Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 1996
Growth hormone (GH) was purified from African catfish (Clarias gariepinus) pituitary extracts in ... more Growth hormone (GH) was purified from African catfish (Clarias gariepinus) pituitary extracts in a single step by use of immunoaffinity chromatography. A monoclonal antibody to chicken GH, which labels the catfish hypophyseal somatotropes in immunocytochemistry, was coupled to CNBr-activated Sepharose, and crude alkaline pituitary extracts were run over the immunoadsorbent. Reversed-phase high-performance liquid chromatography analysis of the eluted material suggested heterogeneity, whereas silver staining upon SDS-polyacrylamide gel electrophoresis showed one single band with an estimated molecular weight between 22,000 and 23,000 Da. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of the same preparation revealed the presence of several components with molecular weights ranging from 20,170 to 20,900 Da. The amino terminus of the protein was homogeneous, and the first 50 residues matched the proposed sequence of GH from two other siluran species (Ictalurus punctatus and Pangasius pangasius), except for one substitution at position 3. These data unequivocally confirm the identity of the purified molecule as suggested by immunochemical evidence. The bioactivity of the GH preparation was demonstrated by the short-term effect of GH on T3 plasma levels in juvenile catfish.
G-protein-coupled receptors (GPCRs) constitute one of the largest families of membrane-spanning p... more G-protein-coupled receptors (GPCRs) constitute one of the largest families of membrane-spanning proteins. Their importance in drug development has been proven over and over again. Therefore, they remain one of the most significant groups of molecules to be characterized. In the postgenomic era, the methods used for the characterization of GPCRs have dramatically changed: the predicted orphan receptors are now often used to ascertain the ligands (reverse pharmacology), whereas, in the past, the bioactive ligand was used to identify the receptor (classic approach). In this review, we will give an overview of the recent postgenomic functional assays that are frequently used to link the orphan GPCR of both vertebrate and invertebrate organisms with their ligands.
Comparative biochemistry and physiology. C, Comparative pharmacology and toxicology, 1992
1. Two novel insect myotropic peptides termed neosulfakinin-I (Neb-SK-I) and neosulfakinin-II (Ne... more 1. Two novel insect myotropic peptides termed neosulfakinin-I (Neb-SK-I) and neosulfakinin-II (Neb-SK-II) were isolated from the heads of 42 thousand fleshflies, Neobellieria bullata (Diptera, Sarcophagidae). 2. A series of four, high-performance liquid chromatographic (HPLC), fractionations performed on columns with different characteristic features yielded two purified biologically active, hindgut motility stimulating fractions, suitable for amino acid sequence analysis. 3. The proposed sequences for the two peptides are: Phe-Asp-Asp-Tyr-Gly-His-Met-Arg-Phe-(NH2), (Neb-SK-I) and X-X-Glu-Glu-Gln-Phe-Asp-Asp-Tyr-Gly-His-Met-Arg-Phe-(NH2), (Neb-SK-II). 4. These sulfakinins exhibit very high homology to putative drosulfakinin sequences which, however, have not yet been isolated, but were deduced from a cloned Drosophila gene encoding these peptides. 5. Here we provide the first evidence for the expression of such peptides present in Dipterans. 6. Insect sulfakinins show structural ide...
A method, based on a combination of mass spectrometry and liquid chromatography, was developed to... more A method, based on a combination of mass spectrometry and liquid chromatography, was developed to investigate the release of neuropeptides from isolated locust corpora cardiaca. Melatonin, octopamine, trehalose and forskolin were administered to the perifused glands. The neuropeptides present in the releasates (spontaneous versus induced) were visualized by either conventional or capillary HPLC. Identification was achieved by means of MALDI-TOF MS and/or nanoflow-LC-Q-TOF MS. The observed effects of these chemicals regarding AKH release were in line with previous studies and validate the method. The most important finding of this study was that administration of melatonin stimulated the release of adipokinetic hormone precursor related peptides (APRP 1 and APRP 2), neuroparsins (NP A1, NP A2 and NP B) and diuretic peptide.
The production and characterisation of transgenic tobacco plantsexpressing a precursor of a regul... more The production and characterisation of transgenic tobacco plantsexpressing a precursor of a regulatory peptide from Aedesaegypti (Trypsin Modulating and Oostatic Factor, Aea-TMOF) whichinterferes with the development of tobacco budworm larvae is described. Tobaccoplants were transformed with a synthetic gene containing 6 TMOF units spaced bydibasic residues, Arg-Arg, as potential post-translational cleavage sites.Peptide extracts from transgenic plants had TMOF activity and inhibitedin vitro the biosynthesis of serine proteases. Thisactivity was consistently present in T1 plants and absent in control plants.Tobacco budworm larvae, fed with transgenic leaves showed a reduced growth ratecompared to those fed with control plants. The low rather than acute toxicityofthis low impact gene is discussed in the context of alternative integrated pestmanagement strategies.
... This protocol produced a 63-fold purification and a specific activity of 4.75 U (mg protein)&... more ... This protocol produced a 63-fold purification and a specific activity of 4.75 U (mg protein)"'. The mass of the enzyme was 69 kDa as estimated by gel filtration. ... The column was washed with the same buiffer until the optical density at 280 ntn was lower thart 0.1. ...
Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 1996
Growth hormone (GH) was purified from African catfish (Clarias gariepinus) pituitary extracts in ... more Growth hormone (GH) was purified from African catfish (Clarias gariepinus) pituitary extracts in a single step by use of immunoaffinity chromatography. A monoclonal antibody to chicken GH, which labels the catfish hypophyseal somatotropes in immunocytochemistry, was coupled to CNBr-activated Sepharose, and crude alkaline pituitary extracts were run over the immunoadsorbent. Reversed-phase high-performance liquid chromatography analysis of the eluted material suggested heterogeneity, whereas silver staining upon SDS-polyacrylamide gel electrophoresis showed one single band with an estimated molecular weight between 22,000 and 23,000 Da. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of the same preparation revealed the presence of several components with molecular weights ranging from 20,170 to 20,900 Da. The amino terminus of the protein was homogeneous, and the first 50 residues matched the proposed sequence of GH from two other siluran species (Ictalurus punctatus and Pangasius pangasius), except for one substitution at position 3. These data unequivocally confirm the identity of the purified molecule as suggested by immunochemical evidence. The bioactivity of the GH preparation was demonstrated by the short-term effect of GH on T3 plasma levels in juvenile catfish.
G-protein-coupled receptors (GPCRs) constitute one of the largest families of membrane-spanning p... more G-protein-coupled receptors (GPCRs) constitute one of the largest families of membrane-spanning proteins. Their importance in drug development has been proven over and over again. Therefore, they remain one of the most significant groups of molecules to be characterized. In the postgenomic era, the methods used for the characterization of GPCRs have dramatically changed: the predicted orphan receptors are now often used to ascertain the ligands (reverse pharmacology), whereas, in the past, the bioactive ligand was used to identify the receptor (classic approach). In this review, we will give an overview of the recent postgenomic functional assays that are frequently used to link the orphan GPCR of both vertebrate and invertebrate organisms with their ligands.
Comparative biochemistry and physiology. C, Comparative pharmacology and toxicology, 1992
1. Two novel insect myotropic peptides termed neosulfakinin-I (Neb-SK-I) and neosulfakinin-II (Ne... more 1. Two novel insect myotropic peptides termed neosulfakinin-I (Neb-SK-I) and neosulfakinin-II (Neb-SK-II) were isolated from the heads of 42 thousand fleshflies, Neobellieria bullata (Diptera, Sarcophagidae). 2. A series of four, high-performance liquid chromatographic (HPLC), fractionations performed on columns with different characteristic features yielded two purified biologically active, hindgut motility stimulating fractions, suitable for amino acid sequence analysis. 3. The proposed sequences for the two peptides are: Phe-Asp-Asp-Tyr-Gly-His-Met-Arg-Phe-(NH2), (Neb-SK-I) and X-X-Glu-Glu-Gln-Phe-Asp-Asp-Tyr-Gly-His-Met-Arg-Phe-(NH2), (Neb-SK-II). 4. These sulfakinins exhibit very high homology to putative drosulfakinin sequences which, however, have not yet been isolated, but were deduced from a cloned Drosophila gene encoding these peptides. 5. Here we provide the first evidence for the expression of such peptides present in Dipterans. 6. Insect sulfakinins show structural ide...
A method, based on a combination of mass spectrometry and liquid chromatography, was developed to... more A method, based on a combination of mass spectrometry and liquid chromatography, was developed to investigate the release of neuropeptides from isolated locust corpora cardiaca. Melatonin, octopamine, trehalose and forskolin were administered to the perifused glands. The neuropeptides present in the releasates (spontaneous versus induced) were visualized by either conventional or capillary HPLC. Identification was achieved by means of MALDI-TOF MS and/or nanoflow-LC-Q-TOF MS. The observed effects of these chemicals regarding AKH release were in line with previous studies and validate the method. The most important finding of this study was that administration of melatonin stimulated the release of adipokinetic hormone precursor related peptides (APRP 1 and APRP 2), neuroparsins (NP A1, NP A2 and NP B) and diuretic peptide.
The production and characterisation of transgenic tobacco plantsexpressing a precursor of a regul... more The production and characterisation of transgenic tobacco plantsexpressing a precursor of a regulatory peptide from Aedesaegypti (Trypsin Modulating and Oostatic Factor, Aea-TMOF) whichinterferes with the development of tobacco budworm larvae is described. Tobaccoplants were transformed with a synthetic gene containing 6 TMOF units spaced bydibasic residues, Arg-Arg, as potential post-translational cleavage sites.Peptide extracts from transgenic plants had TMOF activity and inhibitedin vitro the biosynthesis of serine proteases. Thisactivity was consistently present in T1 plants and absent in control plants.Tobacco budworm larvae, fed with transgenic leaves showed a reduced growth ratecompared to those fed with control plants. The low rather than acute toxicityofthis low impact gene is discussed in the context of alternative integrated pestmanagement strategies.
... This protocol produced a 63-fold purification and a specific activity of 4.75 U (mg protein)&... more ... This protocol produced a 63-fold purification and a specific activity of 4.75 U (mg protein)"'. The mass of the enzyme was 69 kDa as estimated by gel filtration. ... The column was washed with the same buiffer until the optical density at 280 ntn was lower thart 0.1. ...
Uploads
Papers