Activation of membrane-associated procaspase-3 is regulated by Bcl-2.

The mechanism by which membrane-bound Bcl-2 inhibits the activation of cytoplasmic procaspases is unknown. Here we characterize an intracellular, membrane-associated form of procaspase-3 whose activation is controlled by Bcl-2. Heavy membranes isolated from control cells contained a spontaneously activatable caspase-3 zymogen. In contrast, in Bcl-2 overexpressing cells, although the caspase-3 zymogen was still associated with heavy membranes, its spontaneous activation was blocked. However, Bcl-2 expression had little effect on the levels of cytoplasmic caspase activity in unstimulated cells. Furthermore, the membrane-associated caspase-3 differed from cytosolic caspase-3 in its responsiveness to activation by exogenous cytochrome c. Our results demonstrate that intracellular membranes can generate active caspase-3 by a Bcl-2-inhibitable mechanism, and that control of caspase activation in membranes is distinct from that observed in the cytoplasm. These data suggest that Bcl-2 may control cytoplasmic events in part by blocking the activation of membrane-associated procaspases.

Peptide inhibitors of the ICE protease family arrest programmed cell death of motoneurons in vivo and in vitro.

Members of the CED-3/interleukin-1 beta-converting enzyme (ICE) protease family have been implicated in cell death in both invertebrates and vertebrates. In this report, we show that peptide inhibitors of ICE arrest the programmed cell death of motoneurons in vitro as a result of trophic factor deprivation and in vivo during the period of naturally occurring cell death. In addition, interdigital cells that die during development are also rescued in animals treated with ICE inhibitors. Taken together, these results provide the first evidence that ICE or an ICE-like protease plays a regulatory role not only in vertebrate motoneuron death but also in the developmentally regulated deaths of other cells in vivo.

Bax- and Bak-induced cell death in the fission yeast Schizosaccharomyces pombe.

The effects of the expression of the human Bcl-2 family proteins Bax, Bak, Bcl-2, and Bcl-XL were examined in the fission yeast Schizosaccharomyces pombe and compared with Bax-induced cell death in mammalian cells. Expression of the proapoptotic proteins Bax and Bak conferred a lethal phenotype in this yeast, which was strongly suppressed by coexpression of the anti-apoptotic protein Bcl-XL. Bcl-2 also partially abrogated Bax-mediated cytotoxicity in S. pombe, whereas a mutant of Bcl-2 (Gly145Ala) that fails to heterodimerize with Bax or block apoptosis in mammalian cells was inactive. However, other features distinguished Bax- and Bak-induced death in S. pombe from animal cell apoptosis. Electron microscopic analysis of S. pombe cells dying in response to Bax or Bak expression demonstrated massive cytosolic vacuolization and multifocal nuclear chromatin condensation, thus distinguishing this form of cell death from the classical morphological features of apoptosis seen in animal cells. Unlike Bax-induced apoptosis in 293 cells that led to the induction of interleukin-1 beta-converting enzyme (ICE)/CED-3-like protease activity, Bax- and Bak-induced cell death in S. pombe was accompanied neither by internucleosomal DNA fragmentation nor by activation of proteases with specificities similar to the ICE/CED-3 family. In addition, the baculovirus protease inhibitor p35, which is a potent inhibitor of ICE/CED-3 family proteases and a blocker of apoptosis in animal cells, failed to prevent cell death induction by Bax or Bak in fission yeast, whereas p35 inhibited Bax-induced cell death in mammalian cells. Taken together, these findings suggest that Bcl-2 family proteins may retain an evolutionarily conserved ability to regulate cell survival and death but also indicate differences in the downstream events that are activated by overexpression of Bax or Bak in divergent cell types.

In situ immunodetection of activated caspase-3 in apoptotic neurons in the developing nervous system.

