Ethanol-induced gastric damage in the rat: Lack of a correlation with oxygen-derived free radicals

The present study investigated the role of oxygen-derived free radicals as mediators of acute damage to rat gastric mucosae exposed to topically applied absolute ethanol. Although a hydroxyl radical scavenger, Dimethylthiourea, was noted to exhibit profound gastroprotective properties, other pretreatment regimens employing a host of known free radical scavengers, and enzyme inhibitors failed to confirm this hypothesis. Furthermore, no change in mucosal malondialdehyde, an indicator of free radical attack to cell membranes, could be detected in ethanol exposed tissues. Taken together, the present study fails to confirm that oxygen-derived free radicals mediate the gastric damaging effects of topically applied absolute ethanol. ^

Alphaviruses induce apoptosis in Bcl-2-overexpressing cells: evidence for a caspase-mediated, proteolytic inactivation of Bcl-2

Bcl-2 oncogene expression plays a role in the establishment of persistent viral infection by blocking virus-induced apoptosis. This might be achieved by preventing virus-induced activation of caspase-3, an IL-1beta-converting enzyme (ICE)-like cysteine protease that has been implicated in the death effector phase of apoptosis. Contrary to this model, we show that three cell types highly overexpressing functional Bcl-2 displayed caspase-3 activation and underwent apoptosis in response to infection with alphaviruses Semliki Forest and Sindbis as efficiently as vector control counterparts. In all three cell types, overexpressed 26 kDa Bcl-2 was cleaved into a 23 kDa protein. Antibody epitope mapping revealed that cleavage occurred at one or two target sites for caspases within the amino acid region YEWD31 (downward arrow) AGD34 (downward arrow) A, removing the N-terminal BH4 region known to be essential for the death-protective activity of Bcl-2. Preincubation of cells with the caspase inhibitor Z-VAD prevented Bcl-2 cleavage and partially restored the protective activity of Bcl-2 against virus-induced apoptosis. Moreover, a murine Bcl-2 mutant having Asp31, Asp34 and Asp36 substituted by Glu was resistant to proteolytic cleavage and abrogated apoptosis following virus infection. These findings indicate that alphaviruses can trigger a caspase-mediated inactivation of Bcl-2 in order to evade the death protection imposed by this survival factor.

The matrix metalloproteinase inhibitor RS-130830 attenuates brain injury in experimental pneumococcal meningitis.

BACKGROUND Pneumococcal meningitis (PM) is characterized by high mortality and morbidity including long-term neurofunctional deficits. Neuropathological correlates of these sequelae are apoptosis in the hippocampal dentate gyrus and necrosis in the cortex. Matrix metalloproteinases (MMPs) play a critical role in the pathophysiology of PM. RS-130830 (Ro-1130830, CTS-1027) is a potent partially selective inhibitor of MMPs of a second generation and has been evaluated in clinical trials as an anti-arthritis drug. It inhibits MMPs involved in acute inflammation but has low activity against MMP-1 (interstitial collagenase), MMP-7 (matrilysin) and tumour necrosis factor α converting enzyme (TACE). METHODS A well-established infant rat model of PM was used where live Streptococcus pneumoniae were injected intracisternally and antibiotic treatment with ceftriaxone was initiated 18 h post infection (hpi). Treatment with RS-130830 (75 mg/kg bis in die (bid) i.p., n = 40) was started at 3 hpi while control littermates received the vehicle (succinylated gelatine, n = 42). RESULTS Cortical necrosis was significantly attenuated in animals treated with RS-130830, while the extent of hippocampal apoptosis was not influenced. At 18 hpi, concentrations of interleukin (IL)-1β and IL-10 were significantly lower in the cerebrospinal fluid of treated animals compared to controls. RS-130830 significantly reduced weight loss and leukocyte counts in the cerebrospinal fluid of survivors of PM. CONCLUSION This study identifies MMP inhibition, specifically with RS-130830, as an efficient strategy to attenuate disease severity and cortical brain injury in PM.

Activation of a caspase 3-related cysteine protease is required for glutamate-mediated apoptosis of cultured cerebellar granule neurons

