Neurotransmitter release regulated by nitric oxide in PC-12 cells and brain synaptosomes

BACKGROUND: Nitric oxide is a messenger molecule of the nervous system, which is produced by the enzyme nitric oxide synthase, which may regulate cyclic guanosine monophosphate levels and which has been implicated in the control of neurotransmitter release. PC-12 pheochromocytoma cells differentiate to form neuronal cells in culture when they are exposed to nerve growth factor. The levels of cyclic guanosine monophosphate in the cells and their ability to release acetylcholine in response to K(+)-depolarization are both maximal after eight days of treatment with nerve growth factor. We set out to assess a possible role for nitric oxide in the processes that occur in differentiating PC-12 cells. RESULTS: Nitric oxide synthase is first evident in differentiating PC-12 cells eight days after beginning treatment with nerve growth factor, coinciding with the marked increase in K(+)-depolarization-induced release of acetylcholine. The release of both acetylcholine and dopamine in response to K(+)-depolarization is blocked by inhibitors of nitric oxide synthase and by hemoglobin, which binds nitric oxide. Providing l-arginine, a precursor required for nitric oxide synthesis, reverses the effects of the inhibitors. In synaptosomal preparations from the corpus striatum, inhibitors of nitric oxide synthase prevent the release of glutamate in response to the glutamate derivative N-methyl-d-aspartate but not in response to K(+)-depolarization. CONCLUSION: Nitric oxide may mediate the release of acetylcholine and dopamine in response to K(+)-depolarization in PC-12 cells and the release of glutamate in response to N-methyl-d-aspartate in striatal synaptosomes. Nitric oxide synthase expression is induced after eight days of treating PC-12 cells with nerve growth factor, coinciding with a marked enhancement of the release of neurotransmitters in response to K(+)-depolarization.     

Particle size influences decay rates of environmental DNA in aquatic systems

Environmental DNA (eDNA) analysis is a powerful tool for remote detection of target organisms. However, obtaining quantitative and longitudinal information from eDNA data is challenging, requiring a deep understanding of eDNA ecology. Notably, if the various size components of eDNA decay at different rates, and we can separate them within a sample, their changing proportions could be used to obtain longitudinal dynamics information on targets. To test this possibility, we conducted an aquatic mesocosm experiment in which we separated fish-derived eDNA components using sequential filtration to evaluate the decay rate and changing proportion of various eDNA particle sizes over time. We then fit four alternative mathematical decay models to the data, building towards a predictive framework to interpret eDNA data from various particle sizes. We found that medium-sized particles (1–10 μm) decayed more slowly than other size classes (i.e., <1 and > 10 μm), and thus made up an increasing proportion of eDNA particles over time. We also observed distinct eDNA particle size distribution (PSD) between our Common carp and Rainbow trout samples, suggesting that target-specific assays are required to determine starting eDNA PSDs. Additionally, we found evidence that different sizes of eDNA particles do not decay independently, with particle size conversion replenishing smaller particles over time. Nonetheless, a parsimonious mathematical model where particle sizes decay independently best explained the data. Given these results, we suggest a framework to discern target distance and abundance with eDNA data by applying sequential filtration, which theoretically has both metabarcoding and single-target applications.     

Fast optimal genome tiling with applications to microarray design and homology search.

In this paper, we consider several variations of the following basic tiling problem: given a sequence of real numbers with two size-bound parameters, we want to find a set of tiles of maximum total weight such that each tiles satisfies the size bounds. A solution to this problem is important to a number of computational biology applications such as selecting genomic DNA fragments for PCR-based amplicon microarrays and performing homology searches with long sequence queries. Our goal is to design efficient algorithms with linear or near-linear time and space in the normal range of parameter values for these problems. For this purpose, we first discuss the solution to a basic online interval maximum problem via a sliding-window approach and show how to use this solution in a nontrivial manner for many of the tiling problems introduced. We also discuss NP-hardness results and approximation algorithms for generalizing our basic tiling problem to higher dimensions. Finally, computational results from applying our tiling algorithms to genomic sequences of five model eukaryotes are reported.     

Impact of flooding on the densities of selected aquatic insects

Data from a four-year study of five aquatic insect species,Hydropsyche betteni, H. morosa, H. bronta, Isonychia bicolor, andEphoron leucon, were utilized to evaluate the impact of a 60-year flood and a few lesser floods. The survey began in August, 1984 and was terminated in October, 1987 with the 60-year flood occurring in November, 1985. Four sampling sites were established on the South River and six quantitative samples were taken each month from each site. Gauging stations on the South River provided accurate discharge data for the sampling sites and useful historical data. Densities for the five species were utilized in the evaluation of the floods. The importance of timing is pointed out, that is, floods that occur very close together or near the end of the life cycle of an insect make it difficult to evaluate floods as disturbances. The importance of life history traits, such as behavior and egg diapause, are discussed in respect to floods. Densities were reduced to less than 50% of their average values immediately after the 60-year flood for the threeHydropsyche spp. and at three sites forI. bicolor. Ephoron leucon showed no response to the 60-year flood. Densities of the four impacted species returned to previous levels in the following generation. The 60-year flood was considered a disturbance in the near term but not for more than one generation.     

