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S Schiffmann

Publications and source records attributed to S Schiffmann.

18 recordsLinked to original sources
PubMed2003

A force-based protein biochip.

A parallel assay for the quantification of single-molecule binding forces was developed based on differential unbinding force measurements where ligand-receptor interactions are compared with the unzipping forces of DNA hybrids. Using the DNA zippers as molecular force sensors, the efficient discrimination between specific and nonspecific interactions was demonstrated for small molecules binding to specific receptors, as well as for protein-protein interactions on protein arrays. Finally, an antibody sandwich assay with different capture antibodies on one chip surface and with the detection antibodies linked to a congruent surface via the DNA zippers was used to capture and quantify a recombinant hepatitis C antigen from solution. In this case, the DNA zippers enable not only discrimination between specific and nonspecific binding, but also allow for the local application of detection antibodies, thereby eliminating false-positive results caused by cross-reactive antibodies and nonspecific binding.

Base Sequence↗
PubMed2001

The NMR structure of the 47-kDa dimeric enzyme 3,4-dihydroxy-2-butanone-4-phosphate synthase and ligand binding studies reveal the location of the active site.

Recent developments in NMR have extended the size range of proteins amenable to structural and functional characterization to include many larger proteins involved in important cellular processes. By applying a combination of residue-specific isotope labeling and protein deuteration strategies tailored to yield specific information, we were able to determine the solution structure and study structure-activity relationships of 3,4-dihydroxy-2-butanone-4-phosphate synthase, a 47-kDa enzyme from the Escherichia coli riboflavin biosynthesis pathway and an attractive target for novel antibiotics. Our investigations of the enzyme's ligand binding by NMR and site-directed mutagenesis yields a conclusive picture of the location and identity of residues directly involved in substrate binding and catalysis. Our studies illustrate the power of state-of-the-art NMR techniques for the structural characterization and investigation of ligand binding in protein complexes approaching the 50-kDa range in solution.

Amino Acid Sequence↗
PubMed2001

Crystal structure of rat GTP cyclohydrolase I feedback regulatory protein, GFRP.

Tetrahydrobiopterin, the cofactor required for hydroxylation of aromatic amino acids regulates its own synthesis in mammals through feedback inhibition of GTP cyclohydrolase I. This mechanism is mediated by a regulatory subunit called GTP cyclohydrolase I feedback regulatory protein (GFRP). The 2.6 A resolution crystal structure of rat GFRP shows that the protein forms a pentamer. This indicates a model for the interaction of mammalian GTP cyclohydrolase I with its regulator, GFRP. Kinetic investigations of human GTP cyclohydrolase I in complex with rat and human GFRP showed similar regulatory effects of both GFRP proteins.

Amino Acid Sequence↗
PubMed2001

Molecular and catalytic properties of Arabidopsis thaliana adenylyl sulfate (APS)-kinase.

A cDNA clone (Atakn1) from Arabidopsis thaliana encoding APS-kinase (EC 2.7.1.25) was investigated for structural and catalytic properties of the gene product. Recombinant his10-AtAkn1 formed PAPS at a Vmax of 7.35 U x mg(-1). The Km for APS was 0.14 microM and for ATP 147 microM. APS caused a severe substrate inhibition (K(i) 4.5 microM). The type of inhibition is uncompetitive with respect to MgATP. High ionic strength and reducing thiols stabilized the enzyme activity. Plant APS-kinase is regulated in vitro by the redox charge with thioredoxin as essential activator. Mutagenesis of a serine in S182C and S182F presumed to be involved in the transfer of the phosphoryl group had no effect upon catalytic activity. Using a yeast two-hybrid system with AtAkn1 as bait, an interacting clone was detected from a cDNA library of A. thaliana cv. Columbia that codes for an APS-kinase iso-form (Atakn2). Complementation of APS-kinase-deficient Saccharomyces cerevisiae met14 showed that AtAkn2 is functionally active as APS-kinase. It was immunologically related to AtAkn1 and presumably represents a plastidal iso-form of the plant APS-kinase gene family.

Amino Acid Sequence↗
PubMed2000

Germline-activating mutation in the kinase domain of KIT gene in familial gastrointestinal stromal tumors.

