Spermine modulation of the glutamate(NMDA) receptor is differentially responsive to conantokins in normal and Alzheimer's disease human cerebral cortex

The pharmacology of the N -methyl-d-aspartate (NMDA) receptor site was examined in pathologically affected and relatively spared regions of cerebral cortex tissue obtained at autopsy from Alzheimer's disease cases and matched controls. The affinity and density of the [H-3]MK-801 binding site were delineated along with the enhancement of [H-3]MK-801 binding by glutamate and spermine. Maximal enhancement induced by either ligand was regionally variable; glutamate-mediated maximal enhancement was higher in controls than in Alzheimer's cases in pathologically spared regions, whereas spermine-mediated maximal enhancement was higher in controls in areas susceptible to pathological damage. These and other data suggest that the subunit composition of NMDA receptors may be locally variable. Studies with modified conantokin-G (con-G) peptides showed that Ala(7)-con-G had higher affinity than Lys(7)-con-G, and also defined two distinct binding sites in controls. Nevertheless, the affinity for Lys(7)-con-G was higher overall in Alzheimer's brain than in control brain, whereas the reverse was true for Ala(7)-con-G. Over-excitation mediated by specific NMDA receptors might contribute to localized brain damage in Alzheimer's disease. Modified conantokins are useful for identifying the NMDA receptors involved, and may have potential as protective agents.

Roles of fibroblast growth factors in the inner ear

The basic biology of the fibroblast growth factor (FGF) receptors and their splice variants is first reviewed, followed by a review of the known roles of FGFs in the inner ear. They include induction of the otocyst by FGF19, followed by FGF3 in further development of the otocyst. In later development, FGF3 or FGF10 acting on FGF receptor 2b is likely to be involved in development of the walls of the cochlear spaces, while FGF receptor 3 is involved in differentiation of the pillar cells of the organ of Corti. FGF1 and FGF2 act as trophic factors for the developing cochlear nerve fibres. Copyright (C) 2002 S. Karger AG, Basel.

Quantitation of alternatively spliced NMDA receptor NR1 isoform mRNA transcripts in human brain by competitive RT-PCR

The N-methyl-D-aspartate (NMDA)-selective subtype of ionotropic glutamate receptor is of importance in neuronal differentiation and synapse consolidation, activity-dependent forms of synaptic plasticity, and excitatory amino acid-mediated neuronal toxicity [Neurosci. Res. Program, Bull. 19 (1981) 1; Lab. Invest. 68 (1993) 372]. NMDA receptors exist in vivo as tetrameric or pentameric complexes comprising proteins from two families of homologous subunits, designated NR1 and NR2(A-D) [Biochem. Biophys. Res. Commun. 185 (1992) 826]. The gene coding for the human NR1 subunit (hNR1) is composed of 21 exons, three of which (4, 20 and 21) can be differentially spliced to generate a total of eight distinct subunit variants. We detail here a competitive RT-PCR (cRT-PCR) protocol to quantify endogenous levels of hNR1 splice variants in autopsied human brain. Quantitation of each hNR1 splice variant is performed using standard curve methodology in which a known amount of synthetic ribonucleic acid competitor (internal standard) is co-amplified against total RNA. This method can be used for the quantitation of hNR1 mRNA levels in response to acute or chronic disease states, in particular in the glutamatergic-associated neuronal loss observed in Alzheimer's disease [J. Neurochem. 78 (2001) 175]. Furthermore, alterations in hNR1 mRNA expression may be reflected at the translational level, resulting in functional changes in the NMDA receptor. (C) 2003 Elsevier Science B.V. All rights reserved.

Interactions of tumor necrosis factor (TNF) and TNF receptor family members in the mouse and human.

