Functional expression of mammalian bitter taste receptors in Caenorhabditis elegans.

Bitter taste has evolved as a central warning signal against the ingestion of potentially toxic substances appearing in the environment. The molecular events in the perception of bitter taste start with the binding of specific water-soluble molecules to G protein-coupled receptors (GPCR) called T2Rs and expressed at the surface of taste receptor cells. The functional characterisation of T2R receptors is far from been completed due to the difficulty to functionally express them in heterologous systems. Taking advantage of the parallelisms between the Caenorhabditis elegans (C. elegans) and mammalian GPCR signalling pathways, we developed a C. elegans-based expression system to express functional human and rodent GPCRs of the T2R family. We generated transgenic worms expressing T2Rs in ASI chemosensory neurons and performed behavioural assays using a variety of bitter tastants. As a proof of the concept, we generated transgenic worms expressing human T2R4 or its mouse ortholog T2R8 receptors, which respond to two bitter tastants previously characterised as their functional ligands, 6-n-propyl-2-thiouracil and denatoniun. As expected, expression of human T2R4 or its mouse ortholog T2R8 in ASI neurons counteracted the water-soluble avoidance to 6-n-propyl-2-thiouracil and denatoniun observed in control wild-type worms. The expression in ASI neurons of human T2R16, the ligand of which, phenyl-beta-d-glucopyranoside, belong to a chemically different group of bitter tastants, also counteracted the water-soluble avoidance to this compound observed in wild-type worms. These results indicate that C. elegans is a suitable heterologous expression system to express functional T2Rs providing a tool to efficiently search for specific taste receptor ligands and to extend our understanding of the molecular basis of gustation.

Identification and characterization of human taste receptor genes belonging to the TAS2R family.

The sense of taste is a chemosensory system responsible for basic food appraisal. Humans distinguish between five primary tastes: bitter, sweet, sour, salty and umami. The molecular events in the perception of bitter taste are believed to start with the binding of specific water-soluble molecules to G-protein-coupled receptors encoded by the TAS2R/T2R family of taste receptor genes. TAS2R receptors are expressed at the surface of taste receptor cells and are coupled to G proteins and second messenger pathways. We have identified, cloned and characterized 11 new bitter taste receptor genes and four new pseudogenes that belong to the human TAS2R family. Their encoded proteins have between 298 and 333 amino acids and share between 23 and 86% identity with other human TAS2R proteins. Screening of a mono-chromosomal somatic cell hybrid panel to assign the identified bitter taste receptor genes to human chromosomes demonstrated that they are located in chromosomes 7 and 12. Including the 15 sequences identified, the human TAS2R family is composed of 28 full-length genes and 16 pseudogenes. Phylogenetic analyses suggest a classification of the TAS2R genes in five groups that may reflect a specialization in the detection of specific types of bitter chemicals.

Ramón y Cajal: a century after the publication of his masterpiece.

Santiago Ramón y Cajal published the first of the three volumes of his principal life's work in 1899, and published the last volume in 1904. This book remains the definitive work on the morphology of the vertebrate nervous system. In it, Ramón y Cajal describes the structure and organization of virtually all parts of the nervous system and discusses his theories, including the neuron doctrine and the law of functional polarization, which are the cornerstones of modern neurobiology. A century later, Ramón y Cajal's work is still fundamental to understanding the nervous system.

Id genes in nervous system development.

Id genes encode helix-loop-helix proteins that function to mediate processes important for normal development including cellular differentiation, proliferation and apoptosis. Id proteins act as negative regulators of other transcription factors, which are essential for cell determination and differentiation in diverse cell types, and interact with proteins important for cell cycle regulation. Studies of Id gene expression in the nervous system and in neural cells in culture indicate that Id proteins contribute to the regulation of mammalian nervous system development. Also, recognition of a wide variety of proteins with which Id transcription factors are capable of interacting suggests that it will be possible to understand more precisely their specific functions and importantly how these are integrated.

Id gene expression as a key mediator of tumor cell biology.

Id genes encode members of the helix-loop-helix (HLH) family of transcription factors that inhibit transcription by forming inactive heterodimers with basic HLH (bHLH) proteins. There are four members of the Id gene family recognized in mammals, and the proteins they encode share homology primarily in their HLH domain. bHLH proteins typically form heterodimers with other bHLH proteins, and their basic domain binds to a DNA sequence element, the E-box, activating transcription. Products of Id genes lack the basic DNA binding domain of the bHLH transcription factors, and when they heterodimerize with bHLH proteins, the complexes are inactive. Generally, high levels of Id mRNA are detected in proliferative undifferentiated, embryonal cells and lower levels are detected in well-differentiated, mature, adult tissues. In vitro, these genes are generally expressed at lower levels in cells after the induction of differentiation. Recently, high levels of expression of Id genes have been identified in cell lines derived from a wide variety of different tumors and in tumor tissues as well. These findings suggest that not only the inappropriate proliferation of tumors but also the anaplastic characteristics that contribute to their malignant behavior may be regulated by Id gene expression.