The proto-oncogene KIT encodes the receptor tyrosine kinase KIT. Gain-of-function mutations in the juxtamembrane domain of KIT have been reported in human gastrointestinal stromal tumors. In a family with multiple gastrointestinal stromal tumors and diffuse hyperplasia of myenteric plexus layer, we have identified another mutation of KIT, a single base mutation, resulting in the substitution of Glu for Lys(642) in the kinase I domain, and studied its biological effect in a cellular system. The mouse homologue of the human KIT mutant was generated by site-directed mutagenesis and stably transfected into the interleukin-3-dependent Ba/F3 murine cell line. The oncogenic potential of the mutated KIT was assessed in vitro by a proliferation assay and in vivo by transplantation into nude mice. Transfected Ba/F3 cells grew autonomously in absence of growth factors and formed tumors in nude mice. Substitution of Glu for Lys(642) is an oncogenic mutation in the tyrosine kinase domain of KIT. As germline heterozygous mutation, it causes a diffuse hyperplasia of myenteric interstitial cells of Cajal during embryonic development and occurrence of multiple gastrointestinal stromal tumors at adulthood.

Adult↗
PubMed1999

Intracellular visualization of BrdU-labeled plasmid DNA/cationic liposome complexes.

Difficulties in specific detection of transfected DNA in cells represent an important limitation in the study of the gene transfer process. We studied the cellular entry and fate of a plasmid DNA complexed with a cationic lipid, Vectamidine (3-tetradecylamino-N-tert-butyl-N'-tetradecylpropionamidine) in BHK21 cells. To facilitate its detection inside the cells, bromodeoxyuridine (BrdU) was incorporated into plasmid DNA under conditions that minimize plasmid alteration. BrdU was localized in cells incubated with Vectamidine/BrdU-labeled plasmid DNA complexes by immunogold labeling and electron microscopy (EM). Labeling was predominantly associated with aggregated liposome structures at the surface of and inside the cells. EM observations of cells transfected with Vectamidine/DNA complexes showed that the liposome/DNA aggregates accumulate in large vesicles in the cell cytosol. On the other hand, using rhodamine-labeled Vectamidine and revealing BrdU with FITC-conjugated antibodies permitted simultaneous detection in the cells of both components of the complexes with confocal laser scanning microscopy. The DNA and lipids co-localized at the surface of and inside the cells, indicating that the complex is internalized as a whole. Our results show that the BrdU-labeled plasmid DNA detection system can be a useful tool to visualize exogenous DNA entry into cells by a combination of electron and confocal microscopy.

Amidines↗
PubMed1994

APS-sulfotransferase activity is identical to higher plant APS-kinase (EC 2.7.1.25).

A cDNA from Arabidopsis thaliana L. Heynh encoding the APS-kinase (EC 2.7.1.25) was modified by deletion of a plastidic transit peptide to enable its expression in Escherichia coli. The resultant protein (MW 25,761) is enzymatically active as APS-kinase and restores prototrophic growth in an APS-kinase mutant. All transformants harbouring the modified plant DNA also acquired APS-sulfotransferase activity. In the absence of ATP but provided with DTT, a tetrameric form of recombinant APS-kinase exhibits APS-sulfotransferase activity. Monospecific polyclonal antibodies raised against the APS-kinase as immunogen also reacted against APS-sulfotransferase. We propose that APS-sulfotransferase activity is a nonphysiological side reaction of APS-kinase.

Adenosine Triphosphate↗
PubMed1994

A cDNA for adenylyl sulphate (APS)-kinase from Arabidopsis thaliana.

A cDNA clone with an open reading frame of 831 nucleotides was isolated from a lambda ZapII-library of Arabidopsis thaliana. The nucleotide sequence of the cDNA is homologous to the APS-kinase genes from enterobacteria, diazotrophic bacteria, and yeast: Escherichia coli (cys C: 53.2%), Rhizobium meliloti (nod Q: 52.6%), and Saccharomyces cerevisiae (met 14:57.1%). The polypeptide deduced from the plant APS-kinase cDNA is comprised of 276 amino acid residues with a molecular weight of 29,790. It contains an N-terminal extension of 77 amino acids. This extension includes a putative transit peptide of 37 residues separated from the core protein by a VRACV processing site for stromal peptidase; a molecular weight of 26,050 is predicted for the processed protein. The relatedness between bacterial, fungal and plant APS-kinase polypeptides ranges from 47.5% (E. coli), 55.4% (S. cerevisiae), 52.6% (R. meliloti), and 50.3% (Azospirillum brasilense). The plant polypeptide contains eight cysteine residues; two cysteines flank a conserved purine nucleotide binding domain: GxxxxGK. Also conserved are a serine-182 as a possible phosphate transferring group and a K/LARAGxxxxFTG motif described for PAPS dependent enzymes. The identity of the gene was confirmed by analyzing the function of the gene product. The putative transit peptide was deleted by PCR and the truncated gene was expressed in a pTac1 vector system. A polypeptide of MW 25761 could be induced by IPTG. The gene product was enzymatically active as APS-kinase. It produced PAPS from APS and ATP--the absence of ATP but supplemented with thiols, the APS-kinase reacted as APS-sulphotransferase. APS-sulphotransferase is not a separate enzyme but identical with APS-kinase.

