Volume 383, Issue 2

Schwann cells are the major glial population of the vertebrate peripheral nervous system. In the adult, they build a protecting sheath around neuronal processes and myelinate largecaliber axons. Already early in development, Schwann cells and neurons establish close contacts. Later development and the maintenance of peripheral nerves are crucially dependent on the controlled bidirectional dialogue between these two cell types. Several major phases can be distinguished in the life of a Schwann cell: determination, differentiation, and potentially myelination. The aim of this review is to summarize the molecular and cellular characteristics of the first steps in the life of a Schwann cell, the development from a multipotent neural crest cell to a differentiated Schwann cell.

The Wnt signalling cascade plays an important role during embryonic patterning and cell fate determination and is highly conserved throughout evolution. Factors of the TCF/LEF HMG domain family (Tcfs) are the downstream effectors of this signal transduction pathway. Upon Wnt signalling, a cascade is initiated that results in the translocation of βcatenin to the nucleus, where it interacts with Tcf to generate a transcriptionally active complex. This bipartite transcription factor is targeted to the upstream regulatory regions of Tcf target genes. In the absence of Wnt signals, βcatenin is degraded in the cytoplasm via the ubiquitinproteasome pathway. Several proteins are instrumental in achieving this tight regulation of βcatenin levels in the cell, including adenomatous polyposis coli (APC), GSK3 β, and Axin/Conductin. Deregulation of the Wnt signalling pathway is implicated in several forms of cancer, such as colon carcinoma and melanoma. This deregulation is achieved via mutation of APC, βcatenin or Axin, resulting in elevated βcatenin levels and the presence of constitutively active Tcfβcatenin complexes in the nucleus. The accompanying inappropriate activation of target genes is considered to be a critical, early event in this carcinogenesis. In addition to regulating βcatenin levels, normal healthy cells have evolved a second level of regulation, by manipulating the activity of the Tcf proteins themselves. In the absence of Wnt signalling, Tcf complexes with several transcriptional repressor proteins ensuring active repression of Tcf target genes. In this review the dual role of Tcf proteins in the Wnt signalling cascade will be discussed.

Using RACE technology the 5 and 3 ends of human carboxypeptidase M (CPM) mRNA were determined and found to be divergent from the published sequence. With these results the complete structure of the human CPM gene was established based on the human genome sequence in the GenBank database. The gene was shown to contain 9 exons comprising at least 75 kb of genomic sequence. A novel first exon of 30 bp was identified and an upstream promoter sequence containing several transcription factor binding sites was found by computer analysis. Furthermore, the ATG starting codon was detected defining an open reading frame of 1329 bp that codes for a protein of 443 residues. Additionally, the polyadenylation site was discovered, determining a 3 noncoding region of 2000 nucleotides. The exonintron boundaries diverged substantially compared to those of the other basic carboxypeptidases, CPD, CPE, CPN, and AEBP1. Cloning and sequencing of RTPCR products from different tissues revealed alternative splicing of exons 3 and 5, which results in the generation of four different mRNA isoforms. RNA extracted from tumor tissues contained more CPM mRNA than control tissue, suggesting an upregulation of CPM expression in tumors and raising the question of the role of this enzyme in cancer.

The clinical course of Cystic Fibrosis is characterized by recurrent pulmonary infections which ultimately lead to death by respiratory failure. The most common CF causing mutation, δF508-CFTR, produces an incorrectly folded protein, which accumulates within the endoplasmic reticulum. However, the molecular mechanism by which the δF508-CFTR protein facilitates pulmonary infection and inflammation remains unclear. Here we show that the expression of δF508-CFTR causes a constitutive activation of the proinflammatory transcription factor NFκB by eliciting an ER stress reaction, the ERoverload response. This endogenous NFκB activation stimulates the transcription of proinflammatory cytokines thereby commencing an inflammatory cascade within the CF lung.

