Performance assessment of a fully automated electro-chemiluminescence immunoassay system for serum S100B protein.

The altered expression levels of S100 proteins can lead to four different categories of diseases: diseases of the heart and of the central nervous system, inflammatory disorders and cancer. Various studies have shown the lack of harmonization of the results obtained with different methods, mainly due to different performances and measurements of S100B. The purpose of this work was to compare quantitatively the fully automated Elecsys® immunoassay with the reference immunoenzimatic method CanAg® EIA for serum S100B protein. In the study serum samples were analyzed of 161 patients: 85 females (aged 22-83 years) and 76 males (aged 16-90 years), affected by oncological and non-oncological pathologies. Passing–Bablok regression was used to analyze the comparison between the assays; it showed a strong interassay correlation: r = 0.9350 (95% CI =0.9122 – 0.9520), with an intercept of 0.02063 (95% CI=-0.02850 – 0.01400) and a slope of 1.1125 (95% CI=1.0200 – 1.2417). Elecsys® S100 assay should be preferred to CanAg® S100 for better standardization, good reliability and precision but also with the aim to reduce costs and obtain results in a shorter time.

Characterization and role of Helix contactin-related proteins in cultured Helix pomatia neurons.

We report on the structural and functional properties of the Helix contactin-related proteins (HCRPs), a family of closely related glycoproteins previously identified in the nervous system of the land snail Helix pomatia through antibodies against the mouse F3/contactin glycoprotein. We focus on HCRP1 and HCRP2, soluble FNIII domains-containing proteins of 90 and 45 kD bearing consensus motifs for both N- and O-glycosylation. Using the anti-HCRPs serum, we find secreted HCRPs in Helix nervous tissue isotonic extracts and in culture medium conditioned by Helix ganglia. In addition, we demonstrate expression of HCRPs on neuronal soma and on neurite extensions. Functionally, in Helix neurons, the antisense HCRP2 mRNA counteracts neurite elongation, and the recombinant HCRP2 protein exerts a strong positive effect on neurite growth when used as substrate. These data point to HCRPs as novel neurite growth-promoting molecules expressed in invertebrate nervous tissue.

A soluble form of the F3 neuronal cell adhesion molecule promotes neurite outgrowth.

The F3 molecule is a member of the immunoglobulin superfamily anchored to membranes by a glycane-phosphatidylinositol, and is predominantly expressed on subsets of axons of the central and peripheral nervous system. In a previous paper (Gennarini, G., P. Durbec, A. Boned, G. Rougon, and C. Goridis. 1991. Neuron. 6:595-606), we have established that F3 fulfills the operational definition of a cell adhesion molecule and that it stimulates neurite outgrowth when presented to sensory neurons as a surface component of transfected CHO cells. In the present study the question as to whether soluble forms of F3 would be functionally active was addressed in vitro on cultures of mouse dorsal root ganglion neurons. We observed that preparations enriched in soluble F3 had no effect on neuron attachment but enhanced neurite initiation and neurite outgrowth in a dose-dependent manner. By contrast, soluble NCAM-120 does not have any measurable effect on these phenomena. Addition of anti-F3 monovalent antibodies reduced the number of process-bearing neurons and the neuritic output per neuron to control values. Addition of cerebrospinal fluid, a natural source of soluble F3, also stimulated neurite extension, and this effect was partially blocked by anti-F3 antibodies. Our results suggest that the soluble forms of adhesive proteins with neurite outgrowth-promoting properties could act at a distance from their site of release in a way reminiscent of growth and trophic factors.

The mouse neuronal cell surface protein F3: a phosphatidylinositol-anchored member of the immunoglobulin superfamily related to chicken contactin.

