Cyclosporine A- and FK506-induced apoptosis in PC12 cells.

The purpose of this study was to examine, using glycogen synthase kinase (GSK) inhibitors, whether GSK-3 is involved in cyclosporine A (CsA)- and FK506-induced apoptosis in PC12 cells. CsA and FK506 increased apoptotic cell death with morphological changes characterized by cell shrinkage and nuclear condensation or fragmentation. Nerve growth factor (NGF) completely blocked cell death. Caspase-3 activation was accompanied by CsA- and FK506-induced cell death and inhibited by NGF. GSK-3 inhibitors such as alsterpaullone and SB216763 prevented CsA- and FK506-induced apoptosis. These results suggest that CsA and FK506 induce caspase-dependent apoptosis and that GSK-3 activation is involved in CsA- and FK506-induced apoptosis in PC12 cells.

beta-cyanoalanine production by marine bacteria on cyanide-free medium and its specific inhibitory activity toward cyanobacteria.

In screening the culture broth of marine bacteria collected at Yap (Micronesia), Palau (Belau), and Okinawa (the southwest islands of Japan) for antimicroalgal activity, 37 out of 2,594 bacterial isolates tested were found to produce anticyanobacterial substances against Oscillatoria amphibia NIES-361. One strain, C-979, identified as a Vibrio sp., was selected and cultured in 2.4 liters of marine broth 2216 to identify the bioactive compound produced by the strain. The purified very hydrophilic compound (16.4 mg) was determined to be beta-cyano-L-alanine (L-CNAla) by instrumental analyses and the application of the advanced Marfey method. L-CNAla did not inhibit the growth of bacteria, yeast, or eukaryotic microalgae, but some cyanobacteria were found to be sensitive to L-CNAla at a concentration of 0.4 to 25 microg/ml. The effect of L-CNAla on some other environmental organisms, including invertebrates and a macroalgae, is discussed. CNAla production in marine broth was examined by thin-layer chromatography for the 37 bacterial isolates which produced an anticyanobacterial substance. The broth of 36 of these strains contained CNAla, suggesting the wide distribution of CNAla production by marine bacteria. This is the first report on bacteria that produce CNAla without a supply of the cyanide ion in the medium.

Apoptotic cell death and caspase-3 activation induced by N-methyl-D-aspartate receptor antagonists and their prevention by insulin-like growth factor I.

The effect of N-methyl-D-aspartate (NMDA) receptor antagonists on cell viability was studied in rat primary cortical cells. NMDA antagonists [MK-801 and 2-amino-5-phosphonovalerate (APV)] induced cell shrinkage, nuclear condensation or fragmentation, and internucleosomal DNA fragmentation. Treatment of cells with MK-801 (an NMDA antagonist) for 1-2 days induced apoptotic cell death in a dose-dependent manner (1 nM to 10 microM). NMDA (25 microM), however, inhibited the MK-801 (0.1 microM)-induced apoptotic cell death. MK-801 and APV decreased the concentration of intracellular calcium ion. Activation of caspase-3 was accompanied by MK-801-induced cell death in a dose-dependent manner, and an inhibitor of caspase-3 reduced the cell death. Further, cycloheximide (0.2 microg/ml) completely protected the cells from MK-801-induced apoptotic cell death and caspase-3 activation. Insulin-like growth factor I completely attenuated MK-801-induced apoptotic cell death and caspase-3 activation. These results demonstrated that the moderate NMDA receptor activation is probably involved in the survival signal of the neuron.

Apoptotic cell death and CPP32-like activation induced by thapsigargin and their prevention by nerve growth factor in PC12 cells.

Thapsigargin, an endoplasmic reticular Ca2+-ATPase inhibitor, induced apoptotic cell death (chromatin condensation and DNA fragmentation) accompanied by the activation of CPP32-like protease, a member of the interleukin-1beta converting enzyme protease (ICE) family, but not the activation of ICE-like protease. Nerve growth factor (NGF) completely inhibited the cell death and CPP32-like activation induced by thapsigargin while Ac-Asp-Glu-Val-Asp-CHO, an inhibitor of CPP32-like protease, reduced the cell death. PD98059, a specific inhibitor of Map kinase kinase, did not reduce the protective effect of NGF on thapsigargin-induced cell death. These results suggest that calcium ion-induced apoptotic cell death was mediated by CPP32-like, but not ICE-like, protease and was regulated by a neurotrophic factor possibly, through the Map kinase cascade independent pathway.

Apoptotic cell death and caspase 3 (CPP32) activation induced by calcium ionophore at low concentrations and their prevention by nerve growth factor in PC12 cells.

