Design and application of a cytokine-receptor-based interaction trap.

Ligand-induced clustering of type I cytokine receptor subunits leads to trans-phosphorylation and activation of associated cytosolic janus kinases (JAKs). In turn, JAKs phosphorylate tyrosine residues in the receptor tails, leading to recruitment and activation of signalling molecules. Among these, signal transducers and activators of transcription (STATs) are important in the direct transmission of signals to the nucleus. Here, we show that incorporation of an interaction trap in a signalling-deficient receptor allows the identification of protein-protein interactions, using a STAT-dependent complementation assay. Mammalian protein-protein interaction trap (MAPPIT) adds to existing yeast two-hybrid procedures, as originally explored by Fields and Song, and permits the detection of both modification-independent and of phosphorylation-dependent interactions in intact human cells. We also demonstrate that MAPPIT can be used to screen complex complementary DNA libraries, and using this approach, we identify cytokine-inducible SH2-containing protein (CIS) and suppressor of cytokine signalling-2 (SOCS-2) as interaction partners of the phosphotyrosine 402 (Tyr 402)-binding motif in the erythropoietin receptor (EpoR). Importantly, this approach places protein-protein interactions in their normal physiological context, and is especially applicable to the in situ analysis of signal transduction pathways.

Analysis of leptin signalling in hematopoietic cells using an adapted MAPPIT strategy.

The adipocyte-secreted hormone leptin participates in the regulation of hematopoiesis and enhances proliferation of hematopoietic cells. We used an adaptation of the MAPPIT mammalian two-hybrid method to study leptin signalling in a hematopoietic setting. We confirmed the known interactions of suppressor of cytokine signalling 3 (SOCS3) and STAT5 with the Y985 and Y1077 motifs of the leptin receptor, respectively. We also provide evidence for novel interactions at the Y1077 motif, including phospholipase C gamma and several members of the SOCS protein family, further underscoring the important role of the Y1077 motif in leptin signalling.

Characterization of the mouse Men1 gene and its expression during development.

The gene responsible for multiple endocrine neoplasia type 1 (MEN1), a heritable predisposition to endocrine tumours in man, has recently been identified. Here we have characterized the murine homologue with regard to cDNA sequence, genomic structure, expression pattern and chromosomal localisation. The murine Men1 gene spans approximately 6.7 kb of genomic DNA and is comprised of 10 exons with similar genomic structure to the human locus. It was mapped to the pericentromeric region of mouse chromosome 19, which is conserved with the human 11q13 band where MEN1 is located. The predicted protein is 611 amino acids in length and overall is 97% homologous to the human orthologue. The 45 reported MEN1 mutations which alter or delete a single amino acid in human all occur at conserved residues, thereby supporting their functional significance. Two transcripts of approximately 3.2 and 2.8 kb were detected in both embryonal and adult murine tissues, resulting from alternative splicing of intron 1. By RNA in situ hybridization and Northern analysis the spatiotemporal expression pattern of Men1 was determined during mouse development. Men1 gene activity was detected already at gestational day 7. At embryonic day 14 expression was generally high throughout the embryo, while at day 17 the thymus, skeletal muscle, and CNS showed the strongest signal. In selected tissues from postnatal mouse Men1 was detected in all tissues analysed and was expressed at high levels in cerebral cortex, hippocampus, testis, and thymus. In brain the menin protein was detected mainly in nerve cell nuclei, whereas in testis it appeared perinuclear in spermatogonia. These results show that Men1 expression is not confined to organs affected in MEN1, suggesting that Men1 has a significant function in many different cell types including the CNS and testis.

Lathyrogens and the role of collagen in the eruption of rat incisors.

Collagen fibres in the periodontal ligament may have two functions: to resist displacing forces and to cause the tooth to erupt. Their function was examined in the continuously-erupting incisor of the rat using various concentrations and types of lathyrogens. Lathyrogens retarded tooth eruption and increased the quantity of salt-soluble collagen in the ligament, indicating inhibition of the maturation of salt-soluble (young) collagen into salt-insoluble (old) collagen, which would lead to reduction in the tensile strength of the fibres and decrease resistance to occlusal forces. The easy extractability of the teeth is explained by the greater susceptibility to lathyrogens of the fibres in the alveolar-related part of the periodontal ligament, compared with those in the other parts.

