycans and proteoglycans. Furthermore, this enzyme has been shown to play a major role in the physiological development of bone and cartilage as well as in pathophysiological processes such as systemic sclerosis, dilated cardiomyopathy, or fibrosis. Here, we report for the first time the identification and characterization of the XYLT1 gene promoter region and important transcription factors involved in its regulation. Members of the activator protein 1 (AP-1) and specificity protein 1 (Sp1) family of transcription factors are necessary for the transcriptional regulation of the XYLT1 gene, which was proven by curcumin, tanshinone IIA, mithramycin A, and short interference RNA treatment. A stepwise 5' and 3' deletion of the predicted GC-rich promoter region, which lacks a TATA and/or CAAT box, revealed that a 531-bp core promoter element is able to drive the transcription on a basal level. A binding site for transcription factors of the AP-1 family, which is essential for full promoter activity, was identified by site-directed mutagenesis located 730 bp 5' of the translation initiation site. The ability of this site to bind members of the AP-1 family was further verified by electrophoretic mobility shift assays. A promoter element containing this binding site was able to drive the transcription to about 79-fold above control in SW1353 chondrosarcoma cells. Our findings provide a first insight into the regulation of the XYLT1 gene and may contribute to understanding the processes taking place during extracellular matrix formation and remodeling in health and disease.


Subject(s)
Pentosyltransferases/genetics , Promoter Regions, Genetic , Base Sequence , Cell Line, Tumor , Gene Expression Regulation , Humans , Molecular Sequence Data , Pentosyltransferases/metabolism , Protein Binding , Sp1 Transcription Factor/genetics , Sp1 Transcription Factor/metabolism , Transcription Factor AP-1/genetics , Transcription Factor AP-1/metabolism , UDP Xylose-Protein Xylosyltransferase
12.
Clin Chim Acta ; 409(1-2): 123-6, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19755118

ABSTRACT

BACKGROUND: We investigated the xylosyltransferase (XT) activity in the serum of liver fibrotic patients with hepatitis C virus induced liver fibrosis at different stages as determined according to the scoring system of Desmet and Scheuer. METHODS: Measurement of XT activity was performed by liquid chromatography-tandem mass spectrometry. RESULTS: We found that serum XT activity in males (n=59, median+/-SD, 27.2+/-2.8 mU/L, p<0.001) and females (n=54, 23.6+/-3.0 mU/L, p<0.01) with liver fibrosis is significantly elevated in comparison to a corresponding healthy control cohort of males (n=50, 23.9+/-2.8 mU/L) and females (n=52, 21.5+/-3.7 mU/L), respectively. Of note, independent from gender, serum XT activity positively correlated with the stage of fibrosis but declined again in patients with histologically proven cirrhosis. CONCLUSIONS: XT activity is increased in the serum of patients with liver fibrosis at different stages, pointing to a possible pathogenetic role in elevated proteoglycan biosynthesis in fibrotic remodeling of this organ during chronic injury.


Subject(s)
Liver Cirrhosis/blood , Liver Cirrhosis/enzymology , Pentosyltransferases/blood , Pentosyltransferases/metabolism , Adolescent , Adult , Aged , Aged, 80 and over , Case-Control Studies , Female , Hepatitis C/complications , Homeostasis , Humans , Liver Cirrhosis/etiology , Liver Cirrhosis/metabolism , Male , Middle Aged , Proteoglycans/metabolism , Young Adult , UDP Xylose-Protein Xylosyltransferase
13.
J Mol Med (Berl) ; 87(10): 965-70, 2009 Oct.
Article in English | MEDLINE | ID: mdl-19575173

ABSTRACT

Pseudoxanthoma elasticum (PXE) is a rare disorder predominantly affecting the skin, the eyes, and the cardiovascular system. The disease is caused by mutations in the ABCC6 gene and characterized by ectopic calcification and extracellular matrix (ECM) alterations. Matrix metalloproteinases (MMPs) play a pivotal role in the process of ECM remodeling. In the present study, we investigated matrix metalloproteinases MMP-2 and MMP-9 in PXE patients compared to healthy controls. We analyzed the serum concentrations of MMP-2 and MMP-9 in a cohort of 69 German PXE patients and in 69 healthy, age-, and sex-matched control subjects using commercially available ELISA assays. We found elevated concentrations of both MMPs in the sera of PXE patients. MMP-2 levels were significantly higher in patients than controls (231 +/- 5.89 vs 202 +/- 5.17 ng/ml, p = 0.0002), as were MMP-9 levels (841 +/- 65.9 vs 350 +/- 30.8 ng/ml, p < 0.0001). Our findings point to an involvement of matrix metalloproteinases in PXE pathology. ECM remodeling in PXE is reflected by elevated levels of circulating MMP-2 and MMP-9. Those MMPs might, therefore, be applicable as serum markers for the matrix-degradative process in PXE.


