Spironolactone prevents alterations associated with cardiac hypertrophy produced by isoproterenol in rats: involvement of serum- and glucocorticoid-regulated kinase type 1

Persistent beta-adrenergic receptor stimulation with isoproterenol is associated with cardiac hypertrophy as well as cardiac synthesis of angiotensin II. Serum- and glucocorticoid-regulated kinase type 1 (SGK-1) is a key mediator in structural, functional and molecular cardiac effects of aldosterone in rats. This study was designed to investigate the cardiac effects of the mineralocorticoid receptor antagonist spironolactone on the response to isoproterenol treatment in rats, as well as the involvement of the main mediator of cellular aldosterone action, SGK-1, in the heart. Male Wistar rats received isoproterenol (3 mg kg-1 day-1) or vehicle for 15 days. Half of the animals in each group were simultaneously treated with spironolactone (200 mg kg-1 day-1). Systolic and diastolic blood pressures were not significantly different among groups. Treatment with spironolactone normalized the increased left ventricular end-diastolic pressure observed in isoproterenol-treated rats. Isoproterenol treatment induced cardiac hypertrophy and increased collagen content, both of which were normalized by spironolactone treatment. The mRNA levels of transforming growth factor beta, connective tissue growth factor, matrix metalloprotease 2, matrix metalloprotease inhibitor 2, tumour necrosis factor a, interleukin 1 beta, p22phox and xanthine dehydrogenase were increased (P < 0.05) in isoproterenol-treated rats, and this effect was prevented by spironolactone (P < 0.05). Spironolactone also reduced the elevated SGK-1 expression in isoproterenol-treated rats. The observed reduction of the principal mediator of aldosterone cellular actions, SGK-1, by spironolactone in hearts from isoproterenol-treated rats suggests a role of mineralocorticoids in the cardiac hypertrophy, fibrosis, inflammation, oxidation and diastolic dysfunction induced by isoproterenol treatment in rats.

Coffee and Caffeine Protect against Liver Injury Induced by Thioacetamide in Male Wistar Rats

Coffee intake has been inversely related to the incidence of liver diseases, although there are controversies on whether these beneficial effects on human health are because of caffeine or other specific components in this popular beverage. Thus, this study evaluated the protective effects of coffee or caffeine intake on liver injury induced by repeated thioacetamide (TAA) administration in male Wistar rats. Rats were randomized into five groups: one untreated group (G1) and four groups (G2G5) treated with the hepatotoxicant TAA (200 similar to mg/kg b.w., i.p.) twice a week for 8 similar to weeks. Concomitantly, rats received tap water (G1 and G2), conventional coffee (G3), decaffeinated coffee (G4) or 0.1% caffeine (G5). After 8 similar to weeks of treatment, rats were killed and blood and liver samples were collected. Conventional and decaffeinated coffee and caffeine intake significantly reduced serum levels of alanine aminotransferase (ALT) (p similar to<similar to 0.001) and oxidized glutathione (p similar to<similar to 0.05), fibrosis/inflammation scores (p similar to<similar to 0.001), collagen volume fraction (p similar to<similar to 0.01) and transforming growth factor beta-1 (TGF-beta 1) protein expression (p similar to=similar to 0.001) in the liver from TAA-treated groups. In addition, conventional coffee and caffeine intake significantly reduced proliferating cellular nuclear antigen (PCNA) S-phase indexes (p similar to<similar to 0.001), but only conventional coffee reduced cleaved caspase-3 indexes (p similar to<similar to 0.001), active metalloproteinase 2 (p similar to=similar to 0.004) and the number of glutathione S-transferase placental form (GST-P)-positive preneoplastic lesions (p similar to<similar to 0.05) in the liver from TAA-treated groups. In conclusion, conventional coffee and 0.1% caffeine intake presented better beneficial effects than decaffeinated coffee against liver injury induced by TAA in male Wistar rats.