Immunofluorescent localization and extractability of fibronectin in human dental pulp.

Fibronectin, visualized in premolar pulps by indirect immunofluorescence, was abundant in the odontoblast layer, around blood vessels and in the core of the pulp. Similarity of alignment of fibronectin with the argyrophilic fibres and von Korff fibres was evident. Fibronectin was extracted from pulps after first removing blood by washing with water, confirmed by eventual negative reaction on alpha 2-macroglobulin. Extraction of fibronectin from this remaining tissue was most effectively achieved by treatment with collagenase or hyaluronidase, though in all cases some fibronectin remained, indicating that fibronectin in pulp is not exclusively associated with collagen and/or proteoglycans. The fibronectin quantified by electro-immunoassay and expressed as percentage of dry weight was 0.030 per cent in the water extract, 0.094 per cent in the collagenase extract and 0.109 per cent in the hyaluronidase extract. Twice as much fibronectin was extracted from the apical pulp as from the coronal and middle parts, in accord with earlier findings of a higher collagen content in the radicular part. It is suggested that with the loss of collagen type III during odontoblast differentiation and its reappearance with advancing vascularization of the dental papilla, the amount of fibronectin is similarly altered.

The concentration, extractability and characterization of collagen in human dental pulp.

Premolar and third molar dental pulps were studied. The amount of collagen in the dried pulps was 25.7 per cent in premolars and 31.9 per cent in third molars. These percentages are much higher than those reported for pulps in other species. Significant differences were further found in the collagen content and cell distribution (DNA) of the coronal, middle and apical parts of the pulp. Collagen content was the lowest in the coronal part, while the cell content was the lowest in the middle part. The extractability of collagen in a neutral salt solution or 0.5 M acetic acid was found to be extremely low (less than 1 per cent). Pretreatment of the pulp with hyaluronidase in order to remove proteoglycans had no effect on the solubility. It is concluded that human pulp collagen is highly cross-linked and cannot be considered as immature. Characterization of collagen was performed by methods in which limited pepsin digestion or CNBr cleavage was used. The digests were analysed by means of quantitative electrophoresis which revealed an amount of 42.6 per cent type III of the total collagen. Because of the large differences between dental pulps from man and experimental animals, extreme caution should be exercised in drawing conclusions from data of other species to explain phenomena observed in human teeth.

Development and preliminary evaluation of two speech communication aids.

In this paper two projects on speech communication aids for the speech-impaired are discussed. Since the group of speech-impaired people is very diverse, the two projects differ in target group, in the speech technology used and consequently in the complexity of input of the aids. In both projects (Pocketstem and Tiepstem) experimental models have been developed, and in the case of Pocketstem prototypes have been developed and evaluated by potential users. This paper describes the aids, their evaluation, the evaluation results and some conclusions and future plans.

The timing of prehensile movements in subjects with cerebral palsy.

In this study, a paradigm is presented for the assessment of manual dexterity in subjects with cerebral palsy (CP) that divides the prehensile action into a 'time-to-contact' phase and a 'time-in-contact' phase. Two experiments were performed that determined the effect of object weight on the timing of both phases for the impaired hand and non-impaired hand of subjects with spastic hemiparesis (N = 14). In the first experiment, subjects had to reach for and lift a tube at their own preferred speed. The results showed that the prehensile deficit of the impaired limb is to a large degree manifested by a longer time spent in contact with the object before it was lifted. The time-in-contact phase was decreased after repeated lifts, suggesting that subjects with CP can control and modify force output in advance based on weight information from preceding lifts. In the second experiment speed of movement execution was stressed to examine whether the observed timing pattern of the first experiment is characteristic of prehensile movements of the paretic arm or represents a movement strategy adapted to the disorder. The results of the second experiment showed that subjects could comply with the instruction by reducing the absolute duration of both phases of the prehensile movement. Furthermore, the anticipation effects were eliminated to a large degree. In both experiments the time-in-contact phase was longer for the impaired limb. These results indicate a pathological constant in the time-in-contact phase for the impaired limb. This assumption is discussed in relation to the application of grip and lift forces during this phase. It is concluded that the paradigm is well suited for use in a practical setting as a simple and broad clinical test to assess the prehensile decrements of subjects with CP.

