The ligand binding site of NPY at the rat Y1 receptor investigated by site-directed mutagenesis and molecular modeling.

The ligand binding site of neuropeptide Y (NPY) at the rat Y1 (rY1,) receptor was investigated by construction of mutant receptors and [3H]NPY binding studies. Expression levels of mutant receptors that did not bind [3H]NPY were examined by an immunological method. The single mutations Asp85Asn, Asp85Ala, Asp85Glu and Asp103Ala completely abolished [3H]NPY binding without impairing the membrane expression. The single mutation Asp286Ala completely abolished [3H]NPY binding. Similarly, the double mutation Leu34Arg/Asp199Ala totally abrogated the binding of [3H]NPY, whereas the single mutations Leu34Arg and Asp199Ala decreased the binding of [3H]NPY 2.7- and 5.2-fold, respectively. The mutants Leu34Glu, Pro35His as well as Asp193Ala only slightly affected [3H]NPY binding. A receptor with a deletion of the segment Asn2-Glu20 or with simultaneous mutations of the three putative N-terminal glycosylation sites, displayed no detectable [3H]NPY binding, due to abolished expression of the receptor at the cell surface. Taken together, these results suggest that amino acids in the N-terminal part as well as in the first and second extracellular loops are important for binding of NPY, and that Asp85 in transmembrane helix 2 is pivotal to a proper functioning of the receptor. Moreover, these studies suggest that the putative glycosylation sites in the N-terminal part are crucial for correct expression of the rY1 receptor at the cell surface.

The complete cDNA sequence encoding dog gastric lipase.

Oligodeoxyribonucleotide ligation to single-stranded cDNA (SLIC) and polymerase chain reaction (PCR) techniques were used to clone an entire dog gastric lipase (DGL) cDNA. The size of the cDNA is confirmed by Northern blot analysis. The DGL is synthesized as a 379-amino acid mature polypeptide with a molecular mass of 43176 Da which is preceded by a 19-amino acid signal sequence located at the NH2-terminus. Comparison of the signal sequences reveals a high degree of similitude between the DGL, the human gastric lipase (HGL), the rabbit gastric lipase (RGL) and the rat lingual lipase (RLL).

Distinct neuroprotective profiles for sigma ligands against N-methyl-D-aspartate (NMDA), and hypoxia-mediated neurotoxicity in neuronal culture toxicity studies.

Substantiating evidence has raised the possibility that sigma ligands may have therapeutic potential as neuroprotective agents in brain ischemia. It has been suggested that the neuroprotective capacity of sigma ligands is related primarily to their affinity for the NMDA receptor complex and not to any selective action at the sigma binding site. However, sigma specific ligands, devoid of significant affinity for the NMDA receptor, are also neuroprotective via an inhibition of the ischemic-induced presynaptic release of excitotoxic amino acids. In the present study, we have investigated the potential neuroprotective effect of a comprehensive series of sigma ligands, with either significant (sigma/PCP) or negligible (sigma) affinity for the PCP site of the NMDA receptor, in order to delineate a selective sigma site-dependent neuroprotective effect. For this aim, we have employed two different neuronal culture toxicity paradigms implicating either postsynaptic-mediated neurotoxicity, (brief exposure of cultures to a low concentration of NMDA or Kainate) or pre- and postsynaptic mechanisms (exposure to hypoxic/hypoglycemic conditions). Only sigma ligands with affinity for the NMDA receptor [(+) and (-) cyclazocine, (+) pentazocine, (+) SKF-10047, ifenprodil and haloperidol] were capable of attenuating NMDA-induced toxicity whereas the sigma [(+)BMY-14802, DTG, JO1784, JO1783, and (+)3-PPP] and kappa-opioid [CI-977, U-50488H] ligands, with very low affinity for the NMDA receptor, were inactive. The rank order of potency, based on the 50% protective concentration (PC50) value, of sigma/PCP ligands against NMDA-mediated neurotoxicity correlates with their affinity for the PCP site of the NMDA receptor, and not with their affinity for the sigma site. In addition sigma/PCP, sigma or kappa-opioid ligands failed to attenuate kainate-mediated neurotoxicity. On the other hand, sigma/PCP, sigma and kappa-opioid ligands were potent inhibitors of hypoxia/hypoglycemia-induced neurotoxicity, although their neuroprotective potency did not correlate with their affinity for either the sigma or PCP binding sites. In conclusion, the ability of sigma and kappa-opioid ligands to attenuate hypoxia/hypoglycemia, but not NMDA or kainate-induced toxicity, suggests that these drugs exert their neuroprotective role by a predominantly presynaptic mechanism possibly by inhibiting ischemic-mediated glutamate release.