Several members of the Ig superfamily are expressed on neural cells where they participate in surface interactions between cell bodies and processes. Their Ig domains are more closely related to each other than to Ig variable and constant domains and have been grouped into the C2 set. Here, we report the cloning and characterization of another member of this group, the mouse neuronal cell surface antigen F3. The F3 cDNA sequence contains an open reading frame that could encode a 1,020-amino acid protein consisting of a signal sequence, six Ig-like domains of the C2 type, a long premembrane region containing two segments that exhibit sequence similarity to fibronectin type III repeats and a moderately hydrophobic COOH-terminal sequence. The protein does not contain a typical transmembrane segment but appears to be attached to the membrane by a phosphatidylinositol anchor. Antibodies against the F3 protein recognize a prominent 135-kD protein in mouse brain. In fetal brain cultures, they stain the neuronal cell surface and, in cultures maintained in chemically defined medium, most prominently neurites and neurite bundles. The mouse f3 gene maps to band F of chromosome 15. The gene transcripts detected in the brain by F3 cDNA probes are developmentally regulated, the highest amounts being expressed between 1 and 2 wk after birth. The F3 nucleotide and deduced amino acid sequence show striking similarity to the recently published sequence of the chicken neuronal cell surface protein contactin. However, there are important differences between the two molecules. In contrast to F3, contactin has a transmembrane and a cytoplasmic domain. Whereas contactin is insoluble in nonionic detergent and is tightly associated with the cytoskeleton, about equal amounts of F3 distribute between buffer-soluble, nonionic detergent-soluble, and detergent-insoluble fractions. Among other neural cell surface proteins, F3 most resembles the neuronal cell adhesion protein L1, with 25% amino acid identity between their extracellular domains. Based on its structural similarity with known cell adhesion proteins of nervous tissue and with L1 in particular, we propose that F3 mediates cell surface interactions during nervous system development.

The F3/11 cell adhesion molecule mediates the repulsion of neurons by the extracellular matrix glycoprotein J1-160/180.

The oligodendrocyte-derived extracellular matrix protein J1-160/180 displays repellent substrate properties toward neurons. In a search for neuronal ligands mediating the response to J1-160/180, we have identified the F3/11 cell surface protein, a glyco-phosphatidylinositol-anchored member of the immunoglobulin superfamily. F3/11 mediates the initial recognition between a J1-160/180 substrate and cerebellar neurons or F3-transfected CHO cells. In cerebellar neurons, the F3/11-J1-160/180 interaction induces a repulsion consisting of the loss of substrate adhesion with time in culture and inhibition of neurite outgrowth. Antibody blocking experiments show that the avoidance response of neurites at J1-160/180 substrate borders is also mediated by F3/11. Active cell-cell and cell-substrate repulsion is considered a major mechanism governing the extent and directionality of axonal growth, but the ligand-receptor interactions involved have remained unknown. Our results show that F3/11 mediates the neuronal response to the repellent molecule J1-160/180 and may thus be involved in signal transduction leading to cell repulsion.

Transfected F3/F11 neuronal cell surface protein mediates intercellular adhesion and promotes neurite outgrowth.

The mouse neuronal F3 glycoprotein and its chicken homolog F11 belong to a subclass of proteins of the immunoglobulin superfamily with preferential localization on axons and neurites. We have transfected F3 cDNA into CHO cells. Biochemical analysis establishes that the cDNA we have cloned codes for a 130 kd phosphatidylinositol-anchored polypeptide. F3-expressing transfectants exhibited enhanced self-adhesive properties, aggregating with faster kinetics and forming larger aggregates than F3-negative control cells. When used as a culture substrate for sensory neurons, F3-transfected cells showed a markedly enhanced ability to promote neurite outgrowth compared with nontransfected cells. The results support the idea that F3/F11 and other closely similar proteins function as cell adhesion molecules that play a role in axonal growth and guidance.

Transgenic models for studying expression and function of axonal adhesive glycoproteins.

In this study, by using two transgenic models, we address the general topic of the significance of axonal glycoproteins regulated expression in nervous tissue maturation. The immunoglobulin superfamily components F3/Contactin (F3) and TAG-1 are used as the molecular models in this respect. First, a minigene including the relevant regulatory sequences of the F3 gene, deduced by a previous in vitro study, has been fused to an EGFP (Enhanced Green Fluorescent Protein) reporter and expressed in transgenic mice, which provided information about the profile of F3 gene developmental activation. In a complementary model, transgenic mice have been generated which express the F3 cDNA under control of a selected regulatory region from the TAG-1 gene. While leading to ectopic expression of F3, this perturbed neuronal precursor proliferation and differentiation. The arising effects were even stronger than those coming from the overall suppression of the F3 or, respectively, TAG-1 genes, thus supporting the hypothesis that the mechanisms underlying axonal glycoprotein regulated expression are themselves endowed with a key significance in neural development.