Ligands of the tumor necrosis factor superfamily (TNFSF) (4-1BBL, APRIL, BAFF, CD27L, CD30L, CD40L, EDA1, EDA2, FasL, GITRL, LIGHT, lymphotoxin alpha, lymphotoxin alphabeta, OX40L, RANKL, TL1A, TNF, TWEAK, and TRAIL) bind members of the TNF receptor superfamily (TNFRSF). A comprehensive survey of ligand-receptor interactions was performed using a flow cytometry-based assay. All ligands engaged between one and five receptors, whereas most receptors only bound one to three ligands. The receptors DR6, RELT, TROY, NGFR, and mouse TNFRH3 did not interact with any of the known TNFSF ligands, suggesting that they either bind other types of ligands, function in a ligand-independent manner, or bind ligands that remain to be identified. The study revealed that ligand-receptor pairs are either cross-reactive between human and mouse (e.g. Tweak/Fn14, RANK/RANKL), strictly species-specific (GITR/GITRL), or partially species-specific (e.g. OX40/OX40L, CD40/CD40L). Interestingly, the receptor binding patterns of lymphotoxin alpha and alphabeta are redundant in the human but not in the mouse system. Ligand oligomerization allowed detection of weak interactions, such as that of human TNF with mouse TNFR2. In addition, mouse APRIL exists as two different splice variants differing by a single amino acid. Although human APRIL does not interact with BAFF-R, the shorter variant of mouse APRIL exhibits weak but detectable binding to mouse BAFF-R.

LEDGF/p75 TATA-less promoter is driven by the transcription factor Sp1.

PSIP1 (PC4 and SFRS1 interacting protein 1) encodes two splice variants: lens epithelium-derived growth factor or p75 (LEDGF/p75) and p52. PSIP1 gene products were shown to be involved in transcriptional regulation, affecting a plethora of cellular processes, including cell proliferation, cell survival, and stress response. Furthermore, LEDGF/p75 has implications for various diseases and infections, including autoimmunity, leukemia, embryo development, psoriasis, and human immunodeficiency virus integration. Here, we reported the first characterization of the PSIP1 promoter. Using 5' RNA ligase-mediated rapid amplification of cDNA ends, we identified novel transcription start sites in different cell types. Using a luciferase reporter system, we identified regulatory elements controlling the expression of LEDGF/p75 and p52. These include (i) minimal promoters (-112/+59 and +609/+781) that drive the basal expression of LEDGF/p75 and of the shorter splice variant p52, respectively; (ii) a sequence (+319/+397) that may control the ratio of LEDGF/p75 expression to p52 expression; and (iii) a strong enhancer (-320/-207) implicated in the modulation of LEDGF/p75 transcriptional activity. Computational, biochemical, and genetic approaches enabled us to identify the transcription factor Sp1 as a key modulator of the PSIP1 promoter, controlling LEDGF/p75 transcription through two binding sites at -72/-64 and -46/-36. Overall, our results provide initial data concerning LEDGF/p75 promoter regulation, giving new insights to further understand its biological function and opening the door for new therapeutic strategies in which LEDGF/p75 is involved.

MyHits: a new interactive resource for protein annotation and domain identification.

The MyHits web server (http://myhits.isb-sib.ch) is a new integrated service dedicated to the annotation of protein sequences and to the analysis of their domains and signatures. Guest users can use the system anonymously, with full access to (i) standard bioinformatics programs (e.g. PSI-BLAST, ClustalW, T-Coffee, Jalview); (ii) a large number of protein sequence databases, including standard (Swiss-Prot, TrEMBL) and locally developed databases (splice variants); (iii) databases of protein motifs (Prosite, Interpro); (iv) a precomputed list of matches ('hits') between the sequence and motif databases. All databases are updated on a weekly basis and the hit list is kept up to date incrementally. The MyHits server also includes a new collection of tools to generate graphical representations of pairwise and multiple sequence alignments including their annotated features. Free registration enables users to upload their own sequences and motifs to private databases. These are then made available through the same web interface and the same set of analytical tools. Registered users can manage their own sequences and annotations using only web tools and freeze their data in their private database for publication purposes.

Identification of new MHC class I-restricted human tumor antigens for immunotherapy