A23187 (a calcium ionophore) at low concentration (0.1 microM) induced apoptotic cell death (chromatin condensation and DNA fragmentation) accompanied by the activation of caspase-3 (CPP32), a member of the interleukin-1beta-converting enzyme protease. On the other hand, A23187 at high concentration (2 microM) induced necrotic cell death not accompanied by the activation of CPP32. Nerve growth factor inhibited the cell death and CPP32 activation induced by 0.1 microM A23187, but not the cell death induced by 2 microM A23187. Acylaspartyl-glutamyl-valyl-aspartyl-aldehyde, an inhibitor of CPP32, reduced the cell death induced by 0.1 microM A23187. These results suggest that calcium-ion-induced apoptotic cell death was mediated by CPP32 activation in PC12 cells.

New anticancer antibiotics pelagiomicins, produced by a new marine bacterium Pelagiobacter variabilis.

In the course of our screening for new anticancer compounds produced by marine bacteria, we found that a new genus marine bacterium Pelagiobacter variabilis produced new phenazine antibiotics, pelagiomicins A, B and C. Those compounds were labile in water and alcohols. The absolute structure of the main component, pelagiomicin A, and the structures of the minor ones were determined from the spectroscopic data and by synthesis. Pelagiomicin A exhibits activity against Gram-positive and -negative bacteria and antitumor activity in vitro and in vivo.

Korormicin, a novel antibiotic specifically active against marine gram-negative bacteria, produced by a marine bacterium.

A novel antibiotic named korormicin was isolated from the marine bacterium, Pseudoalteromonas sp. F-420. This strain was isolated from the surface of a macro alga Halimeda sp. collected from Palau (the Republic of Belau). The planar structure of korormicin was determined by the result of 2D NMR studies and mass spectral data. Korormicin had specific inhibitory activity against marine Gram-negative bacteria, but was inactive against terrestrial microorganisms.

Reactive oxygen species involved in the glutamate toxicity of C6 glioma cells via xc antiporter system.

We recently demonstrated that continuous L-glutamate exposure led to cell death in C6 glioma cells over a period of 24-36 h, due to inhibition of cystine uptake through the cystine/glutamate (XC) antiporter. The antioxidant vitamin E provided protection against this effect, supporting the hypothesis that depletion of glutathione might be responsible, resulting from insufficient cystine uptake. To clarify the content of oxidative stress after glutathione depletion, the present study was done to investigate accumulation and target molecules of reactive oxygen species induced by glutamate treatment. The accumulation of reactive oxygen species was increased three-fold as compared to a control culture. Membrane oxidation, as judged by lipid peroxidation, was increased two-fold after glutamate treatment. Cellular ATP content was significantly reduced by glutamate exposure. For the two cytosolic enzymes examined, activity of glyceraldehyde 3-phosphate dehydrogenase was slightly enhanced by glutamate treatment, while activity of glutamine synthetase was not changed. Impairment of nuclear DNA after glutamate exposure was also revealed by nuclear chromatin condensation with DNA fragmentation. Thus, the multiple targets (membrane, cytoplasm and nuclei) of oxygen radicals in glutamate toxicity through the xc antiporter system were evaluated for the first time. Furthermore, prevention from cell death and from cellular toxicity induced by oxygen radicals could be seen using three specific oxygen radical scavengers, catalase, 3,3,5,5-tetramethyl-pyrroline N-oxide and alpha-phenyl-N-t-butylnitrone, without restoring the glutathione deficit. This indicates that radical scavengers did not interact with the xc antiporter system, but directly scavenged the oxygen radicals. Taken together, the data strongly suggest that O2-, H2O2 and OH accumulate in response to oxidative stress after glutathione depletion, resulting in glutamate cell death of C6 glioma cells.

A cyclic AMP analog and high potassium prevent the death of cultured septal cholinergic neurons after nerve growth factor withdrawal.

Survival, following the addition of a cAMP analog and high K+ to the medium, of cultured fetal septal cholinergic neurons was examined after nerve growth factor (NGF) deprivation. The number of acetylcholinesterase positive cells, which had progressively grown during 11-13 d of culture with NGF (50 ng/ml), was greatly reduced following 5 d of extended culture without NGF (55% of that with NGF). The degeneration of the cholinergic neurons was markedly reduced by addition of dibutyryl cAMP (dbcAMP, 1 mM), forskolin (10 microM) or KCl (15 mM) to the medium. K252b, which blocks the survival of NGF, had no effect on the action of dbcAMP. H-8 and nifedipine inhibited the survival of dbcAMP and high KCl, respectively. These results suggest that NGF, dbcAMP and high K+ promote the survival of septal cholinergic neurons acting via the receptor tyrosine kinase, protein kinase A and a Ca(2+)-dependent mechanism, respectively.

