The complete nucleotide sequence and gene organization of carp (Cyprinus carpio) mitochondrial genome

The complete sequence of the carp mitochondrial genome of 16,575 base pairs has been determined. The carp mitochondrial genome encodes the same set of genes (13 proteins, 2 rRNAs, and 22 tRNAs) as do other vertebrate mitochondrial DNAs. Comparison of this teleostean mitochondrial genome with those of other vertebrates reveals a similar gene order and compact genomic organization. The codon usage of proteins of carp mitochondrial genome is similar to that of other vertebrates. The phylogenetic relationship for mitochondrial protein genes is more apparent than that for the mitochondrial tRNA and rRNA genes.Correspondence to: F. Huang     

Concentration-dependent modulations of potassium and calcium currents of rat osteoblastic cells by arachidonic acid

We show that the voltage-gated K⁺ and Ca²⁺ currents of rat osteoblastic cells are strongly modulated by arachidonic acid (AA), and that these modulations are very sensitive to the AA concentration. At 2 or 3 μm, AA reduces the amplitude and accelerates the inactivation of the K⁺ current activated by depolarization; at higher concentrations (≥5 μm), AA still blocks this K⁺ current, but also induces a very large noninactivating K⁺ current. At 2 or 3 μm, AA enhances the T-type Ca²⁺ current, close to its threshold of activation, whereas at 10 μm, it blocks that current. AA (1–10 μm) also blocks the dihydropyridine-sensitive L-type Ca²⁺ current. Thus, the effect of AA on Ca²⁺ entry through voltage-gated Ca²⁺ channels can change qualitatively with the AA concentration: at 2 or 3 μm, AA will favor Ca²⁺ entry through T channels, both by lowering the voltage-gated K⁺ conductance and by increasing the T current, whereas at 10 μm, AA will prevent Ca²⁺ entry through voltage-gated Ca²⁺ channels, both by inducing a K⁺ conductance and by blocking Ca²⁺ channels.     

Glutamate Exocytosis and MARCKS Phosphorylation Are Enhanced by a Metabotropic Glutamate Receptor Coupled to a Protein Kinase C Synergistically Activated by Diacylglycerol and Arachidonic Acid

Abstract: 4-Aminopyridine evokes repetitive firing of synaptosomes and exocytosis of glutamate by inhibiting a dendrotoxin-sensitive K⁺ channel responsible for stabilizing the membrane potential. We have shown previously that activation of protein kinase C (PKC) by high concentrations of phorbol ester (4β-phorbol dibutyrate) can increase release by inhibiting a dendrotoxin-insensitive ion channel, whereas the metabotropic glutamate receptor (mGluR) agonist (1 S ,3 R )-1-aminocyclopentane-1,3-dicarboxylate [(1 S ,3 R )-ACPD] mimics the action of 4β-phorbol dibutyrate, but only in the presence of 2 µ M arachidonic acid (AA). In this article, we investigate the role of AA. AA plus (1 S ,3 R )-ACPD is without effect on KCl-induced glutamate exocytosis, indicating that the regulatory pathway acts upstream of the release-coupled Ca²⁺ channel or Ca²⁺-secretion coupling. Diacylglycerol concentrations are greatly enhanced by (1 S ,3 R )-ACPD alone, independently of AA, indicating that AA acts downstream of phospholipase C. Myristoylated alanine-rich C kinase substrate (MARCKS) is the major presynaptic substrate for PKC. mGluR activation by (1 S ,3 R )-ACPD enhances phosphorylation of MARCKS, but only in the presence of AA. These results strongly suggest that AA acts on presynaptic PKC synergistically with diacylglycerol generated by the phospholipase-coupled mGluR, consistent with the known behaviour of certain purified PKC isoforms. The magnitude of the effects observed in a population of rat cerebrocortical synaptosomes suggests that this is a major mechanism regulating the release of the brain's dominant excitatory neurotransmitter and supports the concept that AA, or a related compound with a similar locus of action, may in certain circumstances play a role in synaptic plasticity.     

Cerebrospinal Fluid Nitrite/Nitrate Levels in Neurologic Diseases

Abstract: Nitric oxide has been proposed to mediate cytotoxic effects in inflammatory diseases. To investigate the possibility that overproduction of nitric oxide might play a role in the neuropathology of inflammatory and noninflammatory neurological diseases, we compared levels of the markers of nitric oxide, nitrite plus nitrate, in the CSF of controls with those in patients with various neurologic diseases, including Huntington's and Alzheimer's disease, amyotrophic lateral sclerosis, and HIV infection. We found that there were no significant increases in the CSF levels of these nitric oxide metabolites, even in patients infected with HIV or in monkeys infected with poliovirus, both of which have significantly elevated levels of the neurotoxin quinolinic acid and the marker of macrophage activation, neopterin. However, CSF quinolinic acid, neopterin, and nitrite/nitrate levels were significantly increased in a small group of patients with bacterial and viral meningitis.     