Summary Cancer is a leading cause of morbidity and mortality in Western countries (as an example, colorectal cancer accounts for about 300'000 new cases and 200'000 deaths each year in Europe and in the USA). Despite that many patients with cancer have complete macroscopic clearance of their disease after resection, radiotherapy and/or chemotherapy, many of these patients develop fatal recurrence. Vaccination with immunogenic peptide tumor antigens has shown encouraging progresses in the last decade; immunotherapy might therefore constitute a fourth therapeutic option in the future. We dissect here and critically evaluate the numerous steps of reverse immunology, a forecast procedure to identify antigenic peptides from the sequence of a gene of interest. Bioinformatic algorithms were applied to mine sequence databases for tumor-specific transcripts. A quality assessment of publicly available sequence databanks allowed defining strengths and weaknesses of bioinformatics-based prediction of colon cancer-specific alternative splicing: new splice variants could be identified, however cancer-restricted expression could not be significantly predicted. Other sources of target transcripts were quantitatively investigated by polymerase chain reactions, as cancer-testis genes or reported overexpressed transcripts. Based on the relative expression of a defined set of housekeeping genes in colon cancer tissues, we characterized a precise procedure for accurate normalization and determined a threshold for the definition of significant overexpression of genes in cancers versus normal tissues. Further steps of reverse immunology were applied on a splice variant of the Melan¬A gene. Since it is known that the C-termini of antigenic peptides are directly produced by the proteasome, longer precursor and overlapping peptides encoded by the target sequence were synthesized chemically and digested in vitro with purified proteasome. The resulting fragments were identified by mass spectroscopy to detect cleavage sites. Using this information and based on the available anchor motifs for defined HLA class I molecules, putative antigenic peptides could be predicted. Their relative affinity for HLA molecules was confirmed experimentally with functional competitive binding assays and they were used to search patients' peripheral blood lymphocytes for the presence of specific cytolytic T lymphocytes (CTL). CTL clones specific for a splice variant of Melan-A could be isolated; although they recognized peptide-pulsed cells, they failed to lyse melanoma cells in functional assays of antigen recognition. In the conclusion, we discuss advantages and bottlenecks of reverse immunology and compare the technical aspects of this approach with the more classical procedure of direct immunology, a technique introduced by Boon and colleagues more than 10 years ago to successfully clone tumor antigens.

Inactivation of L-type calcium channels is determined by the length of the N terminus of mutant beta(1) subunits.

Voltage-dependent calcium channel (Ca(v)) pores are modulated by cytosolic beta subunits. Four beta-subunit genes and their splice variants offer a wide structural array for tissue- or disease-specific biophysical gating phenotypes. For instance, the length of the N terminus of beta(2) subunits has major effects on activation and inactivation rates. We tested whether a similar mechanism principally operates in a beta(1) subunit. Wild-type beta(1a) subunit (N terminus length 60 aa) and its newly generated N-terminal deletion mutants (51, 27 and 18 aa) were examined within recombinant L-type calcium channel complexes (Ca(v)1.2 and alpha(2)delta2) in HEK293 cells at the whole-cell and single-channel level. Whole-cell currents were enhanced by co-transfection of the full-length beta(1a) subunit and by all truncated constructs. Voltage dependence of steady-state activation and inactivation did not depend on N terminus length, but inactivation rate was diminished by N terminus truncation. This was confirmed at the single-channel level, using ensemble average currents. Additionally, gating properties were estimated by Markov modeling. In confirmation of the descriptive analysis, inactivation rate, but none of the other transition rates, was reduced by shortening of the beta(1a) subunit N terminus. Our study shows that the length-dependent mechanism of modulating inactivation kinetics of beta(2) calcium channel subunits can be confirmed and extended to the beta(1) calcium channel subunit.

GENCODE: producing a reference annotation for ENCODE

Background: The GENCODE consortium was formed to identify and map all protein-coding genes within the ENCODE regions. This was achieved by a combination of initial manualannotation by the HAVANA team, experimental validation by the GENCODE consortium and a refinement of the annotation based on these experimental results.Results: The GENCODE gene features are divided into eight different categories of which onlythe first two (known and novel coding sequence) are confidently predicted to be protein-codinggenes. 5’ rapid amplification of cDNA ends (RACE) and RT-PCR were used to experimentallyverify the initial annotation. Of the 420 coding loci tested, 229 RACE products have beensequenced. They supported 5’ extensions of 30 loci and new splice variants in 50 loci. In addition,46 loci without evidence for a coding sequence were validated, consisting of 31 novel and 15putative transcripts. We assessed the comprehensiveness of the GENCODE annotation byattempting to validate all the predicted exon boundaries outside the GENCODE annotation. Outof 1,215 tested in a subset of the ENCODE regions, 14 novel exon pairs were validated, only twoof them in intergenic regions.Conclusions: In total, 487 loci, of which 434 are coding, have been annotated as part of theGENCODE reference set available from the UCSC browser. Comparison of GENCODEannotation with RefSeq and ENSEMBL show only 40% of GENCODE exons are contained withinthe two sets, which is a reflection of the high number of alternative splice forms with uniqueexons annotated. Over 50% of coding loci have been experimentally verified by 5’ RACE forEGASP and the GENCODE collaboration is continuing to refine its annotation of 1% humangenome with the aid of experimental validation.