Esempio di NAM 2005 (National Accounting Matrix)

Modulo 2. NAM SEC riempita don i dati dei conti nazionali

Valutazione dell'attività delle metalloproteinasi 2 e 9 nelle urine e nel tessuto renale di cani normali e affetti da nefropatia

Matrix metalloproteinases (MMP) are a large family of proteinases that remodel extracellular matrix (ECM) component. Recent data suggest a role for MMPs in a number of renal pathophysiologies, associated with an imbalance of ECM syntesis and degradation, which may result in an accumulation of ECM molecules and renal fibrosis. The aim of this study is to elucidate the role of pro and activated MMP-2 and 9 in urine and renal tissue of healty and nephropatic dogs. Renal tissue of 8 healty dogs and either renal tissue and urine of 9 nephropatic dogs was collected and analize using zimographic method, which is been validated in this study. Either MMPs zimographic bands were present in almost all samples. In particular, pro and activated MMP-9 zimographic bands were poorly represent in renal tissue of healty dogs, whereas were very represent in nephropatic dogs. Pro and activated MMP-2 was present in either tissue of healty and nephropatic dogs. In urine of nephropatic dogs, pro and activated MMP-9 was more evident than MMP-2, but there was not correlaction with renal tissue levels, therefore urine levels of MMPs have poorly usefulness in diagnostic pratice. The values of Pro and activated MMP-9 in nephropatic dogs were significantly higher compared with normal dogs (p < 0,05), whereas there was not statistically meaningful for Pro and activated MMP-2. In conclusion, in this study we have validated a zimographic method for renal tissue of dogs and we have illustrated the changes in nephropatic dogs, which may be useful for further study.

Mass spectrometry-based protein profiling strategies for biomarker discovery in liver and inflammatory bowel diseases

The study of protein expression profiles for biomarker discovery in serum and in mammalian cell populations needs the continuous improvement and combination of proteins/peptides separation techniques, mass spectrometry, statistical and bioinformatic approaches. In this thesis work two different mass spectrometry-based protein profiling strategies have been developed and applied to liver and inflammatory bowel diseases (IBDs) for the discovery of new biomarkers. The first of them, based on bulk solid-phase extraction combined with matrix-assisted laser desorption/ionization - Time of Flight mass spectrometry (MALDI-TOF MS) and chemometric analysis of serum samples, was applied to the study of serum protein expression profiles both in IBDs (Crohn’s disease and ulcerative colitis) and in liver diseases (cirrhosis, hepatocellular carcinoma, viral hepatitis). The approach allowed the enrichment of serum proteins/peptides due to the high interaction surface between analytes and solid phase and the high recovery due to the elution step performed directly on the MALDI-target plate. Furthermore the use of chemometric algorithm for the selection of the variables with higher discriminant power permitted to evaluate patterns of 20-30 proteins involved in the differentiation and classification of serum samples from healthy donors and diseased patients. These proteins profiles permit to discriminate among the pathologies with an optimum classification and prediction abilities. In particular in the study of inflammatory bowel diseases, after the analysis using C18 of 129 serum samples from healthy donors and Crohn’s disease, ulcerative colitis and inflammatory controls patients, a 90.7% of classification ability and a 72.9% prediction ability were obtained. In the study of liver diseases (hepatocellular carcinoma, viral hepatitis and cirrhosis) a 80.6% of prediction ability was achieved using IDA-Cu(II) as extraction procedure. The identification of the selected proteins by MALDITOF/ TOF MS analysis or by their selective enrichment followed by enzymatic digestion and MS/MS analysis may give useful information in order to identify new biomarkers involved in the diseases. The second mass spectrometry-based protein profiling strategy developed was based on a label-free liquid chromatography electrospray ionization quadrupole - time of flight differential analysis approach (LC ESI-QTOF MS), combined with targeted MS/MS analysis of only identified differences. The strategy was used for biomarker discovery in IBDs, and in particular of Crohn’s disease. The enriched serum peptidome and the subcellular fractions of intestinal epithelial cells (IECs) from healthy donors and Crohn’s disease patients were analysed. The combining of the low molecular weight serum proteins enrichment step and the LCMS approach allowed to evaluate a pattern of peptides derived from specific exoprotease activity in the coagulation and complement activation pathways. Among these peptides, particularly interesting was the discovery of clusters of peptides from fibrinopeptide A, Apolipoprotein E and A4, and complement C3 and C4. Further studies need to be performed to evaluate the specificity of these clusters and validate the results, in order to develop a rapid serum diagnostic test. The analysis by label-free LC ESI-QTOF MS differential analysis of the subcellular fractions of IECs from Crohn’s disease patients and healthy donors permitted to find many proteins that could be involved in the inflammation process. Among them heat shock protein 70, tryptase alpha-1 precursor and proteins whose upregulation can be explained by the increased activity of IECs in Crohn’s disease were identified. Follow-up studies for the validation of the results and the in-depth investigation of the inflammation pathways involved in the disease will be performed. Both the developed mass spectrometry-based protein profiling strategies have been proved to be useful tools for the discovery of disease biomarkers that need to be validated in further studies.

