Molecular cloning and nucleotide sequence of cDNA encoding the entire precursor of rat liver medium chain acyl coenzyme A dehydrogenase

cDNA encoding the precursor of rat liver medium chain acyl-CoA dehydrogenase (EC 1.3.99.3) was cloned and sequenced. The longest cDNA insert isolated was 1866 bases in length. This cDNA encodes the entire protein of 421-amino acids including a 25-amino acid leader peptide and a 396-amino acid mature polypeptide. The identity of the medium chain acyl-CoA dehydrogenase clone was confirmed by matching the amino acid sequence predicted from the cDNA to the NH2-terminal and nine internal tryptic peptide sequences derived from pure rat liver medium chain acyl-CoA dehydrogenase. The calculated molecular masses of the precursor medium chain acyl-CoA dehydrogenase, the mature medium chain acyl-CoA dehydrogenase, and the leader peptide are 46,600, 43,700, and 2,900 daltons, respectively. The leader peptide contains five basic amino acids and only one acidic amino acid; thus, it is positively charged, overall. Cysteine residues are unevenly distributed in the mature portion of the protein; five of six are found within the NH2-terminal half of the polypeptide. Comparison of medium chain acyl-CoA dehydrogenase sequence to other flavoproteins and enzymes which act on coenzyme A ester substrates did not lead to unambiguous identification of a possible FAD-binding site nor a coenzyme A-binding domain. The sequencing of other homologous acyl-CoA dehydrogenases will be informative in this regard.     

Melatonin Biosynthesis in Drosophila: Its Nature and Its Effects

Drosophila melanogaster homogenates incubated with tritiated 5-hydroxytryptophan, 5-hydroxytryptamine, or N-acetylserotonin have the ability of converting them into labelled indolamines, including melatonin. All these compounds were characterized by two-dimensional thin-layer chromatography by comparison with nonradiolabelled standards, and melatonin by mass spectrometry and radioimmunoassay. On the other hand, the injection of pharmacological doses of melatonin into 2-day-old female flies diminishes the mating speed and the oviposition rate.     

Purification and properties of carnitine acetyltransferase from human liver

Carnitine acetyltransferase was purified from the supernatant obtained after centrifugation of human liver homogenate to a final specific activity of 78.75 unit.mg-1 with acetyl-CoA as a substrate. Human carnitine acetyltransferase is a monomer of 60.5 kDa with maximum activity in the presence of propionyl-CoA and a pH optimum of 8.7. Apparent Km values for acetyl-CoA are three times lower than for decanoyl-CoA. Km values for L-carnitine in the presence of acetyl-CoA are six times lower than in the presence of decanoyl-CoA. Km values for acetylcarnitine are three times lower than for octanoylcarnitine. The polyclonal antibodies against human carnitine acetyltransferase recognize a 60.5-kDa peptide in the purified preparation of human liver and brain homogenates and in immunoblots of mitochondrial and peroxisomal fractions from human liver. Immunoprecipitation and SDS/PAGE analysis of 35S-labelled proteins produced by human fibroblasts indicate that mitochondrial carnitine acetyltransferase is synthesized as a precursor of 65 kDa. We also purified carnitine acetyltransferase from the pellet obtained after centrifugation of liver homogenate. The pellet was extracted by sonication in the presence of 0.5% Tween 20. The chromatographic procedures for the purification and the kinetic, physical and immunological properties of pellet-extracted carnitine acetyltransferase are similar to those of carnitine acetyltransferase purified from the supernatant of human liver homogenate.     

Pyrite and Organic Compounds Coexisting in Intrusive Mafic Xenoliths (Hyblean Plateau,Sicily): Implications for Subsurface Abiogenesis

Origins of Life and Evolution of Biospheres - Pyrite and organic matter closely coexist in some hydrothermally-altered gabbroic xenoliths from the Hyblean Plateau, Sicily. The representative sample...     

Fishing new proteins in the twilight zone of genomes: the test case of outer membrane proteins in Escherichia coli K12, Escherichia coli O157:H7, and other Gram-negative bacteria

We address the problem of clustering the whole protein content of genomes into three different categories-globular, all-alpha, and all-beta membrane proteins-with the aim of fishing new membrane proteins in the pool of nonannotated proteins (twilight zone). The focus is then mainly on outer membrane proteins. This is performed by using an integrated suite of programs (Hunter) specifically developed for predicting the occurrence of signal peptides in proteins of Gram-negative bacteria and the topography of all-alpha and all-beta membrane proteins. Hunter is tested on the well and partially annotated proteins (2160 and 760, respectively) of Escherichia coli K 12 scoring as high as 95.6% in the correct assignment of each chain to the category. Of the remaining 1253 nonannotated sequences, 1099 are predicted globular, 136 are all-alpha, and 18 are all-beta membrane proteins. In Escherichia coli 0157:H7 we filtered 1901 nonannotated proteins. Our analysis classifies 1564 globular chains, 327 inner membrane proteins, and 10 outer membrane proteins. With Hunter, new membrane proteins are added to the list of putative membrane proteins of Gram-negative bacteria. The content of outer membrane proteins per genome (nine are analyzed) ranges from 1.5% to 2.4%, and it is one order of magnitude lower than that of inner membrane proteins. The finding is particularly relevant when it is considered that this is the first large-scale analysis based on validated tools that can predict the content of outer membrane proteins in a genome and can allow cross-comparison of the same protein type between different species.     