Toxic effect of a beta-amyloid peptide (beta 22-35) on the hippocampal neuron and its prevention.

A synthetic truncated beta-amyloid peptide, beta 22-35, was shown to have a cytotoxic effect on cultured neurons from the rat hippocampus in serum-free medium. The peptide formed aggregates and typical amyloid fibrils resembling those of the beta-amyloid protein (AP) in neutral buffer solution and showed characteristic staining with Congo red and thioflavin-S. The neurotoxicity of beta 22-35 was suppressed by addition of calf serum, dibutyryl cAMP or insulin to culture medium, but not by addition of NGF or substance P. beta 22-35 had no effect on the glial cells. These results suggest that the AP can induce neurotoxicity in the hippocampal cells in vitro and the toxicity may involve a disorder in the intracellular signal transduction.

Extracellular pH modulates N-methyl-D-aspartate receptor-mediated neurotoxicity and calcium accumulation in rat cortical cultures.

The effect of extracellular pH (pHo) on the excitotoxicity of N-methyl-D-aspartate (NMDA) in cultured rat cortical cells was studied. Treatment of cells with 500 microM NMDA for 15 min at various pH's in a range from 6.5 to 8.0 progressively enhanced staining with Trypan blue and release of lactate dehydrogenase with increased pH after 18 h of culture following treatment. The cytotoxic effect of high concentration of K+ (40 mM) or veratridine (10 microM) was also directly related to the increase in pHo. Free calcium accumulation in cells on addition of NMDA increased parallel to pHo. Changes in intracellular pH were estimated to be minor compared with extracellular changes. Specific NMDA antagonists could block both the NMDA- and membrane depolarization-induced neurotoxicity and calcium accumulation completely. These results suggest that the proton concentration outside of cells attenuates NMDA-induced neurotoxicity by blocking calcium accumulation.

Protection by ethanol of cortical neurons from N-methyl-D-aspartate-induced neurotoxicity is associated with blocking calcium influx.

Effect of ethanol on N-methyl-D-aspartate (NMDA)-induced neurotoxicity in rat dissociated cortical cells (8-12 day cultures) was studied. Treatment of cells with NMDA (50 and 500 microM) for 15 min caused cytotoxic effects on the cells, as examined by microscopic observations and lactate dehydrogenase release from cells 18 h after the treatment. Ca2+ is essential for these effects in medium during treatment. Presence of ethanol (50-300 mM) simultaneously with NMDA protected cells from the cytotoxicity depending on the concentration of ethanol. Calcium accumulation in cells on addition of NMDA, as monitored by fluorescence ratio (F405/F485) of Indo-1-preloaded cortical cells, was also decreased depending on the concentration of added ethanol. APV (200 microM) and ketamine (100 microM) blocked both the cytotoxicity and cellular calcium accumulation due to NMDA. These results suggest that ethanol effects its protection of neurons from NMDA-induced cytotoxicity by blocking the receptor-mediated calcium influx.

Structure of the two promoters of the human lck gene: differential accumulation of two classes of lck transcripts in T cells.

The human T-cell- or lymphocyte-specific gene, lck, encodes a tyrosine kinase and is a member of the src family. In this report we demonstrate that there are two classes of human lck transcripts (types I and II), containing different 5'-untranslated regions, which are expressed from two distinct promoters. No apparent sequence similarity was observed between the 5'-flanking regions of the two promoters. The expression of lck in human T-cell leukemia and carcinoma cell lines and in human peripheral blood T lymphocytes was examined by S1 nuclease and primer extension mapping and by Northern (RNA) blot analysis of total cellular RNA. The following results were obtained. (i) Two RNA start sites in the downstream promoter were used to generate type I transcripts. (ii) The major human type I start site has not been described for the mouse. (iii) At least five RNA start sites in the upstream promoter were used to generate type II transcripts. (iv) In T cells and in two colon carcinoma cell lines, type II transcripts were present in higher amounts than type I transcripts. (v) In T cells treated with phytohemagglutinin, tetradecanoylphorbol acetate, and cyclosporin A, the modulation of lck expression was associated primarily with changes in levels of type II transcripts. The above results suggest that the two human lck promoters are utilized differentially and may be regulated independently during certain physiological states.

Structure of T cell antigen receptor beta chain in synovial fluid cells from patients with rheumatoid arthritis.

We attempted to identify a clonal proliferation of T cells from synovial fluid samples from patients with rheumatoid arthritis, using techniques of restriction fragment length polymorphism. We used probes for the beta chain of the T cell receptor to analyze restriction fragments prepared from the genomic DNA of synovial fluid mononuclear cells from 10 patients and synovial fluid T cell preparations from 5 additional patients. The results demonstrated unarranged (germline) T cell receptor gene fragments of DNA in all cell preparations, indicating the lack of clonality of rheumatoid arthritis synovial fluid T cells.