Ruolo dell'Enantiomero (R)-9-HSA nel controllo della proliferazione in una linea di Adenocarcinoma del Colon umano

9-hydroxystearic acid (9-HSA) is an endogenous lipoperoxidation product and its administration to HT29, a colon adenocarcinoma cell line, induced a proliferative arrest in G0/G1 phase mediated by a direct activation of the p21WAF1 gene, bypassing p53. We have previously shown that 9-HSA controls cell growth and differentiation by inhibiting histone deacetylase 1 (HDAC1) activity, showing interesting features as a new anticancer drug. The interaction of 9-HSA with the catalytic site of the 3D model has been tested with a docking procedure: noticeably, when interacting with the site, the (R)-9-enantiomer is more stable than the (S) one. Thus, in this study, (R)- and (S)-9-HSA were synthesized and their biological activity tested in HT29 cells. At the concentration of 50 M (R)-9-HSA showed a stronger antiproliferative effect than the (S) isomer, as indicated by the growth arrest in G0/G1. The inhibitory effect of (S)-9-HSA on HDAC1, HDAC2 and HDAC3 activity was less effective than that of the (R)-9-HSA in vitro, and the inhibitory activity of both the (R)- and the (S)-9-HSA isomer, was higher on HDAC1 compared to HDAC2 and HDAC3, thus demonstrating the stereospecific and selective interaction of 9-HSA with HDAC1. In addition, histone hyperacetylation caused by 9-HSA treatment was examined by an innovative HPLC/ESI/MS method. Analysis on histones isolated from control and treated HT29 confirmed the higher potency of (R)-9-HSA compared to (S)-9-HSA, severely affecting H2A-2 and H4 acetylation. On the other side, it seemed of interest to determine whether the G0/G1 arrest of HT29 cell proliferation could be bypassed by the stimulation with the growth factor EGF. Our results showed that 9-HSA-treated cells were not only prevented from proliferating, but also showed a decreased [3H]thymidine incorporation after EGF stimulation. In this condition, HT29 cells expressed very low levels of cyclin D1, that didn’t colocalize with HDAC1. These results suggested that the cyclin D1/HDAC1 complex is required for proliferation. Furthermore, in the effort of understanding the possible mechanisms of this effect, we have analyzed the degree of internalization of the EGF/EGFR complex and its interactions with HDAC1. EGF/EGFR/HDAC1 complex quantitatively increases in 9-HSA-treated cells but not in serum starved cells after EGF stimulation. Our data suggested that 9-HSA interaction with the catalytic site of the HDAC1 disrupts the HDAC1/cyclin D1 complex and favors EGF/EGFR recruitment by HDAC1, thus enhancing 9-HSA antiproliferative effects. In conclusion 9-HSA is a promising HDAC inhibitor with high selectivity and specificity, capable of inducing cell cycle arrest and histone hyperacetylation, but also able to modulate HDAC1 protein interaction. All these aspects may contribute to the potency of this new antitumor agent.