Regional distribution and cell type-specific expression of the mouse F3 axonal glycoprotein: a developmental study.

The expression of the mouse axonal adhesive glycoprotein F3 and of its mRNA was studied on sections of mouse cerebellar cortex, cerebral cortex, hippocampus, and olfactory bulb from postnatal days 0 (P0) to 30 (P30). In cerebellar cortex, a differential expression of F3 in granule versus Purkinje neurons was observed. F3 was highly expressed during migration of and initial axonal growth from cerebellar granule cells. The molecule was then downregulated on cell bodies and remained expressed, although at low levels, on their axonal extensions. On Purkinje cells, F3 was strongly expressed on cell bodies and processes at the beginning of the second postnatal week; by P16 it was restricted to neurites of Purkinje cells subpopulations. In the cerebral cortex, the molecule was highly expressed on migrating neurons at P0; by P16, it was found essentially within the neuropil with a diffuse pattern. In the hippocampal formation, where F3 was expressed on both pyramidal and granule neurons, a clear shift from the cell bodies to neurite extensions was observed on P3. In the olfactory pathway, F3 was expressed mainly on olfactory nerve fibers, mitral cells, and the synaptic glomeruli from P0 to P3, with a sharp decline from P11 to P16. As a whole, the data show that F3 protein expression is regulated at the regional, cellular, and subcellular levels and suggest that, in different regions, it can be proposed as a reliable neuronal differentiation marker.

A novel soluble brain-specific protein (Sy-1). Identification and partial characterization.

A novel brain-specific antigen, called Sy-1, has been identified in whole rat brain hypotonic extracts by means of an antiserum produced in rabbits against the synaptosomal cytosol. Sy-1 is an acidic protein (pI 4.7) present exclusively in a soluble form and it does not carry any sugar moiety. Sy-1 is found only in rat brain and, in a partially identical form, in mouse brain. In rat brain the antigen is more concentrated in the brainstem than in the forebrain and cerebellum. On the basis of immunological, physico-chemical and biological criteria Sy-1 differs from other, already described, brain-specific proteins.

Transgenic mice expressing F3/contactin from the transient axonal glycoprotein promoter undergo developmentally regulated deficits of the cerebellar function.

We have shown that transgenic transient axonal glycoprotein (TAG)/F3 mice, in which the mouse axonal glycoprotein F3/contactin was misexpressed from a regulatory region of the gene encoding the transient axonal glycoprotein TAG-1, exhibit a transient disruption of cerebellar granule and Purkinje cell development [Development 130 (2003) 29]. In the present study we explore the neurobehavioural consequences of this mutation. We report on assays of reproductive parameters (gestation length, litter size and offspring viability) and on somatic and neurobehavioural end-points (sensorimotor development, homing performance, motor activity, motor coordination and motor learning). Compared with wild-type littermates, TAG/F3 mice display delayed sensorimotor development, reduced exploratory activity and impaired motor activity, motor coordination and motor learning. The latter parameters, in particular, were affected also in adult mice, despite the apparent recovery of cerebellar morphology, suggesting that subtle changes of neuronal circuitry persist in these animals after development is complete. These behavioural deficits indicate that the finely coordinated expression of immunoglobulin-like cell adhesion molecules such as TAG-1 and F3/contactin is of key relevance to the functional, as well as morphological maturation of the cerebellum.

Characterization of the 5' and promoter regions of the gene encoding the mouse neuronal cell adhesion molecule F3.