Neurotoxicity induced by overstimulation of N-methyl-d-aspartate (NMDA) receptors is due, in part, to a sustained rise in intracellular Ca2+; however, little is known about the ensuing intracellular events that ultimately result in cell death. Here we show that overstimulation of NMDA receptors by relatively low concentrations of glutamate induces apoptosis of cultured cerebellar granule neurons (CGNs) and that CGNs do not require new RNA or protein synthesis. Glutamate-induced apoptosis of CGNs is, however, associated with a concentration- and time-dependent activation of the interleukin 1β-converting enzyme (ICE)/CED-3-related protease, CPP32/Yama/apopain (now designated caspase 3). Further, the time course of caspase 3 activation after glutamate exposure of CGNs parallels the development of apoptosis. Moreover, glutamate-induced apoptosis of CGNs is almost completely blocked by the selective cell permeable tetrapeptide inhibitor of caspase 3, Ac-DEVD-CHO but not by the ICE (caspase 1) inhibitor, Ac-YVAD-CHO. Western blots of cytosolic extracts from glutamate-exposed CGNs reveal both cleavage of the caspase 3 substrate, poly(ADP-ribose) polymerase, as well as proteolytic processing of pro-caspase 3 to active subunits. Our data demonstrate that glutamate-induced apoptosis of CGNs is mediated by a posttranslational activation of the ICE/CED-3-related cysteine protease caspase 3.

A tetracycline derivative, minocycline, reduces inflammation and protects against focal cerebral ischemia with a wide therapeutic window

The only treatment of patients with acute ischemic stroke is thrombolytic therapy, which benefits only a fraction of stroke patients. Both human and experimental studies indicate that ischemic stroke involves secondary inflammation that significantly contributes to the outcome after ischemic insult. Minocycline is a semisynthetic second-generation tetracycline that exerts antiinflammatory effects that are completely separate from its antimicrobial action. Because tetracycline treatment is clinically well tolerated, we investigated whether minocycline protects against focal brain ischemia with a wide therapeutic window. Using a rat model of transient middle cerebral artery occlusion, we show that daily treatment with minocycline reduces cortical infarction volume by 76 ± 22% when the treatment is started 12 h before ischemia and by 63 ± 35% when started even 4 h after the onset of ischemia. The treatment inhibits morphological activation of microglia in the area adjacent to the infarction, inhibits induction of IL-1β-converting enzyme, and reduces cyclooxygenase-2 expression and prostaglandin E2 production. Minocycline had no effect on astrogliosis or spreading depression, a wave of ionic transients thought to contribute to enlargement of cortical infarction. Treatment with minocycline may act directly on brain cells, because cultured primary neurons were also salvaged from glutamate toxicity. Minocycline may represent a prototype of an antiinflammatory compound that provides protection against ischemic stroke and has a clinically relevant therapeutic window.

Involvement of caspase-dependent activation of cytosolic phospholipase A2 in tumor necrosis factor-induced apoptosis

Tumor necrosis factor (TNF)-induced apoptosis is mediated by caspases, which are cysteine proteases related to interleukin 1β-converting enzyme. We report here that TNF-induced activation of caspases results in the cleavage and activation of cytosolic phospholipase A2 (cPLA2) and that activated cPLA2 contributes to apoptosis. Inhibition of caspases by expression of a cowpox virus-derived inhibitor, CrmA, or by a specific tetrapeptide inhibitor of CPP32/caspase-3, acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO), inhibited TNF-induced activation of cPLA2 and apoptosis. TNF-induced activation of cPLA2 was accompanied by a cleavage of the 100-kDa cPLA2 to a 70-kDa proteolytic fragment. This cleavage was inhibited by Ac-DEVD-CHO in a similar manner as that of poly(ADP)ribose polymerase, a known substrate of CPP32/caspase-3. Interestingly, specific inhibition of cPLA2 enzyme activity by arachidonyl trifluoromethylketone (AACOCF3) partially inhibited TNF-induced apoptosis without inhibition of caspase activity. Thus, our results suggest a novel caspase-dependent activation pathway for cPLA2 during apoptosis and identify cPLA2 as a mediator of TNF-induced cell death acting downstream of caspases.

Acute systemic inflammation up-regulates secretory sphingomyelinase in vivo: A possible link between inflammatory cytokines and atherogenesis

Inflammation plays a critical role in atherogenesis, yet the mediators linking inflammation to specific atherogenic processes remain to be elucidated. One such mediator may be secretory sphingomyelinase (S-SMase), a product of the acid sphingomyelinase gene. The secretion of S-SMase by cultured endothelial cells is induced by inflammatory cytokines, and in vivo data have implicated S-SMase in subendothelial lipoprotein aggregation, macrophage foam cell formation, and possibly other atherogenic processes. Thus, the goal of this study was to seek evidence for S-SMase regulation in vivo during a physiologically relevant inflammatory response. First, wild-type mice were injected with saline or lipopolysaccharide (LPS) as a model of acute systemic inflammation. Serum S-SMase activity 3 h postinjection was increased 2- to 2.5-fold by LPS (P < 0.01). To determine the role of IL-1 in the LPS response, we used IL-1 converting enzyme knockout mice, which exhibit deficient IL-1 bioactivity. The level of serum S-SMase activity in LPS-injected IL-1 converting enzyme knockout mice was ≈35% less than that in identically treated wild-type mice (P < 0.01). In LPS-injected IL-1-receptor antagonist knockout mice, which have an enhanced response to IL-1, serum S-SMase activity was increased 1.8-fold compared with LPS-injected wild-type mice (P < 0.01). Finally, when wild-type mice were injected directly with IL-1β, tumor necrosis factor α, or both, serum S-SMase activity increased 1.6-, 2.3-, and 2.9-fold, respectively (P < 0.01). These data show regulation of S-SMase activity in vivo and they raise the possibility that local stimulation of S-SMase may contribute to the effects of inflammatory cytokines in atherosclerosis.