Identification and molecular characterization of TM7SF2 in the FAUNA gene cluster on human chromosome 11q13.

In this report, the identification and molecular characterization of a novel gene, designated TM7SF2, is reported. This gene was found in the FAU neighboring area (FAUNA) to which other genes have been mapped previously. The FAUNA gene cluster is located at chromosome 11q13 between landmarks H4B and D11S2196E. The TM7SF2 gene contains eight coding exons, and their splice site consensus sequences are consistent with AG/GT rule. Northern blot analysis with a cDNA probe corresponding to TM7SF2 revealed varying expression levels of a 1.7-kb transcript in adult human heart, brain, pancreas, lung, liver, skeletal muscle, kidney, ovary, prostate, and testis, but no detectable expression in placenta, spleen, thymus, small intestine, colon (mucosal lining), or peripheral blood leukocytes. The open reading frame in the cDNA sequence codes for a protein of 590 amino acids that is rich in glycine (23%) and arginine (17%) residues in its amino-terminal half and contains seven transmembrane domains in its carboxy-terminal half. The transmembrane region of the putative TM7SF2 protein shows amino acid sequence similarity to those of the lamin B receptor and the C14/C24 sterol reductase.

MAPPIT: a cytokine receptor-based two-hybrid method in mammalian cells.

Identifying novel targets for therapy in allergic disease: protein interactions inside the cell Therapy of allergic disease currently relies on pharmacological manipulation of mediators or immunotherapy. Drugs have been developed to target specific mediators and their receptors: for example antihistamines blocking the H1 receptor have been refined to maximize antagonism and reduce central side-effects or adverse effects of activity on other receptors such as muscarinic cholinergic receptors. Traditional pharmacological approaches identify new surface receptors against which chemists will then design or screen compounds for activity: examples are H3 or H4 histamine receptors. With the advent of the sequenced human genome we are faced with a vast array of genes and proteins that interact to define normal physiology or indeed pathology. A major challenge to biotechnology is to evolve novel techniques to understand the function and interaction of these myriad proteins. One particular area of current interest is the signalling cascades downstream of surface receptors. For many years pathways have appeared overlapping and to offer little chance of specific intervention. However, greater understanding of the complexity and integration of signalling, together with the possibility of directing drugs to specific cells has aroused considerable interest in this area for novel therapeutics. Indeed, targeting events within the cell has been done for many years with steroids. Here, Jan Tavernier and colleagues describe some signalling pathways relevant to allergic disease and potential methods for understanding protein interactions that allow mapping of the cascades. In particular they describe an elegant new system of analysis of protein-protein interactions in a mammalian system, which they have developed, termed MAPPIT. The basis of the system is an engineered receptor with JAK kinase but which lacks STAT activation sites. To the cytoplasmic end of the receptor is added a bait protein of interest, and the cell line can then be transduced with plasmid containing 'prey' cDNA from a library of interest linked to an active STAT binding site. If this cDNA encodes a protein which, upon expression, is activated and recruited to the membrane complex, it will bind to the receptor via the bait, then STAT activation will occur and activate a reporter gene system such as luciferase or puromycin resistance. This novel system allows study of known protein-protein interactions by targeted mutagenesis, or screening for novel interactions. It has the advantage over existing systems such as yeast 2 hybrid that it uses mammalian cells and thus can reproduce the physiological conditions for protein processing or activation. As new genes and proteins are linked to the atopic phenotypes, systems such as this hold promise of rapidly defining their function and interacting proteins and may be important in linking genomics and proteomics with function and pharmacology in the future.

Menin interacts directly with the homeobox-containing protein Pem.