Inhibitors of free radical formation fail to attenuate direct beta-amyloid25-35 peptide-mediated neurotoxicity in rat hippocampal cultures.

The direct neurotoxic action of the beta-amyloid protein, the major constituent of senile plaques, may represent the underlying cause of neuronal degeneration observed in Alzheimer's disease. The apoptotic-mediated neuronal death induced by beta-amyloid appears to reside in its ability to form Ca(2+)-permeable pores in neuronal membranes resulting in an excessive influx of Ca2+ and the induction of neurotoxic cascades. It is possible that during beta-amyloid exposure a Ca(2+)-mediated increase in free radical generation may exceed the defensive capacity of cells and thus lead to cell death. Consequently, in the present study we have investigated the effect of a panoply of antioxidants and inhibitors of free radical formation on the development of beta-amyloid neurotoxicity. Acute exposure of rat hippocampal neurons to "aged" beta-amyloid25-35 peptide (5-50 microM) induced a slow, concentration-dependent apoptotic neurotoxicity (25-85%) during a 6 day exposure. Co-incubation of cultures with beta-amyloid25-35 peptide (25 microM) and inhibitors of nitric oxide synthase and/or xanthine oxidase (NG-monomethyl-L-arginine [1 mM), N omega-nitro-L-arginine [1 mM], oxypurinol [100 microM], allopurinol [100 microM]), important mediators of nitric oxide, superoxide, and hydroxyl radical formation, did not attenuate beta-amyloid neurotoxicity. Similarly, a reduction in free radical generation by selective inhibition of phospholipase-A2 cyclooxygenase, and lipoxygenase activities with quinacrine (0.5 microM), indomethacin (50 microM), and nor-dihydroguaiaretic acid (0.5 microM), respectively, did not reduce the proclivity of beta-amyloid to induce cell death. Exposure of cultures to catalase (25 U/ml) and/or superoxide dismutase (10 U/ml) as well as the free radical scavengers vitamin E (100 microM), vitamin C (100 microM), glutathione (100 microM), L-cysteine (100 microM), N-acetyl-cysteine (100 microM), deferoxamine (5 microM), or haemoglobin (35 micrograms/ml) failed to attenuate the neurotoxic action of beta-amyloid. On the other hand, pre-treatment of cultures with subtoxic concentrations of beta-amyloid peptide significantly increased the vulnerability of neurons to H2O2 exposure and suggest that beta-amyloid peptide renders neurons more sensitive to free radical attack. However, a potential beta-amyloid-mediated increase in free radical formation is not a proximate cause of the neurotoxic mechanism of beta-amyloid in vitro.

Tryptic cleavage of gastric lipases: location of the single disulfide bridge.

Human (HGL) and rabbit (RGL) gastric lipases were cleaved by trypsin and the resulting peptides were characterized. Exposure of HGL to trypsin led to the production of three identified fragments (H1, H2 and H3) resulting from cleavage sites at Lys-4 and Arg-229. Fragments H2 (Lys-4-Arg-229) and H3 (Glu-230-Lys-379) were derived from fragment H1 (Lys-4-Lys-379). The single disulfide bridge (Cys-236-Cys-244) of the molecule is localized in fragment H3. Out of the three cysteine residues conserved in all known gastric lipases, the free sulfhydryl group (Cys-227) was localized in fragment H2. Immunoblots, carried out with the tryptic fragments of HGL and anti-HGL mAbs, revealed that five inhibitory mAbs immunoreacted selectively with the N-terminal fragment H2, whereas two other non inhibitory mAbs immunoreacted exclusively with the C-terminal fragment H3. Trypsin also cleaved RGL at two sites (Arg-55 and Arg-229) leading to four identifiable fragments (R1, R2, R3 and R4). One cleavage site (Arg-229) was found to be identical in both RGL and HGL. We propose that this latter site is localized between the two domains of native gastric lipases.

Dog gastric lipase: stimulation of its secretion in vivo and cytolocalization in mucous pit cells.