Automatisierte Flüssigchromatographie mit Säulenschaltung und Ultraviolettspektroskopie oder Massenspektrometrie für therapeutisches Drug Monitoring von Antipsychotika und Antidepressiva

Therapeutisches Drug Monitoring (TDM) wird zur individuellen Dosiseinstellung genutzt, um die Effizienz der Medikamentenwirkung zu steigern und das Auftreten von Nebenwirkungen zu senken. Für das TDM von Antipsychotika und Antidepressiva besteht allerdings das Problem, dass es mehr als 50 Medikamente gibt. Ein TDM-Labor muss dementsprechend über 50 verschiedene Wirkstoffe und zusätzlich aktive Metaboliten messen. Mit der Flüssigchromatographie (LC oder HPLC) ist die Analyse vieler unterschiedlicher Medikamente möglich. LC mit Säulenschaltung erlaubt eine Automatisierung. Dabei wird Blutserum oder -plasma mit oder ohne vorherige Proteinfällung auf eine Vorsäule aufgetragen. Nach Auswaschen von störenden Matrixbestandteilen werden die Medikamente auf einer nachgeschalteten analytischen Säule getrennt und über Ultraviolettspektroskopie (UV) oder Massenspektrometrie (MS) detektiert. Ziel dieser Arbeit war es, LC-Methoden zu entwickeln, die die Messung möglichst vieler Antipsychotika und Antidepressiva erlaubt und die für die TDM-Routine geeignet ist. Eine mit C8-modifiziertem Kieselgel gefüllte Säule (20 µm 10x4.0 mm I.D.) erwies sich in Vorexperimenten als optimal geeignet bezüglich Extraktionsverhalten, Regenerierbarkeit und Stabilität. Mit einer ersten HPLC-UV-Methode mit Säulenschaltung konnten 20 verschiedene Psychopharmaka einschließlich ihrer Metabolite, also insgesamt 30 verschiedene Substanzen quantitativ erfasst werden. Die Analysenzeit betrug 30 Minuten. Die Vorsäule erlaubte 150 Injektionen, die analytische Säule konnte mit mehr als 300 Plasmainjektionen belastet werden. Abhängig vom Analyten, musste allerdings das Injektionsvolumen, die Flussrate oder die Detektionswellenlänge verändert werden. Die Methode war daher für eine Routineanwendung nur eingeschränkt geeignet. Mit einer zweiten HPLC-UV-Methode konnten 43 verschiedene Antipsychotika und Antidepressiva inklusive Metaboliten nachgewiesen werden. Nach Vorreinigung über C8-Material (10 µm, 10x4 mm I.D.) erfolgte die Trennung auf Hypersil ODS (5 µm Partikelgröße) in der analytischen Säule (250x4.6 mm I.D.) mit 37.5% Acetonitril im analytischen Eluenten. Die optimale Flussrate war 1.5 ml/min und die Detektionswellenlänge 254 nm. In einer Einzelprobe, konnten mit dieser Methode 7 bis 8 unterschiedliche Substanzen gemessen werden. Für die Antipsychotika Clozapin, Olanzapin, Perazin, Quetiapin und Ziprasidon wurde die Methode validiert. Der Variationskoeffizient (VK%) für die Impräzision lag zwischen 0.2 und 6.1%. Im erforderlichen Messbereich war die Methode linear (Korrelationskoeffizienten, R2 zwischen 0.9765 und 0.9816). Die absolute und analytische Wiederfindung lagen zwischen 98 und 118 %. Die für das TDM erforderlichen unteren Nachweisgrenzen wurden erreicht. Für Olanzapin betrug sie 5 ng/ml. Die Methode wurde an Patienten für das TDM getestet. Sie erwies sich für das TDM als sehr gut geeignet. Nach retrospektiver Auswertung von Patientendaten konnte erstmalig ein möglicher therapeutischer Bereich für Quetiapin (40-170 ng/ml) und Ziprasidon (40-130 ng/ml) formuliert werden. Mit einem Massenspektrometer als Detektor war die Messung von acht Neuroleptika und ihren Metaboliten möglich. 12 Substanzen konnten in einem Lauf bestimmt werden: Amisulprid, Clozapin, N-Desmethylclozapin, Clozapin-N-oxid, Haloperidol, Risperidon, 9-Hydroxyrisperidon, Olanzapin, Perazin, N-Desmethylperazin, Quetiapin und Ziprasidon. Nach Vorreinigung mit C8-Material (20 µm 10x4.0 mm I.D.) erfolgte die Trennung auf Synergi MAX-RP C12 (4 µm 150 x 4.6 mm). Die Validierung der HPLC-MS-Methode belegten einen linearen Zusammenhang zwischen Konzentration und Detektorsignal (R2= 0,9974 bis 0.9999). Die Impräzision lag zwischen 0.84 bis 9.78%. Die für das TDM erforderlichen unteren Nachweisgrenzen wurden erreicht. Es gab keine Hinweise auf das Auftreten von Ion Suppression durch Matrixbestandteile. Die absolute und analytische Wiederfindung lag zwischen 89 und 107 %. Es zeigte sich, dass die HPLC-MS-Methode ohne Modifikation erweitert werden kann und anscheinend mehr als 30 verschiedene Psychopharmaka erfasst werden können. Mit den entwickelten flüssigchromatographischen Methoden stehen neue Verfahren für das TDM von Antipsychotika und Antidepressiva zur Verfügung, die es erlauben, mit einer Methode verschiedene Psychopharmaka und ihre aktiven Metabolite zu messen. Damit kann die Behandlung psychiatrischer Patienten insbesondere mit Antipsychotika verbessert werden.