F3 is a 135 kDa neuronal cell surface adhesive glycoprotein belonging to the immunoglobulin supergene family (IgSF) which mediates heterophilic contact formation among neural cells and is involved in the control of neurite growth. F3 expression is regulated, during critical developmental periods, on neuronal subpopulations thus suggesting that control of F3 gene expression could be of morphogenetic relevance. To shed light on the mechanism involved in the control of F3 gene expression we isolated clones covering about 50 kilobases of the F3 gene which also included the promoter region. The study of F3 gene exon/intron organization revealed that, like other neural IgSF molecules, each of the first two F3 C2 domains is encoded by two exons while the N-terminus, the signal peptide and the 5' untranslated region are each encoded by distinct exons. A single transcription start site was identified, surrounded by a short 114 bp sequence able to direct reporter gene expression in both F3-expressing and -non-expressing cells. In addition, a cell type-specific enhancer, only active in F3-expressing cells, was found immediately upstream to it. Structural analysis of the promoter region revealed consensus sequences for binding transcription factors involved in cell type-specific and/or developmental regulations. Most of them are homeobox containing transcription factors thus suggesting that regulation of F3 gene expression could be part of a large developmental program.

Functional organization of the promoter region of the mouse F3 axonal glycoprotein gene.

F3 is a developmentally regulated adhesive glycoprotein expressed by subpopulations of central and peripheral neurons which mediates neurite growth and fasciculation via cis- and trans-interactions with cell-surface or matrix components. We previously reported on the characterization of the F3 gene 5' flanking region in which we identified promoter and enhancer elements. Here, we report on the functional organization of the F3 gene regulatory regions. We show that the F3 promoter is built of linearly arranged positive and negative elements scattered through the 5' flanking region of the F3 gene and the 1st exon (exon 0). Neural- and cell type-specific expression of F3 appears to be governed by elements located in the most proximal promoter region which includes a neural-specific enhancer. In retardation assays, all these cis-acting elements bind nuclear proteins, three of which interact with the identified enhancer element while a single species interacts with sequences located within exon 0. Some of these proteins are also specifically expressed within the brain, indicating that they could correspond to neural-specific trans-acting factors. Elements located immediately upstream of the cell type-specific enhancer and within exon 0 are responsible for regulation of F3 expression by cAMP and retinoic acid.

Alternative promoters drive the expression of the gene encoding the mouse axonal glycoprotein F3/contactin.

F3/Contactin is a neuronal glycoprotein which mediates axonal growth control via complex interactions with a number of cell surface or matrix components. As part of this developmental role, its expression undergoes differential regulation during the maturation of definite neuronal populations within the central and peripheral nervous tissue. To elucidate the underlying molecular mechanisms we study here the organization of the regulatory region of the mouse F3/Contactin gene. We show that this region displays peculiar features in that it spans more than 80 kb, bears very large introns and includes four untranslated exons which undergo complex splicing events leading to 11 potential arrangements of the F3/Contactin mRNA 5' end. Within this region we identify three alternative neurospecific promoters which, as deduced from the developmental profile of the associated 5' exons (A1,C1,0), drive two different patterns of F3/Contactin gene expression. The activity of the A1 exon-associated promoter displays only minor developmental changes and is likely to contribute to the basal level of the F3/Contactin gene expression; by contrast, the activities of the exon C1- and exon 0-associated promoters are significantly upregulated at the end of the first postnatal week. The data indicate that differential regulation of the F3/Contactin expression during development may depend upon alternative utilization of distinct promoter elements and may involve complex splicing events of the 5' untranslated exons. Several consensuses for homeogene transcription factors are scattered within the identified regulatory region, in agreement with the general assumption of homeotic gene regulation of neural morphoregulatory molecules.

Ontogenetic changes of the soluble and membrane-bound D2 glycoprotein in rat forebrain.