Conformational study of proteins by SAXS and EDXD: the case of trypsin and trypsinogen

The radius of gyration (Rg) of bovine trypsinogen and beta-trypsin was measured by an energy-dispersive X-ray technique (EDXD) and by small-angle X-ray scattering (SAXS), under different solvent conditions. Both techniques gave superimposable results. The experimental evidence demonstrated that: (1) no structural modifications and/or damage occurred during the data acquisition by EDXD; (2) at pH 4 the active enzyme has one class of chloride binding sites in common with the zymogen, whereas the latter protease shows an additional class able to reverse the effects on Rg induced by chloride at low concentration; and (3) the pH profile of the Rg of both proteases does not resemble at all the pH effect on beta-trypsin activity, a result in line with the finding that the electrical potentials induced by surface charge are small in absolute magnitude and produce no gradient across the active site.     

Effects of cholinergic muscarinic agents on protein kinase C activity in rat pineal gland.

The role of protein kinase C (PKC) on muscarinic regulation of serotonin release in the pineal gland was investigated by measuring the pineal-PKC activity and serotonin secretion in response to muscarinic agents. Pineal slices, short-term incubated (0-15 min) without additions produced a low serotonin release and 20 to 24 percent PKC activity was found associated with membrane fractions. Prolonged exposure of pineal slices (30-180 min) produced further translocation of PKC activity to the membranes and a significant increase of serotonin release. Short-term treatment with pilocarpine and carbachol, stimulated PKC activity of both cytosolic and particulate fractions and the release of pineal serotonin. The pilocarpine effect was blocked by atropine indicating that it was mediated by muscarinic receptors. The present data support that PKC activation correlates with the increase of serotonin release by muscarinic agonist in pineal gland.     

An Optimized Method for Manufacturing a Clinical Scale Dendritic Cell-Based Vaccine for the Treatment of Glioblastoma

Immune-based treatments represent a promising new class of therapy designed to boost the immune system to specifically eradicate malignant cells. Immunotherapy may generate specific anti-tumor immune responses, and dendritic cells (DC), professional antigen-presenting cells, are widely used in experimental cancer immunotherapy. Several reports describe methods for the generation of mature, antigen-pulsed DC for clinical use. Improved quality and standardization are desirable to obtain GMP-compliant protocols. In this study we describe the generation of DC from 31 Glioblastoma (GB) patients starting from their monocytes isolated by immunomagnetic CD14 selection using the CliniMACS® device. Upon differentiation of CD14+ with IL-4 and GM-CSF, DC were induced to maturation with TNF-α, PGE₂, IL-1β, and IL-6. Whole tumor lysate was obtained, for the first time, in a closed system using the semi-automated dissociator GentleMACS®. The yield of proteins improved by 130% compared to the manual dissociation method. Interestingly the Mean Fluorescence Intensity for CD83 increased significantly in DC pulsed with “new method” lysate compared to DC pulsed with “classical method” lysate. Our results indicate that immunomagnetic isolation of CD14⁺ monocytes using the CliniMACS® device and their pulsing with whole tumor lysate proteins is a suitable method for clinical-scale generation of high quality, functional DC under GMP-grade conditions.     

Substrate stereochemistry of 2-methyl-branched-chain acyl-CoA dehydrogenase: elimination of one hydrogen each from (pro-2S)-methyl and alpha-methine of isobutyryl-CoA

Dehydrogenation of (2S)-[3-13C]isobutyryl-CoA was carried out in vitro using 2-methyl-branched-chain acyl-CoA dehydrogenase purified from rat liver mitochondria. The product was subsequently hydrated by the addition of bovine crotonase. The resulting 3-hydroxyisobutyric acid was predominantly enriched with 13C at the beta-hydroxy position as determined as a methyl ester using electron ionization-gas chromatography-mass spectroscopy. This finding indicates that isobutyryl-CoA is dehydrogenated stereospecifically at the (pro-2S)-methyl and alpha-methine groups to form methacrylyl-CoA, which is later hydrated with the addition of hydrogen on the same side of the molecule from which it was subtracted to produce 3-hydroxyisobutyryl-CoA.