Proteolysis of the receptor for advanced glycation end products by matrix metalloproteinases

RAGE mediates diverse physiological and pathological effects by binding a variety of ligands. Despite incomplete understanding of RAGE-mediated disorders soluble RAGE (sRAGE) has been identified as a potential biomarker for RAGE-related diseases and possibly represents a hopeful pharmaceutical against RAGE-mediated disorders. Nevertheless, the source of sRAGE remains poorly investigated. Currently sRAGE is thought to be derived exclusively from alternative splicing of mRNA. In this thesis it was investigated whether sRAGE can also be released as a result of ectodomain shedding of full-length RAGE. Using cells overexpressing RAGE as a model system, it was demonstrated clearly that RAGE undergoes ectodomain shedding in both constitutive and regulated manner. Several stimuli including PMA, AMPA, calcium and chelerythrine stimulated the release of sRAGE into cell culture medium. Moreover, possible mechanisms that regulate ectodomain shedding of RAGE were investigated and it was found that shedding of RAGE is likely independent from PKC and MAPK pathways. By using gain of function and loss of function approaches MMP9 but not ADAM10, ADAM17 or MT1-MMP was characterized as the metalloproteinase that mediates shedding of RAGE. Furthermore, it was shown that cytoplasmic domain of RAGE is not essential for shedding of RAGE. In addition, the potential cleavage site of RAGE by MMP9 was investigated and a lack of sequence specificity for the RAGE processing proteinase was demonstrated by mutation analysis. Finally the physiopathological significance of shedding of RAGE is discussed. In conclusion, for the first time ectodomain shedding of human RAGE and the underlying regulatory mechanisms were investigated. The data open a new field for modulation of RAGE shedding as a novel intervention approach against RAGE-mediated diseases.

Serum anti-interferon alpha antibodies in chronic hepatitis C patients treated with pegylated interferon alpha containing therapy