Amino Acid Sequence↗
PubMed1993

[Localization and role of cholecystokinin and its receptors in the functional organization of the basal ganglia].

In human, cholecystokinin-immunoreactive nerve fibres are abundantly distributed in the ventral striatum and in other regions of the basal ganglia interconnected with the limbic system. This suggests that this neuropeptide is involved in the initiation of movements following emotional stimuli. The demonstration of an extended cholecystokinin expression in cortico-, thalamo-, and nigrostriatal pathways using in situ hybridization indicates that this neuromodulator may have some important functions either in motor, cognitive or limbic systems. In addition, disruption of the nigrostriatal dopaminergic pathway induces the expression of cholecystokinin in the striatal neurons. This new example of functional plasticity suggests that dopamine exerts a negative tonus on cholecystokinin expression. Finally, cholecystokinin expression is increased in meso-limbic and nigrostriatal dopaminergic neurons of schizophrenic patients.

Basal Ganglia↗
PubMed1992

Expression of members of the putative olfactory receptor gene family in mammalian germ cells.

A series of genomic and complementary DNA clones encoding new putative members of G protein-coupled receptors were isolated using homology cloning and low-stringency polymerase chain reaction. Among the unidentified receptors ('orphan receptors'), a human genomic clone (HGMP07) was characterized by the presence of its transcripts in the testis and by its belonging to a large subfamily of genes sharing extensive sequence similarities. Sequence comparison demonstrated that this gene subfamily is the human counterpart of the putative rat olfactory receptors cloned recently. Another 48 members of the family were cloned. Northern blotting further demonstrated the presence of olfactory receptor transcripts in germ cells. Our finding suggests that a common receptor gene family encodes olfactory receptors and sperm cell receptors that could be involved in chemotaxis during fertilization.

Amino Acid Sequence↗
PubMed1991

Computerized analysis of smooth muscle fibers in potent and impotent patients.

Reduction or dysfunction of the intracavernous smooth muscle fibers can provoke impotence. Computerized digital image analysis of corpus cavernosum biopsies was performed in potent and impotent patients to quantify the percentage of smooth muscle fibers. In 5 normal patients the smooth muscle area represented 40 to 52% of the specimen, in 20 patients with cavernous dysfunction it was 10 to 36% and in 10 patients with arterial disease it was 13 to 25%. This method appears to be important to understand better certain mechanisms of impotence and to approach the potential treatment.

Adolescent↗
PubMed1990

RDC8 codes for an adenosine A2 receptor with physiological constitutive activity.

The cDNA of an unidentified recently cloned G protein-coupled receptor, RDC8, has been expressed in Y1 adrenal cells, in dog thyrocytes in primary culture and in Xenopus oocytes. In all these systems this resulted in the activation of adenylyl cyclase and of the cyclic AMP cascade in the absence of any added external signal. However, this physiologically constitutive activator was inhibited by adenosine deaminase and by inhibitors of the adenosine A2 receptor. Cos 7 cells transfected with RDC8 cDNA constructs acquired binding characteristics of an adenosine A2 receptor. Moreover, RDC8 mRNA and adenosine A2 receptors display a very similar distribution in the brain. RDC8 therefore codes for an A2 adenosine receptor. Whether the physiologically constitutive activation of this receptor is entirely explained by endogeneously produced adenosine is as yet unknown.

Adenosine↗
PubMed1990

[A cloned protein belonging to the G protein-coupled receptor family has an essentially striatal distribution copying that of the major component of the D1 receptor].

RDC8 (correction of RCD8), a recently cloned new putative member of the G protein-coupled receptor family protein, is exclusively present in the medium-sized neurons of the striatum. This restricted localisation mimics the major striatal dopamine D1 receptor localisation and is of major importance for the understanding of basal ganglia physiology and degenerative diseases pathogeny such as Huntington's and Parkinson's disease. RDC7, another putative G protein-coupled receptor chemically closely related to RDC8 (correction of RCD8), is mainly distributed in pyramidal neurons of the cerebral cortex, the hippocampus and the claustrum, and in the amygdala, and may represent the minor extra-striatal variant of the D1 receptor.