Effect of intracellular free Ca2+ on leucine transport in Chang liver cells.

Addition of Ca2+ ionophore (A23187) to the medium stimulated the Na+-independent leucine transport in Chang liver cells, increasing the cytoplasmic free Ca2+ concentration, irrespective of the presence or absence of extracellular Ca2+. Anticalmodulin drugs, such as chlorpromazine, trifluoperazine, and W-7, significantly inhibited the leucine transport in the cells. The stimulatory effect of A23187 on leucine transport was completely blocked in the presence of the anticalmodulin drug. Two microtubule disrupting drugs, colchicine and colcemid, significantly stimulated leucine transport. On the other hand, taxol, a microtubule stabilizing agent, decreased the stimulatory effect of colchicine on the leucine transport. These results strongly suggest the involvement of Ca2+ and calmodulin in regulation of Na+-independent leucine transport, possibly through control of assembly and disassembly of the microtubule network.

Effect of N-ethylmaleimide on leucine transport in the Chang liver cell. II. Effect on the kinetics of Na+-independent transport.

The Na+-independent leucine transport system is resolved into two components by their different affinity (Km about 44 microM and 8.0 mM) for leucine in the Chang liver cell. Treatment of the cells with N-ethylmaleimide (1 mM) specifically stimulates the high-affinity component of the Na+-independent system by greatly increasing its Vmax value, whereas the Vmax value of the low-affinity component is markedly lowered. The stimulatory effect of N-ethylmaleimide on leucine transport is reduced by prior treatment of the cells with 2,4-dinitrophenol, but this phenomenon seems to be irrelevant to the ATP-depleting action of the uncoupler. The treatment with 2,4-dinitrophenol has been found not to be inhibitory on the subsequent Na+-independent leucine uptake itself. Treatment with dibucaine, a phospholipid-interacting drug, also reduces to varying degrees (depending on its concentration) the stimulatory effect of N-ethylmaleimide on the subsequent leucine uptake, although pretreatment with dibucaine can stimulate the Na+-independent leucine uptake itself. We conclude that the stimulatory effect of N-ethylmaleimide on leucine transport is not correlated with the energy level of cell, but involves the perturbation of the membrane bilayer structures.

Effect of N-ethylmaleimide on leucine transport in Chang liver cells. I. stimulatory effect on Na+ -independent transport at low concentrations of leucine.

Pretreatment of Chang liver cells with N-ethylmaleimide (0.5 or 1 mM) stimulated Na+ -independent uptake of leucine at low concentrations (less than or equal to 1 mM). The stimulatory effect of N-ethylmaleimide on the uptake of leucine measured in Na+ -replete medium was completely blocked by the addition of beta-2-aminobicyclo[2,2,1]heptane-2-carboxylate (5 mM), which shows that the L system participates in the stimulation. The Na+ -dependent uptake of glycine was depressed by N-ethylmaleimide pretreatment. The stimulation of the Na+ -independent component of leucine uptake continued for at least 30 min after N-ethylmaleimide treatment, while the inhibition of glycine uptake was progressive with time and the Na+ -dependent uptake of leucine became depressed later, after the treatment. It has been demonstrated that treatment of cells with N-ethylmaleimide is capable of increasing the Na+ -independent influx of leucine and at the same time slightly decreasing the efflux of it. These results suggest that N-ethylmaleimide attacks the Na+ -independent system of amino acid transport at the reactive SH groups(s) of relevant protein(s) in favor of specific activation of that system in this cell.

Change of the reactivity of sulfhydryl groups of the membrane proteins in response to amino acids in Chang liver cells.

Intensity of fluorescence induced by reaction of a SH-directed fluorogenic reagent with whole Chang liver cells or their plasma membrane preparations which were previously treated with 0.5 mM N-ethylmaleimide (NEM) plus 5 mM leucine was enhanced by 30% or more in the presence of 5 mM leucine or valine in assay medium consisting of Tris-Hepes buffer, pH 6.85, compared with the control. The dissociation constant for binding of leucine to the membrane protein(s) was estimated to be approximately 3 mM from the increments of fluorescence intensity with increasing leucine concentration. On the other hand, the amount of [14C]NEM bound to the membrane preparations pretreated with NEM plus leucine was significantly reduced in the presence of leucine in the reaction medium. A decrease of reactivity of SH groups near the binding sites of the membrane proteins and of mobility of the fluorescent adducts on binding leucine was suggested to explain all these results consistently.