Subject(s)
Matrix Metalloproteinase 2/blood , Matrix Metalloproteinase 9/blood , Pseudoxanthoma Elasticum/blood , Pseudoxanthoma Elasticum/enzymology , Adult , Animals , Biomarkers/blood , Extracellular Matrix/metabolism , Female , Humans , Male , Middle Aged , Multidrug Resistance-Associated Proteins/genetics , Multidrug Resistance-Associated Proteins/metabolism , Pseudoxanthoma Elasticum/genetics , Pseudoxanthoma Elasticum/pathology
14.
Cardiovasc Diabetol ; 8: 36, 2009 Jul 18.
Article in English | MEDLINE | ID: mdl-19615091

ABSTRACT

BACKGROUND: We investigated whether macronutrient composition of energy-restricted diets influences the efficacy of a telemedically guided weight loss program. METHODS: Two hundred overweight subjects were randomly assigned to a conventional low-fat diet and a low-carbohydrate diet group (target carbohydrate content: >55% energy and <40% energy, respectively). Both groups attended a weekly nutrition education program and dietary counselling by telephone, and had to transfer actual body weight data to our clinic weekly with added Bluetooth technology by mobile phone. Various fatness and fat distribution parameters, energy and macronutrient intake, and various biochemical risk markers were measured at baseline and after 6, and 12 months. RESULTS: In both groups, energy intake decreased by 400 kcal/d compared to baseline values within the first 6 months and slightly increased again within the second 6 months. Macronutrient composition differed significantly between the groups from the beginning to month 12. At study termination, weight loss was 5.8 kg (SD: 6.1 kg) in the low-carbohydrate group and 4.3 kg (SD: 5.1 kg) in the low-fat group (p = 0.065). In the low-carbohydrate group, triglyceride and HDL-cholesterol levels were lower at month 6 and waist circumference and systolic blood pressure were lower at month 12 compared with the low-fat group (P = 0.005-0.037). Other risk markers improved to a similar extent in both groups. CONCLUSION: Despite favourable effects of both diets on weight loss, the carbohydrate-reduced diet was more beneficial with respect to cardiovascular risk factors compared to the fat-reduced diet. Nevertheless, compliance with a weight loss program appears to be even a more important factor for success in prevention and treatment of obesity than the composition of the diet. TRIAL REGISTRATION: Clinicaltrials.gov as NCT00868387.


Subject(s)
Diet, Carbohydrate-Restricted/methods , Diet, Fat-Restricted/methods , Overweight/diet therapy , Telemedicine/methods , Weight Loss , Adult , Body Composition/physiology , Female , Humans , Male , Middle Aged , Obesity/diet therapy , Obesity/physiopathology , Overweight/physiopathology , Patient Education as Topic/methods , Weight Loss/physiology
15.
Clin Chem ; 55(8): 1492-502, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19498052

ABSTRACT

BACKGROUND: Bacterial contamination is the major infectious hazard associated with transfusion of platelet preparations (PLTs). Routine testing for bacterial contamination in PLTs has become common, but transfusion-transmitted bacterial sepsis has not been eliminated. Here, we describe a novel flow cytometry-based method for point-of-issue screening of PLTs for bacterial contamination. METHODS: We used the BactiFlow flow cytometer to detect and count bacteria based on esterase activity in viable cells. We compared the assay to incubation (BacT/Alert culture system) and rapid nucleic acid-based or immunoassay (reverse transcription PCR, Pan Genera Detection) methods. RESULTS: We established a protocol for bacterial screening of PLTs consisting of enzymatic digestion and centrifugal filtration for the elimination of viable platelets and selective labeling of bacteria with fluorescent esterase substrate (ChemChrome V23). Results from the BactiFlow showed an excellent correlation (r = 0.9923 E. coli, r = 0.9736 S. epidermidis) to traditional plate count results. The lower detection limit of the assay was determined to be 150 CFU/mL, and the time to result was <1 h. CONCLUSIONS: Our study demonstrates that BactiFlow flow cytometry is suitable for rapid screening of PLTs for bacterial contamination and fulfils the requirements for a point-of-issue testing of PLTs with acceptable time to result, specificity, sensitivity, and cost.