Dog gastric lipase (DGL) secretion is stimulated in vivo by urecholine, pentagastrin, histamine, 16,16-dimethyl prostaglandin E2, and secretin. Under fasting conditions, DGL is irreversibly inactivated by gastric acid below pH 1.5; consequently, DGL output can be underestimated. This problem has been resolved by buffering the acid or by using an antisecretory drug such as omeprazole during stimulation. There is a clear parallelism between the secretion of DGL and of gastric mucus. This observation led to the present investigation of the cellular localization of DGL using immunofluorescence techniques. Results showed that DGL is cytolocalized in mucous pit cells of gastric glands. Pepsinogen is found in chief cells. To the authors' knowledge, this is the first description of an enzyme (gastric lipase) secreted by mucous-type gastric cells. In contrast to other species, gastric lipase of the dog is located in cardiac, fundic, and antral mucosae.

Purification and biochemical characterization of dog gastric lipase.

A lipase was found to be present in dog stomach which appeared to be more abundant in the fundic than in the pyloric mucosa. Dog gastric lipase was extracted by soaking the gastric tissue and further purified after cation exchange, anion exchange and gel-filtration using fast protein liquid chromatography. The amino-acid composition, N-terminal amino-acid sequence, substrate specificity, interfacial and kinetic behavior and inactivation by sulfhydryl reagents were determined and compared with those of human and rabbit gastric lipases. We report for the first time that a gastric lipase is 13 times more active on long-chain than on short-chain triacylglycerols at pH 4.0, reaching a maximal specific activity of 950 U/mg on Intralipide emulsion.

Cloning of murine SeGpx cDNA and synthesis of mutated GPx proteins in Escherichia coli.

Glutathione peroxidase (GPx) of mammalian cells and Escherichia coli formate dehydrogenase both contain a selenocysteine (SeCys) in their amino acid (aa) sequence. In these two enzymes, this aa is encoded by a UGA codon, which is usually a stop codon for protein synthesis. We constructed plasmids to test the synthesis of GPx in E. coli. These constructions permitted high-level production of GPx mutants, where the SeCys codon was replaced by cysteine (UGC, UGU) or serine (UCA) codons, but synthesis of selenoprotein could not be detected: our data suggest that signals used for the recognition of the UGA codon as a SeCys codon are not conserved between E. coli and mammalian cells.

Functional analysis of the mouse H-2Kb gene promoter in embryonal carcinoma cells.

Mouse embryonal carcinoma (EC) cells do not express the major H-2 class I transplantation antigens. The latter, however, become detectable upon in vitro differentiation of EC cells. Neither class I H-2 genes nor the gene coding for beta-2 microglobulin (beta 2m) is transcribed in EC cells. We have constructed two hybrid plasmids containing the 5' flanking region of an H-2Kb gene followed by the coding regions of either the herpes simplex virus thymidine kinase (H-2 tk) or the chloramphenicol acetyl transferase (H-2 CAT) genes. Upon transfer into EC cells, the H-2 tk hybrid gene is expressed in F9 tk- cell lines which thus acquire a stable tk+ phenotype. When such transformed clones are induced to differentiate in vitro, tk activity shows a moderate increase, which reflects an increase in transcription of the hybrid gene. In transient transformation experiments, EC cells were found to express the H-2 CAT hybrid gene as well. We conclude that the 2 kilobase pair region of the H-2Kb gene which we used contains an active promoter region, but does not include all the elements required for the correct regulation of the H-2Kb gene.

Functional expression of a mouse H-2Kb gene isolated from non-expressing teratocarcinoma cells.

Embryonal carcinoma cells do not express H-2 antigens or beta 2-microglobulin. Recent studies have suggested that the expression of these antigens is likely to be controlled at the level of transcription. To study the precise organization of the corresponding genes and their possible expression in adult mouse cells, we have isolated H-2-related genes from a genomic cosmid library constructed with PCC4-aza-RI from DNA of EC cells. Clones isolated from the library after stringent hybridization with an H-2 cDNA probe were tested for their ability to direct H-2 antigen synthesis after DNA-mediated gene transfer in a fibroblastic L cell. Four clones have been found to code for the major transplantation antigen H-2Kb. Structural analysis showed that these clones contained the same entire H-2Kb gene, identical to the corresponding gene isolated from differentiated C57Bl/10 cells. Furthermore, the present studies showed that this embryonal carcinoma gene was expressed and was functional when transfected into a differentiated cell.