Poly(ADP-Ribose) Synthetase Activation: An Early Indicator of Neurotoxic DNA Damage

Abstract: DNA damage activates a nuclear enzyme poly(ADP-ribose) synthetase (PARS) that facilitates DNA repair by adding multiple ADP-ribose groups to nuclear proteins such as histones and PARS itself. N -Methyl- d -aspartate neurotoxicity may involve DNA damage excessively activating PARS to deplete its substrate NAD, as PARS inhibitors prevent this toxicity. We now show that PARS is rapidly and markedly activated in PC12 cells following treatment with neurotoxic agents, including the amyloid β-protein, hydrogen peroxide, N -methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and its active metabolite N -methyl-4-phenylpyridine (MPP⁺). With MPP⁺, PARS activity is increased fivefold in 1 h and 20-fold by 3 h. By contrast, direct measurement of DNA damage by the terminal-deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling assay shows no significant increase by 3 h and less than fourfold by 24 h. These findings indicate that PARS activity can provide a simple, sensitive, and early index of DNA damage following neurotoxic insults.     

Spectrum of small mutations in the dystrophin coding region.

Duchenne and Becker muscular dystrophies (DMD and BMD) are caused by defects in the dystrophin gene. About two-thirds of the affected patients have large deletions or duplications, which occur in the 5' and central portion of the gene. The nondeletion/duplication cases are most likely the result of smaller mutations that cannot be identified by current diagnostic screening strategies. We screened approximately 80% of the dystrophin coding sequence for small mutations in 158 patients without deletions or duplications and identified 29 mutations. The study indicates that many of the DMD and the majority of the BMD small mutations lie in noncoding regions of the gene. All of the mutations identified were unique to single patients, and most of the mutations resulted in protein truncation. We did not find a clustering of small mutations similar to the deletion distribution but found > 40% of the small mutations 3' of exon 55. The extent of protein truncation caused by the 3' mutations did not determine the phenotype, since even the exon 76 nonsense mutation resulted in the severe DMD phenotype. Our study confirms that the dystrophin gene is subject to a high rate of mutation in CpG sequences. As a consequence of not finding any hotspots or prevalent small mutations, we conclude that it is presently not possible to perform direct carrier and prenatal diagnostics for many families without deletions or duplications.     

Release of arachidonic acid from 1-alkyl-2-acyl-sn-glycero-3-phosphocholine, a precursor of platelet-activating factor, in rat alveolar macrophages

Platelet activating factor and the bioactive metabolites of arachidonic acid are secreted by alveolar macrophages in response to stimulation by phagocytic agents or calcium ionophore. We have previously shown a deacylation-acetylation sequence in the formation of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF) from alkylacyl-(long chain)-GPC (Albert, D.H. and Snyder, F. (1983) J. Biol. Chem. 258, 97-102). This sequence may be an important source of 20:4 during inflammatory reactions since, in alveolar macrophages, the ether lipid precursor of PAF represents 35% of the choline glycerophospholipids and has a much higher content (35%) of 20:4 in the sn-2 position than does diacyl-GPC (17%). Alveolar macrophages prelabeled with 14C-labeled fatty acids (16:0, 18:1, 18:2 and 20:4) and [1-3H]alkyllyso-GPC were used to study the release of fatty acids from ether-linked and diacyl phospholipids. Each of these fatty acids was incorporated primarily into the choline glycerophospholipids of alveolar macrophages. The release of 20:4 from macrophage phospholipids was increased by treatment of the labeled cells with the calcium ionophore A23187 (2 microM) or zymosan (1 mg/ml), whereas the release of 16:0, 18:1 and 18:2 was not increased above control levels by either stimuli. Although more of the labeled 20:4 is released from the diacyl-GPC (50% of the total released), substantial amounts (44%) of 20:4 are derived from alkylacyl-GPC after incubating the stimulated cells for 60 min. The loss of 20:4 continued from the diacyl species throughout the incubation period studied, whereas a slower net release of 20:4 lost from the alkylacyl-GPC fraction was evident after 2 h. We conclude that the deacylation-reacylation cycle is an important aspect of the metabolism of 20:4 and alkylacyl-GPC during inflammatory stimulation of alveolar macrophages and that the deacylation of this ether-linked phospholipid (which is the first step in the formation of PAF) is responsible for a significant amount of the 20:4 released.     

Vertebrate class distribution of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine acetylhydrolase in serum

Serum from numerous mammals and lower vertebrates contains an enzyme activity that is specific for the hydrolysis of the acetate moiety of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF, platelet activating factor). Acetylhydrolase (EC 3.1.1.47, 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine acetylhydrolase) was found in all mammalian sera with activity ranging from 11 (fetal calf) to 178 (rabbit) pmol acetate liberated/microliter serum/min. The enzyme is not present in avian serum but is a constituent of reptiles and bony fishes.     

Selective labeling of alpha-noradrenergic receptors in rat brain with [3H]dihydroergokryptine.

Using concentrations of [³H] dihydroergokryptine between 0.1 and 5 nM, saturable binding can be demonstrated in rat cerebral cortical membranes with a dissociation constant (K_D) of about 0.8 nM. α-Noradrenergic agonists and antagonists compete for the sites labeled by these low concentrations of [³H] dihydroergokryptine with relative potencies characteristics of classical α-noradrenergic receptors. The very low potency of serotonin in competing for these binding sites indicates that, in contrast to findings with higher concentrations of [³H] DHE, low concentrations do not label serotonin receptors. Moreover, the low potency of dopamine in competing for [³H] dihydroergokryptine binding in both striatal and cortical membranes indicates that no detectable portion of binding is associated with postsynaptic dopamine receptors.