The effect of nitrofurantoin on deoxyribonucleic acid synthesis in plant mitochondria.

Nitrofurantoin (1-([(5-nitrofuran-2-yl)methylene]amino)imidazolidine-2,4-dione), a widely used drug and also a well-known bacterial mutagen, inhibits DNA synthesis in mitochondria from 48 h etiolated seedlings of Vigna sinensis (Linn.) Savi (snake bean). The effect appears at the level of the uptake of radioactive deoxynucleoside triphosphates by the plant mitochondria. Nitrofurantoin does not inhibit DNA synthesis in vitro by homogeneous Escherichia coli DNA polymerase I and DNA polymerase from avian-myeloblastosis virus. No specific nitroreductase activity could be detected in mitochondria.     

Degradation of rRNA in interferon-treated vaccinia virus-infected cells

In interferon-treated mouse L cells, following infection with a DNA-containing virus, vaccinia, synthesis of unique 2'-5'-linked oligonucleotides of the general formula ppp5'A2'(p5'A)n, abbreviated as 2-5A, was detected by competition radiobinding assay. In addition, degradation of rRNA into discrete and characteristic products, similar to those produced by 2-5A-activated endonuclease, was observed. The degradation of rRNA may represent a significant component of antiviral action of interferon in vaccinia virus-infected cells.     

Attempt at gene reversion of a desired phenotype as a rescue against strain degeneration

Mutagenesis may lead to the production of antibiotic producer revertant derivatives from a non-producer degenerate originally active against skin pathogens. All of the producer revertants examined in the present study were phenotypically different from the producer parental mutant. Most of the revertants were active against plant pathogens, the test fungus used for screening being a plant pathogen. The selective pressure of the test fungus for isolation of revertants may prove very useful in producing a revertant derivative of a desired phenotype.     

Postsynaptic complexin controls AMPA receptor exocytosis during LTP

Long-term potentiation (LTP) is a compelling synaptic correlate of learning and memory. LTP induction requires NMDA receptor (NMDAR) activation, which triggers SNARE-dependent exocytosis of AMPA receptors (AMPARs). However, the molecular mechanisms mediating AMPAR exocytosis induced by NMDAR activation remain largely unknown. Here, we show that complexin, a protein that regulates neurotransmitter release via binding to SNARE complexes, is essential for AMPAR exocytosis during LTP but not for the constitutive AMPAR exocytosis that maintains basal synaptic strength. The regulated postsynaptic AMPAR exocytosis during LTP requires binding of complexin to SNARE complexes. In hippocampal neurons, presynaptic complexin acts together with synaptotagmin-1 to mediate neurotransmitter release. However, postsynaptic synaptotagmin-1 is not required for complexin-dependent AMPAR exocytosis during LTP. These results suggest?a complexin-dependent molecular mechanism for regulating AMPAR delivery to synapses, a mechanism that is surprisingly similar to presynaptic exocytosis but controlled by regulators other than synaptotagmin-1.     

Chronic Exposure to κ-Opioids Enhances the Susceptibility of Immortalized Neurons (F-11κ7) to Apoptosis-Inducing Drugs by a Mechanism that May Involve Ceramide

Abstract: Chronic exposure of embryonic brain to opioids leads to microcephaly and developmental abnormalities. An immortalized mouse neuroblastoma × dorsal root ganglion hybrid cell line stably transfected to overexpress κ-opioid receptors (F-11κ7) showed complete loss of κ-receptor binding to [³H]U69,593 after exposure to the κ-agonist U69,593 for 24 h. U69,593 had no measurable effect on cell viability as determined by either cell viability or DNA fragmentation assays. However, when cell death (apoptosis) was induced by either staurosporine or the phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002, cells pretreated with U69,593 for 24 h showed increased apoptosis compared with untreated cells. Thus, staurosporine (50 n M ), wortmannin (4 µ M ), and LY294002 (30 µ M ) treatment for 24 h induced a 50% loss of cell viability and DNA fragmentation in 24 h. U69,593 pretreatment produced the same killing at lower concentrations, namely, 20 n M staurosporine, 2 µ M wortmannin, and 14 µ M LY294002, respectively. The effects of U69,593 were time-, dose-, and naloxone-reversible, suggesting that they are receptor-mediated. However, coaddition of U69,593 at the same time as staurosporine, wortmannin, or LY294002 did not enhance apoptosis. All three drugs that induced apoptosis were found to increase the level of ceramide, and pretreatment with U69,593 further increased the rate of formation of ceramide, a lipid that induces apoptosis in cells. We propose that chronic exposure to κ-receptor agonists promotes increased vulnerability of neurons to apoptosis.     