Ascorbic Acid Inhibits 125I-SCH 23982 Binding but Increases the Affinity of Dopamine for D1 Dopamine Receptors

Abstract: Effects of ascorbic acid (AA) on ¹²⁵I-SCH 23982 binding to D₁ dopaminergic receptors in membrane preparations from rat striatum were investigated. AA in the range of 0.03 µ M –0.33 m M inhibited 75% of specific binding of ¹²⁵I-SCH 23982 in a dose-dependent manner. At higher concentrations, this inhibition of binding activity by AA was less potent, and 3.3 m M AA inhibited only 30% of specific binding. Reduced glutathione did not alter the inhibition of binding by 0.33 m M AA, but reduced the inhibition by 3.3 m M AA to 8% of specific binding. The loss of specific binding by AA was rescued by 1 m M EDTA, an inhibitor of lipid peroxidation. In the absence of AA, competition experiments with the agonist, dopamine, revealed the presence of high-affinity ( K _h = 224.9 ± 48.9 n M ) and low-affinity ( K _l = 21,100 ± 2,400 n M ) binding sites. Although the maximum binding of ¹²⁵I-SCH 23982 decreased to 40% without affecting the K _D value in the presence of 1.67 m M AA, the value of the high-affinity site for dopamine was increased ( K _h = 23.3 ± 9.4 n M ) and that of the low-affinity site was decreased ( K _l = 136,800 ± 40,900 n M ). These results suggest that AA may affect D₁ dopamine receptor function by lipid peroxidation, competition with dopamine for low-affinity sites, and reduced oxidation of dopamine.     

A Novel Photoaffinity Ligand for Kainate Sites Labels Two Polypeptides with Molecular Mass 45 and 33.5 kDa in Chick Cerebellum

Abstract: The synthesis of (2 S ,3 S ,4 S )-4-[1-(4-azidobenzamidomethyl)ethenyl]-2-carboxy-3-pyrrolidineacetic acid (ABCPA) is described. This novel kainic acid analogue, bearing a photolabile functionality on the isopropenyl side chain, was proven to be a good inhibitor of [³H]CNQX and [³H]kainic acid binding on chick cerebellar membranes. [³H]ABCPA was photoaffinity cross-linked on the membrane fraction of chick cerebellum. Electrophoretic analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two major radioactive bands with apparent molecular masses of 45 and 33.5 kDa. [³H]ABCPA incorporation in both bands was completely blocked by 2 m M CNQX. When photoaffinity labeling was performed in the presence of 2 m M kainic acid, incorporation of [³H]ABCPA was blocked by ∼70% in the 45-kDa band and by 18% in the 33.5-kDa band. Incorporation of radioactivity in both bands was blocked by ∼30% with 10 m M glutamate.     

Accumulation of hexoses in leaf vacuoles: Studies with transgenic tobacco plants expressing yeast-derived invertase in the cytosol,vacuole or apoplasm

The subcellular distribution of hexoses, sucrose and amino acids among the stromal, cytosolic and vacuolar compartments was analysed by a nonaqueous fractionation technique in leaves of tobacco (Nicotiana tabaccum L.) wild-type and transgenic plants expressing a yeast-derived invertase in the cytosolic, vacuolar or apoplasmic compartment. In the wild-type plants the amino acids were found to be located in the stroma and in the cytosol, sucrose mainly in the cytosol and up to 98% of the hexoses in the vacuole. In the leaves of the various transformants, where the contents of hexoses were greater than in wild-type plants, again 97–98% of these hexoses were found in the vacuoles. It is concluded that leaf vacuoles contain transporters for the active uptake of glucose and fructose against a high concentration gradient. A comparison of estimated metabolite concentrations in the subcellular compartments of wild-type and transformant plants indicated that the decreased photosynthetic capacity of the transformants is not due to an osmotic effect on photosynthesis, as was shown earlier to be the case in transformed potato leaves, but is the result of a long-term dedifferentiation of tobacco leaf cells to heterotrophic cells.Abbreviations apo-inv tobacco plant with yeast invertase in the apoplasm - Chl chlorophyll - cy-inv tobacco plant with yeast invertase in the cytosol - vac-inv tobacco plant with yeast invertase in the vacuole - WT wild-type tobacco plant The authors thank A. Großpietsch for her able technical assistance. This work has been supported by the Bundesminister für Forschung und Technologie.     

Nuclear replication activities during imbibition of abscisic acid- and gibberellin-deficient tomato (Lycopersicon esculentum Mill.) seeds

The role of cis-abscisic acid (ABA) and gibberellins (GAs) in the induction of cell-cycle activities has been studied during imbibition and subsequent germination of tomato seeds. Using flow cytometry, nuclear replication activity was investigated in embryo root tips isolated from seeds of the ABA-deficient mutant sit ^w , the GA-deficient mutant gib-1, and the wild-type (MM) tomato (Lycopersicon esculentum Mill. cv. Moneymaker) upon imbibition in water, 10 μM GA₄₊₇, 5 μM ABA or 5 μM ABA+10 μM GA₄₊₇. The nuclei of fully matured dry MM, sit ^w and gib-1 seeds predominantly showed 2C DNA signals, indicating that the cell-cycle activity of most root-tip cells had been arrested at the G₁ phase of nuclear division. However, ABA-deficient sit ^w seeds contained a significantly higher amount of G₂ cells (4C DNA) compared with the other genotypes, suggesting that, during maturation, cell-cycle activity in sit ^w seeds is less efficiently arrested in G₁. Upon imbibition in water, an induction of the 4C signal, indicating nuclear replication, was observed in the root tip cells of both MM and sit ^w embroys. The augmentation in the 4C signal occurred before visible germination. Gib-1 seeds did not show cell-cycle activity and did not germinate in water. Upon imbibition in GA₄₊₇, both cell-cycle activity and subsequent germination were enhanced in MM and sit ^w seeds, and were induced in gib-1. In ABA, the germination of MM and sit ^w seeds was inhibited while nuclear replication of these seeds was not affected. It is concluded that GA influences germination by acting upon processes that precede cell-cycle activation, while ABA affects growth by acting upon processes that follow cell-cycle activation.