Activation of caspase-3 requires proteolytic processing of the inactive zymogen into p18 and p12 subunits. We generated a rabbit polyclonal antiserum, CM1, which recognizes the p18 subunit of cleaved caspase-3 but not the zymogen. CM1 demonstrated an apparent specificity for activated caspase-3 by specifically immunolabelling only apoptotic but not necrotic cortical neurons in vitro. In the embryonic mouse nervous system, CM1 immunoreactivity was detected in neurons undergoing programmed cell death and was markedly increased in Bcl-xL-deficient embryos and decreased in Bax-deficient embryos. CM1 immunoreactivity was absent in the nervous system of caspase-3-deficient mouse embryos and in neurons cultured from caspase-3-deficient mice. Along with neuronal somata, extensive neuritic staining was seen in apoptotic neurons. These studies indicate that caspase-3 is activated during apoptosis in the developing nervous system in vivo and that CM1 is a useful reagent for its in situ detection.

Mutational analysis of the interacting cell death regulators CED-9 and CED-4.

The genes ced-3, ced-4 and ced-9 are central components in the cell death pathway of the nematode C. elegans. Ced-9, which functions to inhibit cell death, is homologous to the Bcl-2 family of mammalian anti-apoptotic genes. The ced-3 gene encodes a protein homologous to the caspases, a family of cysteine proteases involved in the execution of programmed cell death. It has recently been demonstrated that CED-4, an inducer of apoptosis for which no mammalian equivalent has been reported, can interact with CED-9 and Bcl-x(L). Here we confirm that CED-9 and CED-4 interact and using a series of deletion mutants, demonstrate that only short N-terminal deletions are tolerated in each molecule without loss-of-interaction. Two loss-of-function point mutations in different regions of CED-4 also lead to a significant loss of interaction suggesting further that the relevant interaction domains are not short linear sequences, but rather, are formed by more complex structural determinants in each molecule. Furthermore, we demonstrate that CED-4 not only interacts with Bcl-x(L) but also with its homologue, Bcl-2, and that the unstructured loop region present in Bcl-x(L) and Bcl-2 can regulate the CED-4 interaction. Lastly, we show that a BH3 peptide that can inhibit Bcl-2 family interactions also inhibits the interaction between Bcl-x(L) and CED-4.

Biochemical similarity of expressed human prorenin and native inactive renin.

Prorenin is secreted by mammalian cells transfected with a human preprorenin expression construct. The purpose of this investigation was to compare the physicochemical properties of expressed prorenin in culture medium with the known characteristics of human inactive renin, which accounts for nearly half the renin in plasma and kidney. We found that expressed human prorenin strongly resembles human renal and plasma inactive renin. The expressed prorenin was inactive and could be equally activated by acid (dialysis to pH 3.3) or trypsin. Acid activation was completely reversible; reexposure to acid could reactivate the expressed inactive renin. Exposure to cold (-5 degrees C for 3 days) could also activate expressed renin. The Michaelis-Menten constant of acid-activated expressed renin with sheep substrate was 0.29 microM, and the pH optimum was 7.8. Expressed inactive renin bound to a cibacron-blue affinity column and could be eluted with 0.5M NaCl. All the above characteristics resemble those of human renal and plasma inactive renin. In addition, the molecular weight of expressed prorenin and human chorionic renin was 47,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and 46,000, as measured by high-performance liquid chromatography. These data, taken together with the published observation that native human inactive renin cross-reacts with antibodies generated against amino acid sequences in the prosegment of renin, provide strong support for the hypothesis that human inactive renin is prorenin.

A monoclonal antibody to alpha 4 integrin suppresses and reverses active experimental allergic encephalomyelitis.

In experimental allergic encephalomyelitis (EAE), circulating leukocytes enter the central nervous system (CNS) producing inflammation, myelin damage and paralysis. Prevention of leukocyte infiltration by an antibody against alpha 4 integrin suppressed clinical and pathological features of EAE in the guinea pig. Rapid clearance of leukocytes from the CNS and reversal of clinical findings were observed when anti-alpha 4 treatment was administered during active disease. Clinical improvement was accompanied by a marked decrease in abnormal pathological findings, including demyelination. Therefore anti-alpha 4 is an effective treatment of EAE and may be similarly useful in the treatment of autoimmune diseases such as multiple sclerosis.