In order to test the hypothesis that an imbalance between energy requirement and energy supply regulates mitochondrial genes and ultimately mitochondrial biogenesis, energy supply was challenged in HepG2 cells by withdrawal of glucose from the culture medium, making the cells exclusively dependent on mitochondrial ATP production. Such cells showed a 2-fold increase of cytochrome c oxidase activity, elevated levels of mitochondrial DNA, mitochondrial DNA encoded mRNAs and proteins, as well as the nuclear encoded mitochondrial transcription factor A. Lactate production was significantly reduced and glutamine was consumed as an alternative substrate for oxidative metabolism. Longterm adapted cells formed exclusively monolayers, while they normally grow in multilayers forming tumor spheroids. Also, longterm adapted cells proliferated significantly faster. No differences for the ATP/ADP ratio were observed, indicating that this is not the primary signal initiating the adaptative processes. These results show that mitochondrial biogenesis and oxidative metabolism are stimulated in HepG2 cells grown in the absence of fermentable glucose, probably in order to compensate for the diminished supply of glycolytic ATP.

A protocol was developed for performing intracellular concentration measurements in flat adherent tissue culture cells by fluorescence correlation microscopy (FCM). Determination of the number of molecules in the confocal detection volume had to account for background fluorescence caused by molecules adsorbed to the surface of the measurement chamber. Such a background signal leads to a decrease in the amplitude of the autocorrelation function, and thereby to the calculation of an erroneously high number of molecules. Because of the spatial heterogeneity of the background intensity, a method was devised by which its contribution to the total fluorescence could be determined directly from each individual autocorrelation measurement. This method was applied to a comparison of the import efficiencies of different cellpermeable peptides at nanomolar concentrations. The Antennapedia homeodomainderived peptide penetratin was imported about three times as efficient as the basic fibroblast growth factorderived MTS peptide. Both peptides equilibrated between cytoplasm and nucleus. A relatively high mobility of these molecules inside the cells indicated that they may be rapidly degraded by cytosolic proteases. Based on these results, it will be possible to determine intracellular concentrations of inhibitors linked to import peptides directly by FCM at nanomolar concentrations and to optimise such constructs for proteolytic stability.

Cystatin C, a major extracellular cysteine proteinase inhibitor, is deposited as amyloid in brain haemorrhage patients with hereditary cystatin C amyloid angiopathy (HCCAA). A diseasecausing mutation on the genetic level results in the substitution Leu68-> Gln (L68Q) in cystatin C, which causes protein instability. Besides carrying the L68Q substitution, cystatin C in amyloid deposits isolated from patients is Nterminally truncated by 10 amino acids. To elucidate the role of the Nterminal truncation for protein stability and aggregation properties, (δ1-10,L68Q)cystatin C was produced in an Escherichia coli expression system and characterised. Unlike wildtype cystatin C, this variant rapidly dimerised under physiological conditions. Two unfolding intermediates of (δ1- 10,L68Q)cystatin C were identified, under the same pH and ionic strength conditions as required to form intermediates of fulllength L68Q cystatin C. No evidence was found that the Nterminal truncation per se alters protein stability and leads to higher forms of aggregation. Monomeric as well as dimeric L68Q cystatin C incubated with neutrophil elastase was truncated as in HCCAA patients amyloid. A protein variant with a thrombin cleavage site placed in front of residue Gly11 in L68Q cystatin C was constructed and used to confirm that the Nterminal segment is similarly accessible to proteinases in the monomeric and dimeric states of L68Q cystatin C. Thus, the Nterminal segment of L68Q cystatin C is exposed to proteolytic attack and does not seem to be involved in intramolecular contacts leading to dimerisation or higherorder aggregation. We conclude that the Nterminal truncation likely is an event secondary to amyloid formation, and of no relevance for the development of HCCAA.

Sialic acids present on human colonic mucins are highly Oacetylated, however, little is known about the underlying enzymatic activity required for Oacetylation in this tissue. Here we report on the substrate specificity, subcellular localization and characterization of the sialate-7(9)Oacetyltransferase in normal human colonic mucosa. Using CMPNeu5Ac, the most efficient acceptor substrate of all those tested, the enzymatic activity was found to be optimal at 37 C, with a pH optimum of 7.0. Activity was also found to be dependent on protein, CMPNeu5Ac (Km: 59.2 M) and AcCoA (Km: 6.1 M) concentrations, as well as membrane integrity. The enzymes activity could be inhibited by CoA with a Ki of 11.9 M. In addition, enzymatic activity was found to be localized in the Golgienriched membrane fraction. The nature of the Oacetylated products formed were verified with the aid of chromatographic and enzymatic techniques. The main product was 9-Oacetylated Neu5Ac, with a significant amount of oligoOacetylated Neu5Ac also being detected. The utilization of CMPNeu5Ac as the acceptor substrate was confirmed by the isolation and characterization of the putative product, CMPNeu5,9Ac2, using ionexchange chromatography. The ability of CMPNeu5,9Ac2 to act as a sialic acid donor for sialyltransferases represents the conclusive demonstration for the formation of CMPNeu5,9Ac2.