Id4 expression induces apoptosis in astrocytic cultures and is down-regulated by activation of the cAMP-dependent signal transduction pathway.

The Id family of helix-loop-helix transcription factors has been implicated in the regulation of cellular differentiation in several different lineages. We have explored the potential regulatory role of the cyclic AMP-dependent signaling pathway on Id gene expression in astroglial primary cultures. We found that primary cultures of mouse forebrain astrocytes constitutively expressed the four known members of the Id gene family, Id1, Id2, Id3, and Id4. During culture in presence of serum for 4 weeks, the expression of Id4 was up-regulated. In these same cultures, treatment with dibutyryl-cyclic AMP, a cyclic AMP analogue known to promote astrocyte differentiation, dramatically and selectively decreased Id4 gene expression. This effect was detectable after short-term treatment and was maintained during long-term treatment. Forskolin and pentoxifylline, two other agents known to elevate intracellular cyclic AMP through different mechanisms, also potently decreased Id4 gene expression. Furthermore, overexpression of Id4 in an astrocyte-derived cell line induced cells to round up and die by apoptosis. These results indicate that the cyclic AMP pathway acts as an inhibitor of Id4 gene expression in astrocytes, identify a new function for Id4, and suggest that Id4 is strategically positioned in the chain of molecular events regulating astrocyte differentiation and apoptosis.

Cloning of human ENC-1 and evaluation of its expression and regulation in nervous system tumors.

We recently identified and characterized a novel murine gene, ENC-1, that is expressed primarily in the nervous system and encodes an actin-binding protein. To gain insight into a potential role for ENC-1 gene in the processes of cell differentiation and malignant transformation in the human nervous system, we first cloned and characterized the human homologue of ENC-1. The human ENC-1 gene appeared to be highly expressed in adult brain and spinal cord, and in a number of cell lines derived from nervous system tumors we detected low steady-state levels of ENC-1 mRNA. We used a neuroblastoma differentiation model, the retinoic acid-induced neuronal differentiation of SMS-KCNR cells, to study the regulation of the ENC-1 gene during neural crest cell differentiation. We found that the expression of ENC-1 increased dramatically in the differentiated SMS-KCNR cells as compared to control undifferentiated cells. These results suggest that ENC-1 expression plays a role during differentiation of neural crest cells and may be down regulated in neuroblastoma tumors.

Injury selectively down-regulates the gene encoding for the Id4 transcription factor in primary cultures of forebrain astrocytes.

Astrogliosis is an important component of the response to injury of the central nervous system (CNS). The Id family of helix-loop-helix (HLH) transcription factors has been implicated in the regulation of cellular differentiation in several different lineages and may contribute to the regulation of astrogliosis. We examined the expression of Id genes in primary cultures of mouse forebrain astrocytes under experimental conditions in which astrogliosis was elicited by mechanical injury. Astrocyte cultures expressed the four known members of the Id gene family, Id1, Id2, Id3, and Id4. After injury, at a time when astrocytes developed the characteristic phenotypic changes of astrogliosis, Id4 expression decreased dramatically. Id1, Id2, and Id3 mRNA levels did not change. These results identify Id4 as a candidate marker of astroglial activation in culture and suggest that Id4 expression plays a role in the process of astrogliosis.

ENC-1: a novel mammalian kelch-related gene specifically expressed in the nervous system encodes an actin-binding protein.

We have identified and characterized a novel murine gene, Ectoderm-Neural Cortex-1 (ENC-1), that is an early and highly specific marker of neural induction in vertebrates. ENC-1, which encodes a kelch family related protein, is expressed during early gastrulation in the prospective neuroectodermal region of the epiblast and later in development throughout the nervous system (NS). ENC-1 expression is highly dynamic and, after neurulation, preferentially defines prospective cortical areas. The only apparent expression of ENC-1 outside the NS is restricted to the rostral-most somitomere of the presomitic mesoderm, at the times corresponding to the epithelialization that precedes somite formation. Cellular expression of epitope-tagged ENC-1 shows extensive co-localization of ENC-1 with the actin cytoskeleton, and immunoprecipitation studies demonstrate a physical association between ENC-1 and actin. ENC-1 functions as an actin-binding protein that may be important in the organization of the actin cytoskeleton during neural fate specification and development of the NS.

