Christof Schild

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 patients blood. The recruited fibronectin should then promote cell adhesion on the implant surface, thereby accelerating osseointegration of the implant.

Reliability of M protein quantification : comparison of two peak integration methods on Capillarys 2

Figure 2 Linearity of M protein quantitation assay using different integration methods. Serum containing 50 g/L monoclonal IgG (squares) or 25 g/L monoclonal IgA (triangles) was diluted in steps of 1:2 with normal serum containing 12.3 g/L polyclonal g-globulins and subjected to capillary zone electrophoresis. M protein peaks were integrated either by the perpendicular drop method (closed symbols) or by the tangent skimming method (open symbols). Absolute concentrations of M protein were calculated from the percentage of the area under the curve attributed to the M protein and the total protein concentration as determined by the biuret method on a Hitachi 917 clinical-chemical analyzer (Roche Diagnostics, Rotkreuz, Switzerland). All experiments were carried out in quadruplicates. Figure 1 Methods of peak integration. (A) Perpendicular drop method and (B) tangent skimming method. Christof Schild*, Bendicht Wermuth, Daniela Trapp-Chiappini, Florence Egger and Jean-Marc Nuoffer

False low holotranscobalamin levels in a patient with a novel TCN2 mutation.

Abstract Background: Measurement of holotranscobalamin (holoTC) is increasingly used as a screening test for cobalamin (Cbl) deficiency. A level well below the reference interval strongly supports a deficient state. We examined a 21-year-old woman diagnosed as Cbl deficient because of an extremely low holoTC level as measured by the Abbott Architect Assay. Methods: The patient was evaluated for Cbl deficiency employing an in-house holoTC method as well as other routine markers of Cbl status. Further analyses included exploration of the Cbl binding proteins employing gel filtration of a serum sample saturated with 57 Co-labeled Cbl and Sanger sequencing of exons 1–9 and the intron-exon boundaries of the TCN2 gene, the gene coding for transcobalamin (TC). Results: The patient had normal hematological variables throughout. Despite initial treatment with Cbl, holoTC as measured by the Abbott assay remained low, while holoTC measured with the in-house assay was normal, and behaved as TC upon gel-filtration. By Sanger sequencing, we detected a homozygous single point mutation c.855T>A in exon 6 of TCN2, corresponding to a asparagine (Asn) to lysine (Lys) substitution in position 267 of the mature protein. Conclusions: We describe a novel point mutation of the TCN2 gene. The mutation does not seem to interfere with the function of TC, but the mutation may well explain the low level of holoTC detected by the Abbott assay. Our results underscores that mutations of TCN2 have to be considered when implausible holoTC results are obtained.

Mitochondrial leucine tRNA level and PTCD1 are regulated in response to leucine starvation

Pentatricopeptide repeat domain protein 1 (PTCD1) is a novel human protein that was recently shown to decrease the levels of mitochondrial leucine tRNAs. The physiological role of this regulation, however, remains unclear. Here we show that amino acid starvation by leucine deprivation significantly increased the mRNA steady-state levels of PTCD1 in human hepatocarcinoma (HepG2) cells. Amino acid starvation also increased the mitochondrially encoded leucine tRNA (tRNALeu(CUN)) and the mRNA for the mitochondrial leucyl-tRNA synthetase (LARS2). Despite increased PTCD1 mRNA steady-state levels, amino acid starvation decreased PTCD1 on the protein level. Decreasing PTCD1 protein concentration increases the stability of the mitochondrial leucine tRNAs, tRNALeu(CUN) and tRNALeu(UUR) as could be shown by RNAi experiments against PTCD1. Therefore, it is likely that decreased PTCD1 protein contributes to the increased tRNALeu(CUN) levels in amino acid-starved cells. The stabilisation of the mitochondrial leucine tRNAs and the upregulation of the mitochondrial leucyl-tRNA synthetase LARS2 might play a role in adaptation of mitochondria to amino acid starvation.

Monoclonal gammopathy missed by capillary zone electrophoresis

Abstract Background: Serum protein electrophoresis is used as a screening test for monoclonal gammopathies. Here, we present a case of a high-concentration monoclonal immunoglobulin (M-protein) that was missed by serum protein electrophoresis on a Capillarys 2 capillary zone electrophoresis system. The aim of our study was to identify the reason for the failure of the system to detect the M-protein. Methods: M-protein solubility was examined in response to temperature, pH, ionic strength, the chaotropic agent urea and the reducing agent 2-mercaptoethanol. Results: Precipitation of the M-protein was not cold-induced, but solubility decreased at pH 8.5 or higher, when the pH approached the apparent isoelectric point. The M-protein also precipitated in alkaline Capillarys 2 electrophoresis buffer (pH 10), which was the reason for the false-negative electrophoresis result. Precipitation of the M-protein was not related to the ionic strength of the buffer. Solubility improved in presence of urea. Pre-treatment of serum with 2-mercaptoethanol revealed the missing M-protein peak of 36 g/L on the electropherogram. Conclusions: This case shows that insolubility of M-proteins in alkaline buffer is one possible cause of false-negative results on capillary zone electrophoresis systems. False-negative results should be considered, especially when accompanying laboratory results are inconsistent with the electropherogram.

[Phosphate disorders: hyperphosphatemia or pseudohyperphosphatemia?].

We report the case of a 79 year old woman presenting with progressive confusion and drowsiness. Renal insufficiency with hyperkalemia as well as hypercalcemia and severe hyperphosphatemia were diagnosed. Renal insufficiency improved with treatment. However, hyperphosphatemia persisted without apparent explanation. We discuss possible causes of hyper- and pseudohyperphosphatemia. Specifically, phosphate analysis may be disturbed by the paraproteins in patients with multiple myeloma, resulting in pseudohyperphosphatemia. We review the standard laboratory phosphate measurement and the mechanisms of interference with paraproteins.

Phosphat(verw)irrungen: tatsächlich «Hyper» oder nur «Pseudo»?

We report the case of a 79 year old woman presenting with progressive confusion and drowsiness. Renal insufficiency with hyperkalemia as well as hypercalcemia and severe hyperphosphatemia were diagnosed. Renal insufficiency improved with treatment. However, hyperphosphatemia persisted without apparent explanation. We discuss possible causes of hyper- and pseudohyperphosphatemia. Specifically, phosphate analysis may be disturbed by the paraproteins in patients with multiple myeloma, resulting in pseudohyperphosphatemia. We review the standard laboratory phosphate measurement and the mechanisms of interference with paraproteins.