Apoptosis of sinusoidal endothelial cells occurs during liver preservation injury by a caspase-dependent mechanism.

BACKGROUND: Cold ischemia/warm reperfusion (CI/WR) liver injury remains a problem in liver transplants. Sinusoidal endothelial cells (SEC) are a target of CI/WR injury, during which they undergo apoptosis. Because caspase proteases have been implicated in apoptosis, our aim was to determine whether liver CI/WR injury induces a caspase-dependent apoptosis of SEC. METHODS: Rat livers were stored in the University of Wisconsin (UW) solution for 24 hr at 4 degrees C and reperfused for 1 hr at 37 degrees C in vitro. Apoptosis was quantitated using the TUNEL assay, and caspase 3 activation determined by immunohistochemical analysis. Rat liver orthotopic liver transplants (OLT) were also performed using livers stored for 30 hr. RESULTS: Terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL) positive hepatocytes were rare and did not increase during CI/WR injury. In contrast, TUNEL positive SEC increased 6-fold after reperfusion of livers stored under cold ischemic conditions, compared with controls or livers stored but not reperfused. Immunohistochemical analysis demonstrated active caspase 3 only in endothelial cells after CI/WR injury. When IDN-1965, a caspase inhibitor, was given i.v. to the donor animal and added to UW solution and the reperfusion media, TUNEL positive endothelial cells were reduced 63+/-11% (P<0.05). Similarly, the duration of survival after OLT was significantly increased in the presence of the inhibitor. CONCLUSION: During liver CI/WR injury: 1) selective apoptosis of endothelial cells occurs; 2) caspase 3 is activated only in endothelial cells; and 3) a caspase inhibitor reduces endothelial cell apoptosis and prolongs animal survival after OLT. The pharmacologic use of caspase inhibitors could prove useful in clinical transplantation.

The effect of diamide on transmitter release and on synaptic vesicle population at vertebrate synapses.

Diamide, a sulfhydryl-oxidizing agent, has previously been shown to cause acetylcholine release in two preparations in the absence of added Ca2+. Similarities in action between diamide and alpha-latrotoxin, a component of black widow spider venom which causes transmitter release with no added Ca2+, and which seems to require a disulfide bond for its action, led us to study further the transmitter-releasing properties of diamide. In rat cerebral cortical slices we show that diamide, like alpha-latrotoxin, released all transmitters studied; GABA, acetylcholine, norepinephrine and dopamine. The response reached a peak after a delay (5-15 min), in contrast to the much faster release evoked by high K+ (within 3 min). Diamide-induced GABA release was found to occur equally well in the absence of added Ca2+, and was blocked when diamide was reduced prior to addition. Our ultrastructural studies of the frog neuromuscular junction showed that whereas alpha-latrotoxin caused the elimination of synaptic vesicles, diamide did not. Dithiothreitol, a disulfide-reducing agent, also caused GABA release, but this effect was Ca2+-dependent, blocked by high Mg2+, and occurred without delay. These observations comparing the 3 transmitter-releasing agents have further delineated the sulfhydryl/disulfide-group involvement in transmitter release and have demonstrated that dithiothreitol is operating at a different site from either alpha-latrotoxin or diamide.