Id genes encoding inhibitors of transcription are expressed during in vitro astrocyte differentiation and in cell lines derived from astrocytic tumors.

Id proteins belong to a class of nuclear transcription factors known as helix-loop-helix proteins. It has been reported that Id genes function as negative regulators of differentiation, and Id gene expression is down-regulated during cell differentiation. We examined the regulation of Id genes during astrocyte differentiation in a murine nervous system precursor cell line, NSEHip2-28, which is able to differentiate along the astroglial lineage, as well as in human astroglial tumor cell lines. Upon induction of NSEHip2-28 differentiation, at a time when glial fibrillary acidic protein expression became detectable, the expression of all four Id family members initially increased dramatically, and subsequently decreased. Furthermore, varying levels of Id gene expression were found in astroglial tumor cell lines displaying variable degrees of lineage-specific differentiation. These results suggest that the expression of Id family members may play an important role in the control of astrocyte differentiation.

Molecular cloning of the cDNA encoding a helix-loop-helix protein, mouse ID1B: tissue-specific expression of ID1A and ID1B genes.

A cDNA encoding a 168 amino acid mouse ID1B helix-loop-helix protein, the longest among the ID family of proteins so far identified, was cloned and its nucleotide sequence determined. Mouse ID1B mRNA is distinguishable from the mRNA encoding ID1A at its 3' end, and the relative level of expression of these two different mRNAs is similar in most tissues, with exception of skeletal muscle and kidney. Comparison of the most carboxyl terminal predicted amino acid sequences of ID1 proteins reveals 100% identity for ID1A, but the predicted Id1B proteins of several species are different.

Expression of glial fibrillary acidic protein and glutamine synthetase genes in the natural scrapie of sheep.

Gene expression of two astroglial markers, glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS), was investigated in cerebellum and brainstem from scrapie-affected sheep. The GFAP and GFAP-mRNA concentrations were increased in the two cerebral regions studied in the scrapie-affected animals as compared to the controls. The good correlation between the increase in GFAP and GFAP-mRNA concentrations found in scrapie-affected sheep indicates a significant de novo synthesis of GFAP in this pathology. In contrast to these results, in scrapie no significant differences in GS-mRNA content appeared in either brain area from scrapie-affected sheep as compared to the controls. This fact could suggest some specificity of GFAP expression changes in this pathology. The overexpression of GFAP gene could be related to a possible interaction between GFAP and scrapie infectious agent in astrocytes. The relative increase in the GFAP and its encoding message in affected animals was higher in the cerebellum than in the brainstem, which would suggest regional comparative differences in the effect here described.

Thyroid hormones influence the astroglial plasticity: changes in the expression of glial fibrillary acidic protein (GFAP) and of its encoding message.

Normal development of the brain requires the presence of thyroid hormones. To progress in the understanding of the contribution of astrocytes to brain pathophysiology we investigated the effect of T3, on the astroglial plasticity through the expression of two astroglial proteins: the Glial fibrillary acidic protein (GFAP) and the glutamine synthetase (GS). Western and northern blots were performed using astroglial primary cultures initiated from neocortex and cerebellum of new-born mice. Treatment with T3 caused a decrease of GFAP and of its encoding message level in both areas, suggesting a transcriptional regulation of its expression, whereas it had no apparent effect on GS expression. This reduction in GFAP expression was developmentally regulated; it was significant in proliferating but not in more mature astrocytes. T3 effect on astrocytes was higher in the cerebellum compared to the neocortex, suggesting the presence of astroglial subpopulations differing by their sensitivity to T3. The astroglial specific response to T3, corresponds to a precise, targetted and regulated adaptation of the cell. Factors of the microenvironment may modulate this specific astroglial response in vivo.

Quantification of glial fibrillary acidic protein in developing sheep brain cortex, cerebellum and stem.

1. The glial fibrillary acidic protein (GFAP) content of foetal, young (lamb) and adult sheep brain white (stem and cerebellum) and grey (cortex) matter-enriched regions has been determined by means of an improved ELISA using one layer of anti-human GFAP monoclonal antibody. 2. The order of GFAP concentration in brain regions was as follows: brain stem greater than cerebellum greater than cortex. 3. Postnatal brain development accounts for an increase of GFAP in all the regions. The most important increase in GFAP was observed in the adult brain and was proportionally more significant in the grey matter-enriched cortex.