A soluble form of the D2 glycoprotein, detected in the rat brain hypotonic extract, is described. Its specific relative concentration did not differ significantly in the three examined cerebral regions (forebrain, brainstem and cerebellum), while in the cerebellum the membrane-bound form was about three and four times more concentrated than in the forebrain and brainstem, respectively. No sizeable developmental variations of the soluble D2 concentration could be detected in forebrain, whereas the amount of the membrane-bound protein rose from birth to postnatal day 6 and then decreased to the adult value (about 40% of the newborn concentration). Ontogenetic modifications of the membranous D2 glycans (studied through the binding of the molecule to several lectins) occur around postnatal day 18 when the binding to Ricinus communis lectin, specific for galactose, becomes evident. At all ages both soluble and membrane-bound forms bind to Concanavalin A, specific for mannose and glucose, and to wheat germ agglutinin, specific for N-acetylglucosamine, while the lack of binding to Ulex europeus lectin suggests the absence of discrete amount of fucose. The results are discussed in relation to the possible involvement of D2 glycoprotein in cell-to-cell adhesion.

Functional studies and cellular distribution of the F3 GPI-anchored adhesion molecule.

Many adhesion molecules of the immunoglobulin superfamily expressed in the nervous system are attached to the neuronal membrane by a glycan-phosphatidylinositol. Using neuronal glycoprotein F3 as a model we will discuss how this lipid modification might confer on molecules specific properties which may be particularly well suited to a role in modulating neuronal interactions. In particular, the following data dealing with the question of how the glycosylphosphatidylinositol (GPI) anchor influences the function, transport and localization of this molecule will be presented. 1) When anchored to the plasma membrane, F3 fulfills the operational criteria of an adhesion molecule while its soluble form is able to stimulate neurite outgrowth of sensory neurons in culture. 2) In the hypothalamo-hypophyseal system, immunoblot analysis indicates that there is more F3 in the neurohypophysis where secretory axons terminate than in the hypothalamic nuclei where the molecule is synthesized. In addition, GPI-linked forms predominate in the nuclei while there are mainly soluble forms in the neurohypophysis, suggesting that there is conversion of the GPI-bearing form to the soluble form during axonal transport. 3) In the cerebellum, F3 is polarized to the tips of the axons of granule cells, the major neuronal population in this system, as an indication that indeed GPI might be a signal for targeting molecules to axons. However, some neurons such as Golgi cells express F3 over all their surface.

Neurobehavioral studies, in transgenic F3/CONTACTIN (C57BL/6J × CBA) mice, on cognitive and anxiety aspects during late-adolescential period.

OBJECTIVE: Besides than in the control of developmental events, axonal adhesive glycoproteins may be also involved in functions requiring fine organization and connectivity of the nervous tissue. We previously demonstrated morphological alterations and functional cerebellar deficits in transgenic mice (TAG/F3 mice) ectopically expressing the F3/Contactin axonal glycoprotein under the control of a selected regulatory region from the Transient Axonal Glycoprotein (TAG-1) gene. In the present study, the hippocampal function was explored by evaluating the ability of TAG/F3 mice to encode spatial and non-spatial relationships between discrete stimuli and to analyze an anxiety-related behavior. MATERIALS AND METHODS: To the first end, mice were placed in an "open-Field" containing five objects and, after three sessions of habituation (S2-S4), their reactivity to objects displacement (S5-S4) and object substitution (S7-S6) was examined.To the second end, mice were placed in the "elevated zero maze", a standard test to explore the anxiety-related behavior, in order to study, in transgenic mice, the effects of F3 misexpression on emotional reactivity by measuring the avoidance of the unsheltered open sectors. RESULTS: Statistical evaluations of reactivity to object novelty, TAG-F3 mice showed a lower DO exploration with respect to wild-type mice and, regarding DOs, TAG/F3 mice interacted less than wild-type mice, showing an impaired spatial change response. Furthermore, the number of HDIPS in transgenic TAG/F3 mice resulted significantly lower with respect to the controls (wild type). CONCLUSIONS: These results indicate that the coordinated expression of axonal adhesive glycoproteins may be relevant for the functional maturation of the hippocampus.

F3/F11 cell surface molecule expression in the developing mouse cerebellum is polarized at synaptic sites and within granule cells.

