The short 5′ untranslated region of the βA3/A1-crystallin mRNA is responsible for leaky ribosomal scanning

Leaky ribosomal scanning allows the expression of multiple proteins from a single mRNA by occasionally skipping the first start codon, and initiating translation at a subsequent one. A3- and A1-crystallin, two members of the -crystallin family of vertebrate eye lens proteins, are produced via this mechanism, of which, until now, only very few examples have been found in eukaryotic genes. Since the two start codons on the A3/A1 messenger lie in the same reading frame, the two translated proteins are identical, except for the 17 residues shorter N-terminal extension of A1-crystallin. It has been suggested that the very short leader (5–7 nucleotides) of the A3/A1 messenger might cause slippage at the first start codon, although the unfavorable context of this start codon might also be responsible. Using transient transfections, we now demonstrate that increasing the length of the leader sequence to 67 nucleotides indeed completely abolishes translation initiation at the second start codon, and thus expression of the A1-crystallin protein. Messengers having a leader of 5, 7 or 14 nucleotides all express both A3- and A1-crystallin at very similar relative levels.     

β-Alanine Release from the Adult and Developing Hippocampus Is Enhanced by Ionotropic Glutamate Receptor Agonists and Cell-Damaging Conditions

The release of the inhibitory amino acid -alanine was investigated in hippocampal slices from adult (3-month-old) and developing (7-day-old) mice, using a superfusion system. The release was enhanced by -alanine itself and the structural analogs taurine and -aminobutyrate. It was dependent on Na⁺, but independent of Ca²⁺ in both mature and immature hippocampus, being thus mostly mediated by uptake carriers operating in an outward direction. The release was potentiated in the developing mice, but not affected in the adults, by the ionotropic glutamate receptor agonists N-methyl-D-aspartate, kainate, 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate and tetrazolylglycine in a receptor-mediated manner. Cell-damaging conditions, including hypoxia, hypoglycemia, ischemia, oxidative stress and the presence of free radicals, greatly enhanced -alanine release at both ages, but more markedly in the adults. The great amounts of -alanine, together with the inhibitory amino acids taurine and -aminobutyrate, released simultaneously with the excitatory amino acids in the hippocampus may constitute an important protective mechanism against excitotoxicity, which leads to neuronal death.     

Peroxisomal β-Oxidation Enzymes

The enzymes involved in -oxidation spiral are schematically classified into two groups. The first group consists of palmitoyl-CoA oxidase, the L-bifunctional protein, which has been called as the bifunctional protein, and 3-ketoacyl-CoA thiolase. The second group consists of the newly confirmed enzymes, branched chain oxidase, the D-bifunctional protein, and sterol carrier protein x. The enzymes of the first group are inducible and act on the straight chain acyl-CoA substrates. But the enzymes of the second group are non-inducible and act on branched chain acyl-CoAs. Accordingly, bile acid formation and oxidation of pristanic acid derived from phytol are catalyzed by the enzymes of the second group but not by those of the first group. The functions of the peroxisomal system and methods of analysis of the enzymes are briefly summarized.     

GM2 Promotes Ciliary Neurotrophic Factor-Dependent Rescue of Immortalized Motor Neuron-Like Cell (NSC-34)

We have examined whether ciliary neurotrophic factor (CNTF) can alter serum-free cell survival of immortalized motor neuron-like cells, which were established by fusing mouse neuroblasoma N18TG2 with mouse motor neurons. One of the cell lines, NSC-34 exhibited cell survival in the presence of CNTF. NSC-34 preserves the most characteristics of motor neurons, such as the formation of neuromuscular junctions on co-cultured myotube. GM2 ganglioside is characteristic of motor neurons, and expressed highly in NSC-34. When NSC-34 was cultured with exogenous GM2 ganglioside and CNTF, GM2 facilitated the cell survival effect of CNTF. In the addition, 1,4 N-acetylgalactosaminyltransferase (GM2 synthase) activity was enhanced up to 3.9-fold by culture in the presence of CNTF. GM2 might be a functional modulator of CNTF in motor neurons. It might be presented to cell surface by its enzyme activation, and become a signal of early stage, when CNTF rescues motor neurons.     

Effect of Dietary Lipids on Phospholipase D Activity in Rat Brain

Phospholipase D (PLD) is emerging as a major player in many novel signaling pathways. Based on recent studies correlating membrane composition with enzyme function, we speculated that feeding of dietary lipids to the newborns has a major impact on brain PLD activity. To test this hypothesis, the rat dams were fed fat-free powder containing either safflower oil or fish oil, and a control powdered chow. The pups were weaned onto the diet and sacrificed at 30 days of age. PLD activity was measured by transphosphatidylation assays using rat brain membranes. This study shows that microsome GTPS-dependent PLD activity in rats fed safflower oil or fish oil was significantly reduced by 38% and 30% respectively compared to controls. Oleate-dependent PLD activity in the safflower oil group, however, was significantly increased by 38%. In contrast, synaptosome membrane (P2) GTPS-dependent PLD activity in rats consuming safflower oil was significantly increased by 29%, but there was no difference in oleate-dependent PLD activity. Likewise, no difference was observed in microsome oleate-dependent PLD and P2 GTPS-dependent PLD activity between the fish oil and the control groups. These results indicate that dietary lipid intake appears to modulate phospholipid metabolism and differential expression of PLD isozymes in the brain.     