Subject(s)
Bacteria/isolation & purification , Blood Platelets/microbiology , Colony Count, Microbial/methods , Flow Cytometry/methods , Time Factors , Bacteria/growth & development , Colony Count, Microbial/economics , Flow Cytometry/economics , Humans , Sensitivity and Specificity
16.
Hum Mol Genet ; 18(17): 3344-51, 2009 Sep 01.
Article in English | MEDLINE | ID: mdl-19483196

ABSTRACT

Pseudoxanthoma elasticum (PXE) is a heritable disorder affecting the skin, eyes and cardiovascular system. It is caused by mutations in the ABCC6 gene and its clinical picture is highly variable. PXE often leads to severe visual impairment due to the development of choroidal neovascularisation (CNV). CNV in PXE-associated retinopathy is believed to be mediated by the action of vascular endothelial growth factor (VEGF). The objective of the present study was to evaluate a possible impact of variations in the VEGFA gene on ocular manifestations of PXE. For this purpose, we evaluated the distribution of 10 single nucleotide polymorphisms (SNPs) in the promoter and coding region of the VEGFA gene in DNA samples from 163 German patients affected by PXE and in 163 healthy control subjects. Haplotype analysis of SNPs c.-1540A>C, c.-460C>T, c.-152G>A, c.405C>G, c.674C>T, c.1032C>T, c.4618C>T and c.5092C>A revealed that the haplotype CTGGCCCC was associated with PXE (OR 2.05, 95% CI 1.33-3.15, P(corrected) = 0.01). Furthermore, five SNPs showed significant association with severe retinopathy. The most significant single SNP association was c.-460C>T (OR 3.83, 95% CI 2.01-7.31, P(corrected) = 0.0003). Logistic regression analysis identified the c.-460T and the c.674C alleles as independent risk factors for development of severe retinopathy. Our findings suggest an involvement of VEGF in the pathogenesis of ocular PXE manifestations. VEGF gene polymorphisms might prove useful as prognostic markers for the development of PXE-associated retinopathy and permit earlier therapeutic intervention in order to prevent loss of central vision, one of the most devastating consequences of this disease.


Subject(s)
Eye Diseases, Hereditary/genetics , Polymorphism, Single Nucleotide , Pseudoxanthoma Elasticum/genetics , Vascular Endothelial Growth Factor A/genetics , Adult , Aged , Aged, 80 and over , Case-Control Studies , Eye Diseases, Hereditary/diagnosis , Female , Genotype , Germany , Humans , Male , Middle Aged , Prognosis , Promoter Regions, Genetic , Pseudoxanthoma Elasticum/diagnosis , Young Adult
17.
Clin Biochem ; 42(13-14): 1413-9, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19560454

ABSTRACT

OBJECTIVES: Lipopolysaccharide-binding protein (LBP) was recently demonstrated to be a supportive biomarker for the diagnostic and therapeutic monitoring of infective endocarditis (IE) with a considerable variability in the individual LBP response of IE patients. DESIGN AND METHODS: LBP was measured by chemiluminescence assay in sera from 78 IE patients. Moreover, three new LightCycler PCR assays have been developed for sequence variation analysis and the distribution of the five LBP polymorphisms c.-1978C>T, c.-836T>C, c.291C>T, c.613A>G and c.1306C>T was determined in IE patients and healthy blood donors. RESULTS: A weak association with IE was determined for the two single nucleotide polymorphisms c.291C>T and c.613A>G; the frequencies of the other polymorphisms did not differ significantly between IE patients and controls. Elevated serum LBP concentrations of infective patients did not correlate with genotypes. CONCLUSIONS: The association of the polymorphisms c.291C>T and c.613A>G suggest a role of LBP in the disease manifestation of IE.


Subject(s)
Acute-Phase Proteins/genetics , Carrier Proteins/genetics , Endocarditis, Bacterial/genetics , Membrane Glycoproteins/genetics , Polymorphism, Single Nucleotide , Staphylococcal Infections/genetics , Adult , Aged , Aged, 80 and over , Carrier Proteins/blood , Chi-Square Distribution , DNA Mutational Analysis/methods , Endocarditis, Bacterial/blood , Female , Gene Frequency , Genotype , Gram-Positive Bacterial Infections/blood , Gram-Positive Bacterial Infections/genetics , Humans , Luminescent Measurements/methods , Male , Membrane Glycoproteins/blood , Middle Aged , Odds Ratio , Polymerase Chain Reaction , Staphylococcal Infections/blood , Young Adult
18.
Cell Biol Int ; 33(8): 848-52, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19427387