The tumour suppressor gene causing multiple endocrine neoplasia type 1 (MEN1) encodes a 610 amino acid protein, menin. In order to identify menin-interacting proteins we used a yeast two-hybrid assay to screen a 12.5-dpc mouse embryo library with partial menin encompassing amino acids 278 to 476. This identified a homeobox containing protein encoded by a placenta and embryonic expression gene, referred to as Pem. GST-pull-down and coimmunoprecipitation experiments confirmed the interaction. Both proteins colocalised predominantly in the nucleus but were occasionally also found in the cytoplasm. Furthermore, in situ hybridisation studies revealed similarities in their expression patterns in mouse embryos and adult tissues. In adult mice both Men1 and Pem yielded strong signals in testis, Sertoli cells and particularly in seminiferous tubules. Thus, our study has identified that menin interacts with Pem, and the high expression of these proteins in the testis suggests a role in spermatogenesis.

Construction of a 1.2-Mb sequence-ready contig of chromosome 11q13 encompassing the multiple endocrine neoplasia type 1 (MEN1) gene. The European Consortium on MEN1.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant familial cancer syndrome characterized by parathyroid, pancreatic, and anterior pituitary tumors. The MEN1 locus has been previously localized to chromosome 11q13, and a 2-Mb gene-rich region flanked by D11S1883 and D11S449 has been defined. We have pursued studies to facilitate identification of the MEN1 gene by narrowing this critical region to a 900-kb interval between the VRF and D11S1783 loci through melotic mapping. This was achieved by investigating 17 cosmids for microsatellite polymorphisms, which defined two novel polymorphisms at the VRF and A0138 loci, and utilizing these to characterize recombinants in MEN1 families. In addition, we have established a 1200-kb sequence-ready contig consisting of 26 cosmids, eight BACs, and eight PACs that encompass this region. The precise locations for 19 genes and three ESTs within this contig have been determined, and three gene clusters consisting of a centromeric group (VRF, FKBP2, PNG, and PLCB3), a middle group (PYGM, ZFM1, SCG1, SCG2 (which proved to be the MEN1 gene), and PPP2R5B), and a telomeric group (H4B, ANG3, ANG2, ANG1, FON, FAU, NOF, NON, and D11S2196E) were observed. These results represent a valuable transcriptional map of chromosome 11q13 that will help in the search for disease genes in this region.

Identification of the multiple endocrine neoplasia type 1 (MEN1) gene. The European Consortium on MEN1.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterised by tumours of the parathyroids, pancreas and anterior pituitary that represents one of the familial cancer syndromes. The MEN1 locus has been previously localised to chromosome 11q13, and a <300 kb gene-rich region flanked centromerically by PYGM and telomerically by D11S1783 defined by combined meiotic and tumour deletion mapping studies. Two candidate genes, ZFM1 and PPP2R5B, from this region have been previously excluded, and in order to identify additional candidate genes we used a BAC to isolate cDNAs from a bovine parathyroid cDNA library by direct selection. One of the novel genes that we identified, SCG2, proved to be identical to the recently published MEN1 gene, which is likely to be a tumour suppressor gene. The SCG2 transcript was 2.9 kb in all tissues with an additional 4.2 kb transcript also being present in the pancreas and thymus. Mutational analysis of SCG2 in 10 unrelated MEN1 families identified one polymorphism and nine different heterozygous mutations (one missense, four non-sense, one insertional and three deletional frameshifts) that segregated with the disease, hence providing an independent confirmation for the identification of the MEN1 gene.

A putative human zinc-finger gene (ZFPL1) on 11q13, highly conserved in the mouse and expressed in exocrine pancreas. The European Consortium on MEN 1.

In the process of identification of the multiple endocrine neoplasia type 1 gene, which was recently published, we isolated a novel gene in the 11q13 region. This gene (named ZFPL1, for zinc-finger protein-like 1) is expressed strongly in the exocrine pancreas as a 1.4-kb polyadenylated RNA encoding a putative protein of 310 amino acids. A mouse EST contig predicts an equally sized murine protein with 91% amino acid sequence identity to the human protein. No significant homology with known proteins could be found through database screening. However, zinc-finger-like domains and leucine-zipper-like motifs in the predicted ZFPL1 protein were identified, suggesting the presence of DNA-binding and dimerization domains possibly involved in transcription regulation. This notion is supported by the presence of a putative bipartite nuclear localization signal. This paper presents the full-length cDNA sequence for this gene, its genomic structure and chromosomal orientation, and expression studies by Northern blot hybridization and RNA in situ hybridization.