The development of anti-IFNα antibodies is an occurrence described in chronic hepatitis C patients during treatment with Interferonα/PEG-Interferonα. However, its relevance, especially in difficult-to treat patients, has not been defined. Methods: We retrospectively measured the serum levels of anti-IFNα antibodies (baseline and week 12) and IFNα levels (week 12) by ELISA in 76 previous non-responders, and in 14 naive patients treated with Pegylated-IFNα and Ribavirin. A group of 57 healthy donors (HD) was also assessed as control. Positivity to anti-IFNα antibodies was established on the values of HD. Results: Baseline anti-IFNα antibodies were detected in 15.5% of patients and in 7% of HD, with significantly higher concentrations in patients than HD (181.5±389.9 vs 95.9±143.0 ng mL−1, p=0.0023). All positive patients were IFNα-experienced. At week 12, the prevalence of positivity increased to 22.3 and 28.5% in experienced and naïve patients, respectively, and the levels of anti-IFNα antibodies did not differ between the two groups (391±792.3 vs 384.7±662.6 ng mL−1, respectively). IFNα concentrations were significantly lower in antibody-positive patients than in antibody-negatives (988.2±1402 vs 3462±830.8 pg mL−1, p≤0.0001) and the levels of antibodies and IFNα were inversely correlated (r=-0.405, p=0.0001). The antibody-positive population clustered in null responders (67%) and 19/21 patients (90%) did not achieve SVR. Conclusions: The development of anti-IFNα antibodies is a non-negligible occurrence in patients treated with PEG-IFNα, is stable over time, and has a relevant clinical impact when associated with low levels of circulating PEG-IFNα. It should be considered in patients undergoing treatments including PEG-IFNα.

Healing of two and three wall intrabony periodontal defects following treatment with an enamel matrix derivative combined with autogenous bone

BACKGROUND: There are still limited data on the outcomes of regenerative periodontal surgery using a combination of an enamel matrix protein derivative (EMD) and autogenous bone (AB). AIM: To evaluate the healing of deep intrabony defects treated with either a combination EMD+AB or EMD alone. MATERIALS AND METHODS: Forty patients with advanced chronic periodontitis, with one deep intrabony defect, were randomly treated with either EMD+AB (test) or EMD (control). Clinical assessments were performed at baseline and at 1 year after treatment. The primary outcome variable was relative attachment level (RAL). RESULTS: Healing was uneventful in all patients. The test sites showed a reduction in the mean probing pocket depth (PPD) of 5.6 +/- 0.9 mm (p<0.001), a gain in the mean RAL of 4.2 +/- 1.1 mm (p<0.001) and a gain in the mean probing bone level (PBL) of 3.9 +/- 1.0 mm (p<0.001). The control group displayed a mean PPD reduction of 4.6 +/- 0.4 mm (p<0.001), a mean RAL gain of 3.4 +/- 0.8 mm (p<0.001) and a mean PBL gain of 2.8 +/- 0.8 mm (p<0.001). RAL gains of > or =4 mm were measured in 90% of the test defects and in 55% of the controls. PBL gains of > or =4 mm were obtained in 85% of the test defects and in 25% of the control ones. The test treatment resulted in statistically higher PPD reductions, RAL gains and PBL gains compared with the control (p<0.01). CONCLUSIONS: Within their limits, the present results indicate that: (i) at 1 year after surgery, both therapies resulted in statistically significant clinical improvements compared with baseline and (ii) although the combination of EMD+AB resulted in statistically significant higher soft and hard tissue improvements compared with treatment with EMD, the clinical relevance of this finding is unclear.

Meta-analyses qualify metzincins and related genes as acute rejection markers in renal transplant patients

Definition of acute renal allograft rejection (AR) markers remains clinically relevant. Features of T-cell-mediated AR are tubulointerstitial and vascular inflammation associated with excessive extracellular matrix (ECM) remodeling, regulated by metzincins, including matrix metalloproteases (MMP). Our study focused on expression of metzincins (METS), and metzincins and related genes (MARGS) in renal allograft biopsies using four independent microarray data sets. Our own cases included normal histology (N, n = 20), borderline changes (BL, n = 4), AR (n = 10) and AR + IF/TA (n = 7). MARGS enriched in all data sets were further examined on mRNA and/or protein level in additional patients. METS and MARGS differentiated AR from BL, AR + IF/TA and N in a principal component analysis. Their expression changes correlated to Banff t- and i-scores. Two AR classifiers, based on METS (including MMP7, TIMP1), or on MARGS were established in our own and validated in the three additional data sets. Thirteen MARGS were significantly enriched in AR patients of all data sets comprising MMP7, -9, TIMP1, -2, thrombospondin2 (THBS2) and fibrillin1. RT-PCR using microdissected glomeruli/tubuli confirmed MMP7, -9 and THBS2 microarray results; immunohistochemistry showed augmentation of MMP2, -9 and TIMP1 in AR. TIMP1 and THBS2 were enriched in AR patient serum. Therefore, differentially expressed METS and MARGS especially TIMP1, MMP7/-9 represent potential molecular AR markers.