Analysis of the in vitro coding properties of the 3' region of turnip yellow mosaic virus genomic RNA.

The genomic RNA of turnip yellow mosaic virus (TYMV) codes in vitro for two proteins of 195,000 (195K) and 150,000 (150K) daltons initiated at the same site of the RNA. In the presence of yeast amber suppressor tRNA, synthesis of the 195K protein is reduced in favor of a 210,000-dalton (210K) protein. Using TYMV RNA sequence data and comparison of tryptic peptides of the 150K, 195K, 210K, and coat proteins, we postulate that the silent region between the 195K gene and the coat protein gene located in the 3' region of TYMV genomic RNA is only 14 nucleotides long.

Post-Translational Proteolytic Cleavage of In Vitro-Synthesized Turnip Yellow Mosaic Virus RNA-Coded High-Molecular-Weight Proteins.

In a reticulocyte lysate, turnip yellow mosaic virus genomic RNA directs the synthesis of two proteins with molecular weights of 150,000 (150K) and 195K. We present evidence that the larger protein is processed in vitro, after its completion, in at least three fragments. The NH(2)-terminal fragment (82K) and the COOH-terminal fragment (78K) have been well characterized by different methods. The fact that the 150K protein is not cleaved in vitro, although it contains the regions that are processed in the 195K protein, could be of fundamental biological significance for the expression of the viral genes: a single polypeptide chain could be processed in several ways, leading to different peptides with distinct biological activities.

Polyamines stimulate suppression of amber termination codons in vitro by normal tRNAs.

Polyamines, such as spermine and spermidine, are able to stimulate reading of amber termination codons on viral messenger RNAs in vitro. This phenomenon is not due to an overall increase of error frequency during translation, but to a specific effect on a normal tRNA that is present in various eukaryotic cell preparations. The enhancement of reading of termination codons by normal tRNAs should be of major importance for the expression of specific genes in eukaryotic cells.

Translational studies with turnip yellow mosaic virus RNAs isolated from major and minor virus particles.

The major and minor nucleoprotein components in turnip yellow mosaic virus (TYMV) preparations have been fractionated by four cycles of CsCl gradient equilibrium centrifugations. RNAs isolated from the various fractions were translated in a rabbit reticulocyte cell-free system. Three classes of RNAs were studied: genomic RNA with an Mr of 2.0 x 10(6), the subgenomic coat protein messenger with an (r) of 2.4 x 10(5) (H. Guilley and J. P. Briand, 1978, Cell15, 113-122) and RNA molecules of intermediate sizes. Genomic RNA is found in the major TYMV components. These particles do not contain the coat protein messenger. The latter messenger has mainly been detected in two types of minor components, those which have an RNA content exceeding that of the major TYMV virions and particles which contain the coat protein messenger as their sole RNA constituent. RNAs of intermediate sizes are found in minor components less dense than the major particles; the full-length translational products of these RNAs and of genomic RNA overlap with one another and share a common NH2 terminus. It is concluded that a number of intermediate-sized RNAs and the genomic RNA have a common site for initiation of translation located close to their 5'-termini.

In vitro synthesis of turnip yellow mosaic virus coat protein in a wheat germ cell-free system.

Turnip Yellow Mosaic Virus RNA directs the synthesis in vitro of its coat protein in a wheat germ cell-free extract. Optimum conditions for synthesis have been defined, and the effect of spermine on specifically enhancing coat protein formation has been examined. Identity between the in vitro synthesized coat protein and authentic coat protein of Turnip Yellow Mosaic Virus was established by analysis on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, peptide mapping, and immunoprecipitation.

tRNA nucleotidyltransferase-catalyzed incorporation of CMP and AMP into RNA-bacteriophage genome fragments.

Fragments of bacteriophage RNAs R17, MS2 and Qbeta obtained by incubation with commercial snake venom phosphodiesterase become substrates of the Escherichia coli tRNA nucleotidyltransferase. The transferase adds back CMP and AMP in conditions in which it remains highly specific of CCA-deprived tRNAs. The results suggest that the fragment from the 3' end of the viral genome and/or possibly one or more internal fragment(s) are recognized by the transferase. These observations might indicate that bacteriophage RNAs contain certain features probably present in all tRNAs and which are recognized by the transferase.