A tumor-specific cellular environment at the brain invasion border of adamantinomatous craniopharyngiomas.

Craniopharyngiomas (CP) are benign epithelial tumors of the sellar region and can be clinicopathologically distinguished into adamantinomatous (adaCP) and papillary (papCP) variants. Both subtypes are classified according to the World Health Organization grade I, but their irregular digitate brain infiltration makes any complete surgical resection difficult to obtain. Herein, we characterized the cellular interface between the tumor and the surrounding brain tissue in 48 CP (41 adaCP and seven papCP) compared to non-neuroepithelial tumors, i.e., 12 cavernous hemangiomas, 10 meningiomas, and 14 metastases using antibodies directed against glial fibrillary acid protein (GFAP), vimentin, nestin, microtubule-associated protein 2 (MAP2) splice variants, and tenascin-C. We identified a specific cell population characterized by the coexpression of nestin, MAP2, and GFAP within the invasion niche of the adamantinomatous subtype. This was especially prominent along the finger-like protrusions. A similar population of presumably astroglial precursors was not visible in other lesions under study, which characterize them as distinct histopathological feature of adaCP. Furthermore, the outer tumor cell layer of adaCP showed a distinct expression of MAP2, a novel finding helpful in the differential diagnosis of epithelial tumors in the sellar region. Our data support the hypothesis that adaCP, unlike other non-neuroepithelial tumors of the central nervous system, create a tumor-specific cellular environment at the tumor-brain junction. Whether this facilitates the characteristic infiltrative growth pattern or is the consequence of an activated Wnt signaling pathway, detectable in 90% of these tumors, will need further consideration.

Mutations leading to X-linked hypohidrotic ectodermal dysplasia affect three major functional domains in the tumor necrosis factor family member ectodysplasin-A.

Mutations in the epithelial morphogen ectodysplasin-A (EDA), a member of the tumor necrosis factor (TNF) family, are responsible for the human disorder X-linked hypohidrotic ectodermal dysplasia (XLHED) characterized by impaired development of hair, eccrine sweat glands, and teeth. EDA-A1 and EDA-A2 are two splice variants of EDA, which bind distinct EDA-A1 and X-linked EDA-A2 receptors. We identified a series of novel EDA mutations in families with XLHED, allowing the identification of the following three functionally important regions in EDA: a C-terminal TNF homology domain, a collagen domain, and a furin protease recognition sequence. Mutations in the TNF homology domain impair binding of both splice variants to their receptors. Mutations in the collagen domain can inhibit multimerization of the TNF homology region, whereas those in the consensus furin recognition sequence prevent proteolytic cleavage of EDA. Finally, a mutation affecting an intron splice donor site is predicted to eliminate specifically the EDA-A1 but not the EDA-A2 splice variant. Thus a proteolytically processed, oligomeric form of EDA-A1 is required in vivo for proper morphogenesis.

Mouse GLUT9: evidences for a urate uniporter.

GLUT9 (SLC2A9) is a newly described urate transporter whose function, characteristics, and localization have just started to be elucidated. Some transport properties of human GLUT9 have been studied in the Xenopus laevis oocyte expression system, but the type of transport (uniport, coupled transport system, stoichiometry ... .) is still largely unknown. We used the same experimental system to characterize in more detail the transport properties of mouse GLUT9, its sensitivity to several uricosuric drugs, and the specificities of two splice variants, mGLUT9a and mGLUT9b. [(14)C]urate uptake measurements show that both splice variants are high-capacity urate transporters and have a K(m) of approximately 650 microM. The well-known uricosuric agents benzbromarone (500 microM) and losartan (1 mM) inhibit GLUT9-mediated urate uptake by 90 and 50%, respectively. Surprisingly, phloretin, a glucose-transporter blocker, inhibits [(14)C]urate uptake by approximately 50% at 1 mM. Electrophysiological measurements suggest that urate transport by mouse GLUT9 is electrogenic and voltage dependent, but independent of the Na(+) and Cl(-) transmembrane gradients. Taken together, our results suggest that GLUT9 works as a urate (anion) uniporter. Finally, we show by RT-PCR performed on RNA from mouse kidney microdissected tubules that GLUT9a is expressed at low levels in proximal tubules, while GLUT9b is specifically expressed in distal convoluted and connecting tubules. Expression of mouse GLUT9 in the kidney differs from that of human GLUT9, which could account for species differences in urate handling.