Animals↗
PubMed1988

Immunocytochemical detection of GABAergic nerve cells in the human temporal cortex using a direct gamma-aminobutyric acid antiserum.

Recently, an immunocytochemical method using glutaraldehyde fixation and an antiserum developed against a GABA--glutaraldehyde--protein conjugate has permitted direct visualization of GABAergic structures in the brains of perfused animals. This paper reports a successful use of this technique on human temporal cortex fixed by immersion. The cerebral tissue was obtained from patients operated for focal epilepsy. GABA-positive somata, fibres and terminals are observed in all layers of the temporal cortex. Terminals are particularly abundant in the superficial portion of layer I and in layers II, III and IV. Dense plexuses of fibres are located in layers II, III, IV and VI and in the underlying white matter. Somata are found in all cortical layers and in the underlying white matter; they are round, oval, fusiform or triangular and exhibit a multipolar, bitufted or bipolar dendritic pattern. This technique for the visualization of GABAergic structures in the human brain may allow a better understanding of the pathogeny of epilepsy in which the GABAergic transmission has been implicated.

Humans↗
PubMed1988

Study of neuropeptide Y-containing nerve fibers in the human penis.

Neuropeptide Y 1-36 (IR-NPY) immunoreactive nerve-fiber processes have been observed in tunicae of veins and arteries and in smooth muscles of the human penis taken at autopsy or during surgery by use of light- and electron-microscopic immunohistochemical techniques. Numerous IR-NPY nerve fibers were mostly concentrated in the inner part of the adventitia close to the media of the arterial and venous vessels and among the intracavernous smooth muscle cells. IR-NPY nerve fibers were less abundant in veins than in arteries. Positive somata were not observed in the penises. At the ultrastructural level, IR-NPY were localized exclusively in large, dense granules of nerve terminals by means of the postembedding immunogold technique. In the deep dorsal vein, IR-NPY nerve fibers were also located in the media formed by an outer circular and an inner longitudinal layer. In the intracavernous and dorsal arteries, they showed the highest density in the inner part of the adventitia. In the corpora cavernosa and in the corpus spongiosum, IR-NPY nerve processes were intermingled between the smooth-muscle fibers around the sinusoid spaces. IR-NPY nerve fibers were present in the cavernous nerves close to the central arteries. The urethra did not show any IR-NPY-positive nerve fibers. This peculiar distribution of IR-NPY nerve fibers suggested that they could participate in regulating arterial and venous blood flow and intracavernous smooth-muscle tone. NPY may therefore be of importance in some of the mechanisms of penile erection especially during detumescence.

Adolescent↗
PubMed1985

Co-existence of cholecystokinin- or gastrin-like peptides with other peptides in the hypophysis and the hypothalamus.

The presence of cholecystokinin and gastrin has been reported in the hypothalamohypophyseal system. These peptides present a peculiar distribution in the hypothalamic nuclei, the median eminence, and the neurohypophysis. CCK and gastrin have close relationships with other peptides like oxytocin, CRF, vasopressin, and the enkephalins; these relationships vary in different projecting areas and in different types of hypothalamic neurons. The functional role of G-CCK in neurosecretion seems to be linked to the role of these closely associated peptides and certainly deserves further investigation.

Animals↗
PubMedDate not supplied

Distribution of neuropeptide Y immunoreactivity in human visual cortex and underlying white matter.

Immunocytochemical techniques have been used to study neuropeptide Y (NPY) distribution in the human visual cortex (Brodman's areas 17, 18 and 19) NPY cell bodies belong mostly to inhibitory (multipolar and bitufted) but also to excitatory (bipolar and some pyramidal) neuronal types. Their distribution is similar in the three cortical areas studied: 20 to 40% of the NPY perikarya are located in the cortical gray matter, mostly in the deep layers, while the remaining 60 to 80% are located in the underlying white matter. Immunoreactive NPY processes form a rich network of intersecting fibers throughout the entire visual cortex. A superficial plexus (layers I and II) and a deep plexus (deep layer V and layer VI) of NPY fibers are present in areas 17, 18 and 19. In area 17, an additional well developed plexus is present in layers IVb and IVc. These plexuses receive branches from long parallel fibers arising from deep cortical layers or underlying white matter and terminating in superficial layers. Local or extrinsic NPY terminals wind around vessels in the cortex as well as in the white matter, and either penetrate them or form clusters of club endings on their walls. Our results suggest a role for NPY in human visual circuitry and in cortical blood flow regulation.

Aged↗