The distribution of the F3/F11 neuronal cell surface molecule was investigated in the developing and adult mouse cerebellum by immunocytochemistry at the light and electron microscopic levels. F3/F11 was confined to subsets of neuronal types, since the Purkinje cell body and dendritic arborization as well as the stellate cells were not immunoreactive. In the young developing cerebellum, the granule cell axons strongly express F3/F11 as soon as they begin to grow, consistent with a functional role in promoting directional outgrowth of neuronal processes. In 10-d-old and adult cerebella, the granule cell bodies and dendrites were not immunoreactive whereas the parallel fibers, which are the granule cell axons, were labeled including in their presynaptic varicosities. By contrast, dendrites, cell bodies, and axons of Golgi cells were labeled by anti-F3 antibodies. Hence, F3/F11 can either be expressed throughout the cell or be polarized to the axons. This raises the question of how segregation of the glypiated F3/F11 molecule between different subcellular compartments depending on the type of neuron is achieved. F3/F11 was found to be present at three types of synaptic sites, suggesting that it might play a role in the formation and maintenance of synapses. However, in each type of synpase, F3/F11 was present at only the pre- or postsynaptic site, never at both: the parallel fiber varicosities contained F3/F11 whereas the postsynaptic compartment in contact, that is, the Purkinje cell dendritic spines, did not. The granule cell dendrites were unlabeled while the mossy fiber terminals contacting them were immunoreactive, and finally, the Golgi cell dendrites and dendritic spines were labeled while the presynaptic compartment contacting them was not. If F3/F11 functions as an adhesion molecule in vivo as indicated by in vitro assays, F3/F11-mediated adhesion is likely to be heterophilic.

Studies on the transmembrane disposition of the neural cell adhesion molecule N-CAM. The use of liposome-inserted radioiodinated N-CAM to study its transbilayer orientation.

The transmembrane orientation of the polypeptide chains present in preparations of adult and neonatal mouse N-CAM was studied using, as a model system, liposome-inserted purified N-CAM preparations. N-CAM purified from adult or neonatal mouse brain was 125I-labeled and reconstituted into artificial lipid vesicles. After trypsin digestion, the peptides that remained associated with the liposomes were isolated by floatation of the vesicles on sucrose gradients. In control experiments the liposomes were lysed before trypsin treatment. Large, overlapping peptides were obtained after this treatment, several of which were protected by the liposome membrane. Sialic-acid-bearing peptides were revealed by their sensitivity to neuraminidase. To distinguish between peptides corresponding to intracellular or extracellular domains use was made of the P61 and H28.123 monoclonal antibodies, which recognize determinants located on the cytoplasmic and the extracellular part of the molecules respectively. There was no indication that the N-CAM chains were inserted in an inside-out configuration. Peptides protected from trypsin attack by the liposomes and recognized only by P61 had Mr values of 92 000, 42 000 and 35 000. The H28.123 determinant could be mapped to a 32 000-Mr peptide located close to the membrane at the vesicle's exterior. The bulk of the sialic acid seemed to be carried by a rather short sequence distal to the H28.123-reactive peptide but at some distance from the N terminus. Fragments of very similar Mr were generated from young and adult material. However, a 45 000-Mr peptide from neonatal N-CAM appeared to migrate in the higher-Mr region of sodium dodecyl sulfate/polyacrylamide gels in its fully sialylated form. It is concluded that (a) identical polypeptide chains are present in young and adult preparation, (b) the 180 000-Mr, 140 000-Mr and 120 000-Mr chains differ by the length of their cytoplasmic extensions and (c) the largest cytoplasmic sequences have a Mr close to 90 000. A tentative linear model of the transmembrane topography of the N-CAM polypeptides is presented.

[Analysis of cerebellar proteins and glycoproteins in adult cattle. I. Con A positive glycoproteins]. / Analisi di proteine e glicoproteine del cervelletto di bovino adulto. Nota I. Glicoproteine con A positive.

The cerebellar glycoproteins of bovine have been separated by affinity chromatography on Con A-Sepharose and analyzed by poliacrylamide gel electrophoresis. Some soluble Con A binding glycoproteins are common to the insoluble (membrane bound) glycoproteins suggesting a relationship between the two classes of molecules. The data support the hypothesis that some of the soluble glycoproteins can be considered precursors of the insoluble.