Detection of Cannabinoid CB1, Adenosine A1, Muscarinic Acetylcholine, and GABAB Receptor-Dependent G Protein Activity in Transducin-Deactivated Membranes and Autoradiography Sections of Rat Retina

1. Several G-protein-coupled receptors (GPCRs) have been localized to various layers of the vertebrate retina, using autoradiographic and immunohistochemical techniques, but the functional data concerning G protein activation are limited. Here, we establish optimized assay conditions to detect receptor-dependent G protein activity in membranes and tissue sections of the rat retina. 2. Agonist-stimulated [35S]GTPgammaS-binding responses were characterized for the Gi/o-linked adenosine A1, cannabinoid CB1, m2/m4 muscarinic acetylcholine, and GABA(B) receptors. Initial assumption was that G protein activity under "basal conditions" is high due to enrichment and activity of rhodopsin and transducin in this tissue. 3. We found that pretreatment of retina membranes with hydroxylamine (10 mM), a rhodopsin-inactivating drug, substantially (up to 60%) reduced basal G protein activity, thereby improving signal-to-noise ratio to detect agonist-stimulated G protein activation for all studied receptors. [35S]GTPgammaS autoradiography revealed that hydroxylamine specifically reduced basal binding in the transducin-enriched photoreceptor layer. In contrast, hydroxylamine did not affect GPCR signaling in brain membranes, indicating specific action on retinal transducin. 4. For all studied receptors, [35S]GTPgammaS autoradiography allowed localization of G protein activity to different retinal layers, with the bulk of signal detected in the ganglion cell layer. Strongest responses were observed for adenosine and muscarinic receptor agonists. Additional G protein activity was detected in the inner plexiform layer. 5. Responses to all tested agonists were reversed in the presence of appropriate receptor-selective antagonists, indicating receptor-mediated G protein activation.     

Cloning and characterization of a human cDNA ACAD10 mapped to chromosome 12q24.1

Mitochondrial fatty acid -oxidation is an important energy resource for many mammal tissues. Acyl-CoA dehydrogenases (ACADs) are a family of flavoproteins that are involved in the -oxidation of the fatty acyl-CoA derivatives. Deficiency of these ACADs can cause metabolic disorders including muscle fatigue, hypoglycaemia, hepatic lipidosis and so on. By large scale sequencing, we identified a cDNA sequence of 3960 base pairs with a typical acyl-CoA dehydrogenase function domain. RT-PCR result shows that it is widely expressed in human tissues, especially high in liver, kidney, pancreas and spleen. It is hypothesized that this is a novel member of ACADs family. Abbreviations: ACADs – acyl-CoA dehydrogenases, FAD – flavinadenine dinucleotide, SCAD – short-chain acyl-CoA dehydrogenase,MCAD – medium-chain acyl-CoA dehydrogenase, LCAD – long-chain acyl-CoAdehydrogenase, VLCAD – very long- chain acyl-CoA dehydrogenase, IVD –isocalery-CoA dehydrogenase, SBCAD – short/branched chain acyl-CoAdehydrogenase, GCD – glutaryl- CoA dehydrogenase, ETF – electron transferflavoprotein, ACAD8 – acyl-CoA dehydrogenase 8, ACAD9 – acyl-CoAdehydrogenase 9, ACAD10 – acyl-CoA dehydrogenase 10.     

Complexity of phenotypes and symbiotic behaviour of <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> biovar <Emphasis Type="Italic">trifolii</Emphasis> exopolysaccharide mutants

Quantitative in vitro antibacterial activities, i.e., minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs), of 12 -lactam antibiotics against Agrobacterium tumefaciens strains LBA4404 and EHA101 were examined, in order to identify antibiotics effective in eliminating the bacteria in Agrobacterium-mediated plant genetic transformation. The antibacterial activities of -lactams tested against strain EHA101 were equal to or less than those tested against strain LBA4404. Cefotaxime, cefbuperazone, and meropenem had high activities against strain LBA4404 (MBC <1 mg l^–1). Against strain EHA101, however, only meropenem showed activity comparable to that against strain LBA4404. The production of -lactamase was observed only in strain EHA101.Abbreviations CFU Colony-forming unit - MBC Minimum bactericidal concentration - MIC Minimum inhibitory concentration - PBP Penicillin-binding protein     

Acidophilic granulocytes of the marine fish gilthead seabream (<Emphasis Type="Italic">Sparus aurata</Emphasis> L.) produce interleukin-1β following infection with<Emphasis Type="Italic"> Vibrio anguillarum</Emphasis>

The fish immune response to Gram-negative bacteria is poorly understood. In this study, we use a monoclonal antibody (mAb) specific to acidophilic granulocytes from the marine fish gilthead seabream (Sparus aurata L.), together with an antiserum specific to interleukin-1 (IL-1) from this species, in order to investigate whether these cells are involved in the immune response against the pathogenic bacterium Vibrio anguillarum and, in particular, in the production of the pro-inflammatory cytokine IL-1. We found that gilthead seabream head-kidney, peritoneal exudate and peripheral blood leukocytes accumulated proIL-1 intracellularly when challenged in vitro with V. anguillarum, whereas only peritoneal exudate and blood leukocytes were able to accumulate proIL-1 following infection. Importantly, the blood leukocytes from infected animals that accumulated proIL-1 were shown to be the acidophilic granulocytes. A rapid mobilization of such cells from the head-kidney to the site of inflammation following infection with V. anguillarum was also observed. This work was supported by the Spanish Ministry of Science and Technology (grants BIO2001-2324-C02-02 and AGL2002-03529, and fellowship to J. García-Castillo), Spanish Ministry of Education, Culture and Sport (fellowship to E. Chaves-Pozo) and Fundación Séneca, Coordination Centre for Research (grant PI-51/00782/FS/01 and fellowship to P. Pelegrín).E. Chaves-Pozo and P. Pelegrín contributed equally to this work.