Humanization of a mouse antibody against human alpha-4 integrin: a potential therapeutic for the treatment of multiple sclerosis.

alpha 4 beta 1 integrin (VLA-4) is crucial for the adhesion of leukocytes to human vascular cell adhesion molecule-1 (VCAM-1) on inflamed endothelium. This cell adhesion event is the first step in leukocyte extravasation across the blood-brain barrier in inflammatory diseases of the central nervous system (CNS) such as experimental autoimmune encephalomyelitis (EAE). Prevention of leukocyte infiltration by antibodies against the alpha 4 integrin, which block the alpha 4 beta 1 integrin/VCAM-1 interaction, have been shown to suppress clinical and pathological features of EAE. In this study, two mouse monoclonal antibodies (MAb) directed against human alpha 4 integrin were analyzed in vitro for their ability to block the interaction of leukocytes with VCAM-1 under different assay conditions. The best blocking MAb, AN100226m, was humanized by complementarily-determining region grafting, associated with human C regions and expressed. We found that modification of two structural determinants (H27 and H29) for the heavy chain CDR1 loop in one hand, and modification of framework amino acid H38, H40 and H44 in the other hand, had no effect on antigen binding. In contrast, modification of a structural determinant (H71) for the heavy chain CDR2 loop resulted in loss of binding. The humanized antibody. AN100226, was equivalent to the murine antibody. AN100226m, in binding to alpha 4 beta 1 integrin and in blocking cell adhesion. More importantly, AN100226 was as effective as AN100226m in the reversal of active EAE in guinea pigs and thus may be useful in the treatment of autoimmune diseases such as multiple sclerosis. AN100226 is currently in phase II clinical trials in the UK for the treatment of multiple sclerosis exacerbations.

Immunochemical properties and cytochemical localization of the voltage-sensitive sodium channel from the electroplax of the eel (Electrophorus electricus).

Immunochemical methods have been used to investigate questions concerning the relatedness of sodium channels from different sources and their distribution in the eel electroplax. The reagents employed were two monoclonal antibodies, 5D10 (Moore, H. -P. H., L. C. Fritz, M. A. Raftery, and J. P. Brockes (1982) Proc. Natl. Acad. Sci. U.S.A. 79: 1673-1677) and 5F3, and a rabbit antiserum; all three were directed against determinants present on the 250,000-dalton component of the eel sodium channel. In quantitative adsorption assays, the three reagents were effectively adsorbed by eel electroplax membranes but not by brain membranes from rat, frog, or chick. The rabbit antiserum bound to immobilized membranes of rat brain at a level only approximately 0.1% of that seen with electroplax membranes. The reactivity of the three reagents with the eel electroplax was further investigated by indirect immunofluorescence on frozen sections. Whereas 5D10 showed no detectable reactivity, the rabbit antiserum and, especially, 5F3 stained the electrically excitable caudal face of the electrocytes but not the inexcitable rostral face. The reactivity of 5F3 was examined in greater detail and showed occasional abrupt discontinuities where the membrane was not stained. The presence of positive 5F3 immunoreactivity appeared to be correlated with extracellular filamentous material.

Irreversible caspase inhibitors: tools for studying apoptosis.

Irreversible inhibitors of caspase proteases are often used in studies of apoptosis. However, vigorous interpretation of data generated with irreversible inhibitors requires quantitative analysis of their effects on enzyme kinetics. A simple method for the quantitative analysis of affinity irreversible inhibitors is introduced. The method allows simultaneous measurement of the dissociation constant Ki for the reversible binding to a caspase and the first-order rate constant k3 for the subsequent in situ covalent reaction that follows the noncovalent binding. The Ki value provides information regarding the affinity of an inhibitor for the enzyme, whereas the k3 value provides a measure of the in situ reactivity between the reactive functional groups of the bound inhibitor and the nearby nucleophilic side chain at the protease active site. This two-step kinetic analysis offers a more complete description of the characteristics of an irreversible inhibitor than does the commonly used second-order rate constant. The method has been applied to a library of irreversible caspase inhibitors. We demonstrate how the resulting quantitative inhibitory constants can be used to identify key caspase activities responsible for apoptosis in specific cellular models.

The ionic dependence of black widow spider venom action at the stretch receptor neuron and neuromuscular junction of crustaceans.