G1shortening following unbalanced growth a specific v. nonspecific effect

The synthesis and abundance of proteins were examined in synchronous populations of HeLa cells under conditions in which the lengthening of S phase, by inhibiting DNA synthesis, resulted in shortening of G1 in the subsequent generation. Mitotically collected cells were resynchronized by incubation with 3 microM aphidicolin from 3 to 12 h after mitotic selection; they were blocked again at various times thereafter to induce unbalanced growth. Cells were labelled with [35S]-methionine before and after release from the block to study the changes in protein synthesis. Triton X-100 soluble and insoluble proteins were analysed by 7-15% gradient SDS-PAGE, and radioactivity incorporation was quantified by liquid-scintillation counting. The degree of G1 shortening correlated with S phase position, increasing gradually from early S and reaching maximum when cells were blocked half-way through S phase. Synthesis of proteins of 120, 66, and 51 kDa was stimulated, and synthesis of a new protein of 57kDa was observed, in cells in which DNA synthesis had been blocked in mid-S. These proteins also showed increased accumulation. These results suggest that the shortening of G1, induced by the prior arrest of cell-cycle progression, is associated with synthesis of specific proteins rather than the non-specific accumulation of cellular proteins through unbalanced growth.     

Cyclic AMP Protects Against Staurosporine and Wortmannin-Induced Apoptosis and Opioid-Enhanced Apoptosis in Both Embryonic and Immortalized (F-11κ7) Neurons

Abstract: The mechanism by which opiates affect fetal development is unknown, but one potential target is the programmed cell death (apoptosis) pathway of neurons. Apoptosis was induced in both primary neuronal cultures from embryonic day 7 cerebral hemispheres of chick brain (E7CH) and the F-11κ7 cell line (an immortalized mouse neuroblastoma × dorsal root ganglion hybrid stably transfected to overexpress κ-opioid receptors) by either staurosporine or the phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002. Cells pretreated with either the μ-specific opioid agonist morphiceptin (E7CH) or the κ-specific opioid agonist U69,593 (F-11κ7) for 24 h showed increased apoptosis in response to staurosporine or wortmannin when compared with nonpretreated cells. The effects of morphiceptin and U69,593 were time- and dose-dependent and antagonist-reversible, suggesting that they were receptor-mediated. Neither morphiceptin nor U69,593 by themselves had any measurable effect on cell viability or DNA fragmentation, and coaddition of opiates at the same time as staurosporine, wortmannin, or LY294002 did not enhance apoptosis. Time course studies indicated a maximal opioid effect at a time (16–24 h) when inhibition of adenylate cyclase had been maximal for many hours. Addition of dibutyryl cyclic AMP either before or at the time of opioid addition protected against apoptosis and reduced fragmentation to levels seen for staurosporine plus dibutyryl cyclic AMP alone. The specificity for cyclic AMP was confirmed by showing protection with the specific agonist Sp -adenosine 3',5'-cyclic monophosphothioate and increased killing with the antagonist Rp -adenosine 3',5'-cyclic monophosphothioate. We conclude that the opioid enhancement of apoptosis is based on the inhibition of adenylate cyclase and that the effect is time-dependent.