Five moeA mutants were generated by replacing some conserved amino acids of MoeA by sitedirected mutagenesis. The mutants were assayed for the ability to restore in vivo nitrate reductase activity of the moeA mutant Escherichia coli JRG97 and in vitro Neurospora crassa nit-1 nitrate reductase activity. The replacements Asp59AlaGly60Ala, Asp259Ala, Pro298AlaPro301Ala abolished the function of MoeA in Momolybdopterin formation and stabilization, reflected in the inability to restore nitrate reductase activity. The replacements Gly251AlaGly252Ala reduced, and that of Pro283Ala had no effect, on nitrate reductase activity. E. coli JRG97 cells transformed with mutants that failed to restore nitrate reductase activity showed by HPLC analysis a decreased level of molybdopterinderived dephospho FormA as compared to bacteria transformed with wildtype moeA. The effects of the amino acid replacements on MoeA function may be explained in correlation with the MoeA crystal structure.

The structure of the major tetrahydropterin in Escherichia coli was determined as (6R)-5,6,7,8-tetrahydroLmonapterin, i. e. (6R)-2-amino-5,6,7,8-tetrahydro 6-[(1S,2S)-1,2,3-trihydroxypropyl]pteridin-4(3H)one. Although the stereochemical structure of the trihydroxypropyl side chain has been determined previously by fluorescence detected circular dichroism analysis on its aromatic derivative, the most important configuration at C(6) has not been clarified. The major difficulties for the determination of the chirality were instability toward air oxidation and very low concentration of the tetrahydropterin derivative. In the present study, the C(6)configuration was determined as R by comparing its stable hexaacetyl derivative with authentic (6R) and (6S)hexaacetyl-5,6,7,8- tetrahydroLmonapterins by high performance liquid chromatography (HPLC) and HPLCmass spectrometry (LCMS). (6R)-5,6,7,8-TetrahydroLmonapterin is a new unconjugated tetrahydropterin from natural sources.

We demonstrate that fluorescence correlation spectroscopy (FCS) can be employed to follow the conformational changes of DNA molecules induced by the addition of a cationic condensing compound (spermine). In our experiments the plasmid pHβAPr-1-neo (10 kbp; contour length 3.4 m) was labeled with propidium iodide (PrIo) and then titrated with spermine to induce its condensation. When spermine was applied at concentrations above 5 M (spermine/DNAphosphate =0.375), the diffusion time of the labeled plasmid dropped from 15 ms down to 3 ms (its diffusion coefficient, D, increased from 1.010 exp 12 square m/s to 6.010 exp 12 square m/s). The application of spermine was also accompanied by decreasing count rate and particle number, reflecting the dyes dissociation. The data presented show that FCS may become a valuable tool in studying supramolecular aggregate formation, especially when association is followed by a change in the hydrodynamic size of the resulting complex.

We demonstrate for the first time that ellipsometry and confocal fluorescence correlation spectroscopy (FCS) are complementary methods for the characterisation of supported planar phospholipid bilayers (SPBs) formed on mica, a mineral used in atomic force microscopy investigations of SPBs. Addition of small unilamellar vesicles containing 20% dioleoylphosphatidylserine (DOPS) and 80% dioleoylphosphatidylcholine (DOPC) to an oxidised borosilicate surface, on the other hand, results in a planar lipid system characterised by lateral diffusion coefficients which are three time smaller than those obtained for SPBs. Moreover, seven labelled phospholipids were tested for their suitability in the FCS characterisation of vesicles as well as of SPBs.