The effects of black widow spider venom (BWSV) on the crayfish stretch receptor and the lobster neuromuscular junction were examined. In crayfish stretch receptor neurons, BWSV caused a slight hyperpolarization followed by a large depolarization. The venom-induced depolarization of the stretch receptor was caused by an increase in membrane conductance to Na+ and Ca2+. Black widow spider venom also caused an increase in the frequency of miniature inhibitory postsynaptic potentials recorded in the stretch receptor. The ability of BWSV to increase the frequency of miniature excitatory postsynaptic potentials (MEPSPs) at the lobster neuromuscular junction was dependent on the divalent cation composition of the bathing medium. Ringer solutions containing Ca2+ supported the greatest venom-induced increase in MEPSP frequency, Mg2+ and Mn2+ supported a moderate increase in MEPSP frequency, while Co2+ and Zn2+ blocked this venom effect entirely. Black widow spider venom did not block axonal conduction in lobster walking leg axons or in the axon of the crayfish stretch receptor. The results suggest that in crustaceans, BWSV interacts specifically with membrane of the soma-dendritic region of the stretch receptor and with nerve terminal membrane, causing an increase in Na+ and Ca2+ conductance.

Avermectin B1a irreversibly blocks postsynaptic potentials at the lobster neuromuscular junction by reducing muscle membrane resistance.

Avermectin B1a, a macrocyclic lactone with broad spectrum anthelmintic activity, affects neuromuscular transmission in the lobster stretcher muscle. Perfusion of the muscle with 1-10 microgram of the drug per ml eliminates inhibitory postsynaptic potentials within a few minutes. Intracellularly recorded excitatory postsynaptic potentials are gradually reduced in amplitude over 20-30 min, and their falling phases become faster; there is no effect, however, on extracellularly recorded excitatory potentials. Avermectin B1a reduced the input resistance of the muscle fibers with a time course similar to that of the reduction of excitatory potentials. Washing for up to 2 hr with drug-free solution fails to reverse the drug's effects. However, perfusion with 20 microgram of picrotoxin per ml results in recovery of the excitatory potentials and input resistance. Avermectin B1a also blocks the firing of the crayfish stretch receptor neuron, and this block is also reversed by picrotoxin. We hypothesize that the reduction in excitatory postsynaptic potentials after avermectin B1a treatment is caused solely by reduction in membrane resistance; additional experiments suggest that the reduction in membrane resistance is due to the opening of membrane Cl- channels, perhaps including those regulated by gamma-aminobutyric acid at the inhibitory synapse.

Lobster neuromuscular junctions treated with black widow spider venom: correlation between ultrastructure and physiology.

Black widow spider venom (BWSV) causes marked physiological and morphological alterations at the lobster neuromuscular junction. BWSV is also active at vertebrate neuromuscular junctions but the component which acts on the lobster preparation is different from the one which affects vertebrates. Following exposure to BWSV, lobster neuromuscular junctions showed elevated frequencies of spontaneous miniature synaptic potentials for 15-30 min. Nerve-evoked synaptic potentials became blocked during this period. Subsequently, spontaneous miniature potentials disappeared and less frequent 'giant' spontaneous potentials appeared. Ultrastructural examination of excitatory and inhibitory nerve terminals showed that both types were affected by venom treatment. In untreated terminals, synaptic vesicles were grouped near the dense specialized membranes of the synapses. Soon after venom treatment, the synaptic vesicles were dispersed throughout the terminals and many larger and elongated vesicular structures were apparent. At the time of appearance of 'giant' spontaneous potentials, few synaptic vesicles were seen in the terminals, but large irregular vacuoles were present. Many mitochondria within the nerve terminals were swollen or disrupted, while nearby muscle mitochondria remained normal in size and appearance. Very few presynaptic dense bodies ('active zones') were seen at synapses of affected terminals. The observations are consistent with the hypothesis that BWSV allows an abnormal amount of Ca2+ to enter the nerve terminals, causing the various physiological and morphological changes.

T-lymphocyte subsets in acute illness.

