Acid sphingomyelinase activation requires caspase-8 but not p53 nor reactive oxygen species during Fas-induced apoptosis in human glioma cells

During apoptosis of human glioma cells induced by anti-Fas antibody, ceramide formation with activation of acid, but not neutral sphingomyelinase (SMase), was observed. A potent inhibitor of acid SMase, SR33557, effectively inhibited ceramide formation and apoptosis. Fas-induced apoptosis and ceramide formation proceeded regardless of p53 status. The agents, which modify intracellular levels of reactive oxygen species (ROS) and reduced glutathione (GSH), failed to modulate Fas-induced acid SMase activation and apoptosis. Moreover, expression of functional p53 protein using a temperature-sensitive human p53val(138) induced ceramide generation by activation of neutral SMase but not acid SMase through ROS formation. Peptide inhibitors for caspases-8 (z-IETD-fmk) and -3 (z-DEVD-fmk) suppressed Fas-induced apoptosis. However, activation of acid SMase was inhibited only by z-IETD-fmk. Thus, ceramide generated by acid SMase may take a part in Fas-induced apoptosis of human glioma cells and acid SMase activation may be dependent on caspase-8 activation, but not on p53 nor ROS.     

Carbachol-Induced Cosecretion of Immunoreactive Atrial Natriuretic Peptides with Catecholamines from Cultured Bovine Adrenal Medullary Cells

The distribution and secretion of atrial natriuretic peptides (ANPs) were investigated in bovine adrenal medulla. (1) Cultured bovine adrenal medullary cells (2 x 10(6)/dish) contained 100.4 +/- 6.0 fmol of immunoreactive ANP (IR-ANP) and 207.3 +/- 6.6 nmol of catecholamines as epinephrine plus norepinephrine. (2) Stimulation of nicotinic but not muscarinic acetylcholine receptors caused a cosecretion of IR-ANP and catecholamines corresponding to the ratio of IR-ANP to catecholamines in cultured bovine adrenal medullary cells. (3) Carbachol-stimulated secretion of IR-ANP was dependent on the presence of extracellular Ca2+. (4) Chromaffin granules isolated from bovine adrenal medulla contained large amounts of IR-ANP and catecholamines, in the same ratio as did cultured adrenal medullary cells. (5) Reverse-phase HPLC analysis showed that both stored and secreted IR-ANP consisted of two components, which eluted at the position of ANP(99-126) or ANP(1-126). These results indicate that ANPs are stored as ANP(99-126) and ANP(1-126) in chromaffin granules, and are cosecreted in parallel with catecholamines in a Ca2+-dependent manner by the stimulation of nicotinic acetylcholine receptors.     

Hexokinase-binding properties of the mitochondrial VDAC protein: Inhibition by DCCD and location of putative DCCD-binding sites

The outer mitochondrial membrane receptor for hexokinase binding has been identified as the VDAC protein, also known as mitochondrial porin. The ability of the receptor to bind hexokinase is inhibited by pretreatment with dicyclohexylcarbodiimide (DCCD). At low concentrations, DCCD inhibits hexokinase binding by covalently labeling the VDAC protein, with no apparent effect on VDAC channel-forming activity. The stoichiometry of [¹⁴C]-DCCD labeling is consistent with one to two high-affinity DCCD-binding sites per VDAC monomer. A comparison between the sequence of yeast VDAC and a conserved sequence found at DCCD-binding sites of several membrane proteins showed two sites where the yeast VDAC amino acid sequence appears to be very similar to the conserved DCCD-binding sequence. Both of these sites are located near the C-terminal end of yeast VDAC (residues 257–265 and 275–283). These results are consistent with a model in which the C-terminal end of VDAC is involved in binding to the N-terminal end of hexokinase.     

Interferon and cytotoxic factor (cytotoxin) released in the blood of mice infected with Mycobacterium bovis BCG. III. Interferon and cytotoxin induced by the specific antigen as compared with those induced by bacterial lipopolysaccharide

The time course of development and decline of the ability of BCG-infected mice to produce interferon in the serum in response to the intravenous infection of purified protein derivative of tuberculin (PPD) was very similar to that of their systemic hypersensitivity to PPD. A cytotoxic factor (cytotoxin) was produced in parallel with interferon in the serum of BCG-infected mice after stimulation with PPD. The duration of the period in which cytotoxin-production responsiveness to PPD was definitely detectable was much shorter than that for interferon-production responsiveness although the periods for the maximum production of interferon and cytotoxin coincided. The kinetics of release of interferon in the serum of BCG-infected mice after stimulation with PPD did not parallel that of release of cytotoxin. The four kinds of activities, interferons and cytotoxins induced by PPD and lipopolysaccharide (LPS) in the serum of BCG-infected mice, were compared for their stability to heating at 56 C and to treatment at pH 2. The kinetics of inactivation of these four activities differed significantly, when the serum was either heated at 56 C or treated at pH 2. Interferon produced in response to LPS could be neutralized by anti-L cell(NDV) interferon rabbit serum as easily as L cell (NDV) interferon, 16 times as much antiserum was required to neutralize the same amount of interferon in response to PPD, but cytotoxins induced by PPD and LPS were not neutralized at all by the antiserum. From these findings it is thought likely that interferons and cytotoxins induced by PPD and LPS in the serum of BCG-infected mice are different substances, although the antigenic relationship between cytotoxins induced by PPD and LPS remains unknown.     

Phase determination of the circadian rhythm of conidiation in heterocaryons between two out-of-phase mycelia in Neurospora crassa

Neurospora grows vegetatively as a syncytium in which multiple nuclei exist within a connected cytoplasm. Because of the ability of separate and distinct mycelia to fuse, the possibility exists of generating heterocaryotic cultures in which the nuclei and cytoplasms of two different strains are comingled into the same syncytium. We have used such heterocaryons, in which the component parts differed with respect to their circadian clock phase, to examine whether or not clock-dominant phases exist in the circadian cycle. To this end, the phase subsequent to the formation of heterocaryons by pairs of mycelial discs that are initially at different circadian phases was examined in Neurospora crassa. The resulting phase was an average of the parent phases in many cases, but was sometimes observed to correspond more closely to just one of the original parental phases. In these cases, we did not observe any dominant phases in the circadian cycle; the phase of a particular parent disc was more dominant in the heterocaryon when the proportion of the nuclei from that parent was greater in the heterocaryon. In some instances, which occurred mostly when the difference in phase of the parental discs was large, the resultant phase could not be related in a simple way to the parental phases. An interpretation based on a limit cycle model of the circadian oscillation is possible.     

Induction of ornithine decarboxylase activity in mouse tissues by phorbol ester is effectively blocked by methylglyoxal bis(butylamidinohydrazone)

Ornithine decarboxylase (ODC) was induced in the liver, lung and brain of the mouse injected intraperitoneally with 12-O-tetradecanoylphorbol 13-acetate (TPA), showing maximal enzyme activity four hours after the injection. The increase of ODC activity was due to the enhanced syntheses of mRNA and protein. The induction of ODC activity by TPA was specifically blocked by methylglyoxal bis(butylamidinohydrazone) (MGBB), a competitive inhibitor of ODC and S-adenosylmethionine decarboxylase, but not by the analog methylglyoxal bis(guanylhydrazone) (MGBG).