Delayed gadolinium-enhanced MRI of cartilage in the ankle at 3 T: feasibility and preliminary results after matrix-associated autologous chondrocyte implantation

To demonstrate the feasibility of delayed gadolinium-enhanced magnetic resonance imaging (MRI) of cartilage (dGEMRIC) in the ankle at 3 T and to obtain preliminary data on matrix associated autologous chondrocyte (MACI) repair tissue.

Identification of a fibronectin interaction site in the extracellular matrix protein ameloblastin

Mammalian teeth are composed of hydroxyapatite crystals that are embedded in a rich extracellular matrix. This matrix is produced by only two cell types, the mesenchymal odontoblasts and the ectodermal ameloblasts. Ameloblasts secrete the enamel proteins amelogenin, ameloblastin, enamelin and amelotin. Odontoblasts secrete collagen type I and several calcium-binding phosphoproteins including dentin sialophosphoprotein, dentin matrix protein, bone sialoprotein and osteopontin. The latter four proteins have recently been grouped in the family of the SIBLINGs (small integrin-binding ligand, N-linked glycoproteins) because they display similar gene structures and because they contain an RGD tripeptide sequence that binds to integrin receptors and thus mediates cell adhesion. We have prepared all the other tooth-specific proteins in recombinant form and examined whether they might also promote cell adhesion similar to the SIBLINGs. We found that only ameloblastin consistently mediated adhesion of osteoblastic and fibroblastic cells to plastic or titanium surfaces. The activity was dependent on the intact three-dimensional structure of ameloblastin and required de novo protein synthesis of the adhering cells. By deletion analysis and in vitro mutagenesis, the active site could be narrowed down to a sequence of 13 amino acid residues (VPIMDFADPQFPT) derived from exon 7 of the rat ameloblastin gene or exons 7-9 of the human gene. Kinetic studies and RNA interference experiments further demonstrated that this sequence does not directly bind to a cell surface receptor but that it interacts with cellular fibronectin, which in turn binds to integrin receptors. The identification of a fibronectin-binding domain in ameloblastin might permit interesting applications for dental implantology. Implants could be coated with peptides containing the active sequence, which in turn would recruit fibronectin from the patient's blood. The recruited fibronectin should then promote cell adhesion on the implant surface, thereby accelerating osseointegration of the implant.

Determination of voriconazole in human serum and plasma by micellar electrokinetic chromatography

The micellar electrokinetic capillary chromatography (MEKC) separation and analysis of voriconazole and UK 115794 (internal standard) were examined and an assay for determination of voriconazole in human plasma and serum was developed. The MEKC medium comprises a 2:15 (v/v) mixture of methanol and a pH 9.3 buffer composed of 5mM Na(2)B(4)O(7), 7 mM Na(2)HPO(4) and 54 mM SDS. Sample preparation is based upon liquid/liquid extraction with ethylacetate and dichloromethane (75%/25%) at physiological pH. Using this approach with 250 microl serum or plasma and reconstitution of the dried extract into 100 microl of a buffer composed of 0.5mM Na(2)B(4)O(7) and 0.7 mM Na(2)HPO(4) (pH 9.3), the detection and quantitation limits were determined to be 0.1 and 0.2 microg/ml, respectively, a sensitivity that is suitable for therapeutic drug monitoring of voriconazole (provisional therapeutic range: 1-6 microg/ml) in human plasma and serum samples. The method was validated and compared to an HPLC method, showing excellent agreement between the two for a set of 91 samples that stemmed from patients being treated with voriconazole. The MEKC assay is also demonstrated to be suitable to explore pharmacokinetic data of voriconazole.