ABSTRACT

Changes in proteoglycan and glycosaminoglycan (GAG) content and distribution may play an important role in the development of many diseases, atherosclerosis, cancer and diabetes. Human cell lines act as models for the underlying pathomechanisms. Despite the importance of proteoglycans for cell functioning, information on the GAG composition of most human cell lines is limited. Comparative analysis of the GAG Deltadisaccharide amount in 22 human cell lines yielded a mean value of 94 +/- 58 pmol/10(6) cells (mean+/-SEM). Total GAG amount and heparan sulfate/heparin Deltadisaccharide composition, but not chondroitin sulfate/dermatan sulfate Deltadisaccharide composition, differed significantly between the investigated adherent and suspension cell lines. We provide a novel overview of GAG Deltadisaccharide composition in 22 different human cell lines.


Subject(s)
Disaccharides/analysis , Glycosaminoglycans/chemistry , Cell Line , Chromatography, High Pressure Liquid , Glycosaminoglycans/analysis , Humans
19.
Clin Chem ; 55(6): 1163-70, 2009 Jun.
Article in English | MEDLINE | ID: mdl-19359534

ABSTRACT

BACKGROUND: Evidence is accumulating that vitamin D supplementation of patients with low 25-hydroxyvitamin D concentrations is associated with lower cardiovascular morbidity and total mortality during long-term follow-up. Little is known, however, about the effect of low concentrations of the vitamin D hormone calcitriol on total mortality. We therefore evaluated the predictive value of circulating calcitriol for midterm mortality in patients of a specialized heart center. METHODS: This prospective cohort study included 510 patients, 67.7% with heart failure (two-thirds in end stage), 64.3% hypertension, 33.7% coronary heart disease, 20.2% diabetes, and 17.3% renal failure. We followed the patients for up to 1 year after blood collection. For data analysis, the study cohort was stratified into quintiles of circulating calcitriol concentrations. RESULTS: Patients in the lowest calcitriol quintile were more likely to have coronary heart disease, heart failure, hypertension, diabetes, and renal failure compared to other patients. They also had low 25-hydroxyvitamin D concentrations and high concentrations of creatinine, C-reactive protein, and tumor necrosis factor alpha. Eighty-two patients (16.0%) died during follow-up. Probability of 1-year survival was 66.7% in the lowest calcitriol quintile, 82.2% in the second quintile, 86.7% in the intermediate quintile, 88.8% in the fourth quintile, and 96.1% in the highest quintile (P < 0.001). Discrimination between survivors and nonsurvivors was best when a cutoff value of 25 ng/L was applied (area under the ROC curve 0.72; 95% CI 0.66-0.78). CONCLUSIONS: Decreased calcitriol levels are linked to excess midterm mortality in patients of a specialized heart center.


Subject(s)
Calcitriol/blood , Mortality , Aged , Cohort Studies , Enzyme-Linked Immunosorbent Assay , Female , Humans , Male , Middle Aged
20.
Mol Cell Biochem ; 328(1-2): 85-92, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19266166

ABSTRACT

Chemotherapy failure was reported in treatment of retinoblastoma suggesting a role for ATP-binding cassette (ABC) proteins. Little is known about the expression pattern of ABC proteins in this cancer type. We investigated the gene expression profile of 47 ABC proteins in the human retinoblastoma cell line Y79 by TaqMan low-density array. Analysis revealed 31 ABC transporter genes expressed in this tumor cell line. Y79 cells demonstrate high gene expression of ABCA7, ABCA12, ABCB7, ABCB10, ABCC1, ABCC4, ABCD3, ABCE1, ABCF1, ABCF2, and ABCF3 (more than twofold compared to pooled RNA from different tissues). Moreover, we show that Y79 cells exhibit an active calcein efflux pointing to multidrug resistance protein (MRP)-like transporter activity. In summary, we present for the first time an ABC transporter gene expression profile in cells derived from retinoblastoma. Most of the highly expressed ABC transporter genes are typical markers of cancer cells and might exhibit potential targets for medical treatment of retinoblastoma.


Subject(s)
ATP-Binding Cassette Transporters/genetics , Gene Expression Regulation, Neoplastic , Retinoblastoma/metabolism , Cell Line, Tumor , Fluoresceins/metabolism , Gene Expression Profiling , Humans , Neoplasm Proteins/genetics , Retinoblastoma/pathology
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