Practical considerations for improving the productivity of mass spectrometry-based proteomics.

Mass spectrometry (MS) is currently the most sensitive and selective analytical technique for routine peptide and protein structure analysis. Top-down proteomics is based on tandem mass spectrometry (MS/ MS) of intact proteins, where multiply charged precursor ions are fragmented in the gas phase, typically by electron transfer or electron capture dissociation, to yield sequence-specific fragment ions. This approach is primarily used for the study of protein isoforms, including localization of post-translational modifications and identification of splice variants. Bottom-up proteomics is utilized for routine high-throughput protein identification and quantitation from complex biological samples. The proteins are first enzymatically digested into small (usually less than ca. 3 kDa) peptides, these are identified by MS or MS/MS, usually employing collisional activation techniques. To overcome the limitations of these approaches while combining their benefits, middle-down proteomics has recently emerged. Here, the proteins are digested into long (3-15 kDa) peptides via restricted proteolysis followed by the MS/MS analysis of the obtained digest. With advancements of high-resolution MS and allied techniques, routine implementation of the middle-down approach has been made possible. Herein, we present the liquid chromatography (LC)-MS/MS-based experimental design of our middle-down proteomic workflow coupled with post-LC supercharging.

Permanent correction of an inherited ectodermal dysplasia with recombinant EDA.

X-linked hypohidrotic ectodermal dysplasia (XLHED; OMIM 305100) is a genetic disorder characterized by absence or deficient function of hair, teeth and sweat glands. Affected children may experience life-threatening high fever resulting from reduced ability to sweat. Mice with the Tabby phenotype share many symptoms with human XLHED patients because both phenotypes are caused by mutations of the syntenic ectodysplasin A gene (Eda) on the X chromosome. Two main splice variants of Eda, encoding EDA1 and EDA2, engage the tumor necrosis factor (TNF) family receptors EDAR and XEDAR, respectively. The EDA1 protein, acting through EDAR, is essential for proper formation of skin appendages; the functions of EDA2 and XEDAR are not known. EDA1 must be proteolytically processed to a soluble form to be active. Here, we show that treatment of pregnant Tabby mice with a recombinant form of EDA1, engineered to cross the placental barrier, permanently rescues the Tabby phenotype in the offspring. Notably, sweat glands can also be induced by EDA1 after birth. This is the first example of a developmental genetic defect that can be permanently corrected by short-term treatment with a recombinant protein.

Changes of MAP2 phosphorylation during brain development.

The microtubule-associated protein MAP2 is essential for development of early neuronal morphology and maintenance of adult neuronal morphology. Several splice variants exist, MAP2a-d, with a lack of MAP2a in cat brain. MAP2 is widely used as a neuronal marker. In this study we compared five monoclonal antibodies (MAbs) against MAP2. They show differences in the immunocytochemical distribution of MAP2 isoforms during development of the visual cortex and cerebellum of the cat. Local and temporal differences were seen with MAb AP18, an antibody directed against a phosphorylation-dependent epitope near the N-terminal end. In large pyramidal dendrites in visual cortex, the AP18 epitope remained in parts immunoreactive after treatment with alkaline phosphatase. Three MAbs, AP14, MT-01, and MT-02, recognized the central region of the MAP2b molecule, which is not present in MAP2c and 2d, and reacted with phosphorylation-independent epitopes. During the first postnatal week the immunostaining in cerebellum differed between antibodies in that some cellular elements in external and internal granular layers and Purkinje cells were stained to various degrees, whereas at later stages staining patterns were similar. At early stages, antibody MT-02 stained cell bodies and dendrites in cerebral cortex and cerebellum. With progressing maturation, immunoreactivity became restricted to distal parts of apical dendrites of pyramidal cells and was absent from perikarya and finer proximal dendrites in cortex. MT-02 did not stain MAP2 in cerebellum of adult animals. This study demonstrates that the immunocytochemical detection of MAP2 depends on modifications such as phosphorylation and conformational changes of the molecule, and that MAP2 staining patterns differ between MAbs. Phosphorylation and specific conformations in the molecule may be essential for modulating function and molecular stability of MAP2, and monoclonal antibodies against such sites may provide tools for studying the functional role of modifications.