OBJECTIVES: To determine the range of T-lymphocyte subsets (CD4, CD8, and CD4/CD8 ratios) in acutely ill, hospitalized patients and to determine whether these concentrations correlate with illness severity, survival rate, or immunodepression. DESIGN: Cross-sectional study, comparing Acute Physiology and Chronic Health Evaluation II (APACHE II) scores and the calculated, disease-specific, predicted mortality rate with T-lymphocyte subsets. SETTING: Urban county hospital intensive care unit (ICU), serving as the designated trauma center. PATIENTS: One hundred two consecutively admitted ICU patients (72 medical and 30 surgical). INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Patient clinical data, APACHE II scores, and their associated predicted mortality rate were recorded. Blinded human immunodeficiency virus (HIV) and lymphocyte testing was performed on samples from all patients on ICU admission. Despite only three (2.9%) of 102 patients testing positive for HIV antibodies, 41% (42/102) of patients had CD4 concentrations of < 400 cells/microL, and 29% (29/102) had CD4 concentrations of < 300 cells/microL. Mean CD8 concentrations were even lower, compared with normal laboratory values, resulting in a slight increase in CD4/CD8 ratios, although 16% (16/102) of patients had a CD4/CD8 ratio of < 1. CD4 counts were linearly related to total lymphocyte concentrations (Pearson correlation coefficient = 0.948), but no relationship was found between total lymphocyte or lymphocyte subset counts and APACHE II score, predicted mortality rate, or survival rate. CONCLUSIONS: Acute illness alone, in the absence of HIV infection, can be associated with profound decreases of T-lymphocyte populations. This problem is unpredictable and does not correlate with severity of illness, predicted mortality rate, or actual mortality rate. No conclusions regarding HIV serostatus or survival can be made based on single measurements of T-cell concentrations in acutely ill hospitalized patients.

Isolation and characterization of a monoclonal antibody against the saxitoxin-binding component from the electric organ of the eel Electrophorus electricus.

A monoclonal hybridoma cell line secreting antibody against the saxitoxin-binding component from the eel Electrophorus electricus has been isolated. The specificity of this monoclonal antibody was established by (i) its ability to immunoprecipitate bound [3H]saxitoxin from a detergent extract of electroplax membranes in a dose-dependent manner, (ii) the inability of unrelated monoclonal antibodies to immunoprecipitate the toxin-binding activity in a similar assay, and (iii) the ability of excess unlabeled tetrodotoxin to displace [3H]saxitoxin from the immunoprecipitated component. The antibody is of the subclass IgG1 and binds specifically to a polypeptide component of Mr approximately 250,000 on NaDodSO4/polyacrylamide gels. The antigenic determinant is associated with the same polypeptide component throughout the purification procedure, indicating that this component is not a result of artifactual aggregation or degradation during isolation. We conclude that the 250,000-dalton polypeptide is part of the saxitoxin binding/sodium channel protein in the native electroplax membrane.

The beta-amyloid precursor protein is not processed by the regulated secretory pathway.

The beta-amyloid peptide is derived from a larger membrane bound protein and accumulates as amyloid in Alzheimer's diseased brains. beta-amyloid precursor protein (beta APP) proteolytically processed during constitutive secretion cannot be a source of deposited amyloid because this processing results in cleavage within the amyloidogenic peptide. To see if other secretory pathways could be responsible for generating potentially amyloidogenic molecules we tested the possibility that beta APP is targeted to the regulated secretory pathway. Stable AtT20 cell lines expressing exogenous human beta APP were genetically engineered. These cells were labeled with [35S]-methionine, and chased in the presence or absence of secretagogue. The beta APP both inside the cells and released from the cells was analyzed by immunoprecipitation and gel analysis. Quantitation of autoradiograms showed that virtually all of the synthesized beta APP was secreted by the constitutive pathway, and that no detectable (less than 1%) beta APP was targeted to the regulated secretory pathway.