Selective activation of JNK1 is necessary for the anti-apoptotic activity of hILP

The balance between the inductive signals and endogenous anti-apoptotic mechanisms determines whether or not programmed cell death occurs. The widely expressed inhibitor of apoptosis gene family includes three closely related mammalian proteins: c-IAP1, c-IAP2, and hILP. The anti-apoptotic properties of these proteins have been linked to caspase inhibition. Here we show that one member of this group, hILP, inhibits interleukin-1β-converting enzyme-induced apoptosis via a mechanism dependent on the selective activation of c-Jun N-terminal kinase 1. These data demonstrate that apoptosis can be inhibited by an endogenous cellular protein by a mechanism that requires the activation of a single member of the mitogen-activating protein kinase family.

Caspase-1 is activated in neural cells and tissue with amyotrophic lateral sclerosis-associated mutations in copper-zinc superoxide dismutase

The mechanism by which mutations in the superoxide dismutase (SOD1) gene cause motor neuron degeneration in familial amyotrophic lateral sclerosis (ALS) is unknown. Recent reports that neuronal death in SOD1-familial ALS is apoptotic have not documented activation of cell death genes. We present evidence that the enzyme caspase-1 is activated in neurons expressing mutant SOD1 protein. Proteolytic processing characteristic of caspase-1 activation is seen both in spinal cords of transgenic ALS mice and neurally differentiated neuroblastoma (line N2a) cells with SOD1 mutations. This activation of caspase-1 is enhanced by oxidative challenge (xanthine/xanthine oxidase), which triggers cleavage and secretion of the interleukin 1β converting enzyme substrate, pro-interleukin 1β, and induces apoptosis. This N2a culture system should be an instructive in vitro model for further investigation of the proapoptotic properties of mutant SOD1.

Tetracyclines inhibit microglial activation and are neuroprotective in global brain ischemia

Ischemic stroke is the most common life-threatening neurological disease and has limited therapeutic options. One component of ischemic neuronal death is inflammation. Here we show that doxycycline and minocycline, which are broad-spectrum antibiotics and have antiinflammatory effects independent of their antimicrobial activity, protect hippocampal neurons against global ischemia in gerbils. Minocycline increased the survival of CA1 pyramidal neurons from 10.5% to 77% when the treatment was started 12 h before ischemia and to 71% when the treatment was started 30 min after ischemia. The survival with corresponding pre- and posttreatment with doxycycline was 57% and 47%, respectively. Minocycline prevented completely the ischemia-induced activation of microglia and the appearance of NADPH-diaphorase reactive cells, but did not affect induction of glial acidic fibrillary protein, a marker of astrogliosis. Minocycline treatment for 4 days resulted in a 70% reduction in mRNA induction of interleukin-1β-converting enzyme, a caspase that is induced in microglia after ischemia. Likewise, expression of inducible nitric oxide synthase mRNA was attenuated by 30% in minocycline-treated animals. Our results suggest that lipid-soluble tetracyclines, doxycycline and minocycline, inhibit inflammation and are neuroprotective against ischemic stroke, even when administered after the insult. Tetracycline derivatives may have a potential use also as antiischemic compounds in humans.

Inhibition of caspase 1 reduces human myocardial ischemic dysfunction via inhibition of IL-18 and IL-1β

The proinflammatory cytokine IL-18 was investigated for its role in human myocardial function. An ischemia/reperfusion (I/R) model of suprafused human atrial myocardium was used to assess myocardial contractile force. Addition of IL-18 binding protein (IL-18BP), the constitutive inhibitor of IL-18 activity, to the perifusate during and after I/R resulted in improved contractile function after I/R from 35% of control to 76% with IL-18BP. IL-18BP treatment also preserved intracellular tissue creatine kinase levels (by 420%). Steady-state mRNA levels for IL-18 were elevated after I/R, and the concentration of IL-18 in myocardial homogenates was increased (control, 5.8 pg/mg vs. I/R, 26 pg/mg; P < 0.01). Active IL-18 requires cleavage of its precursor form by the IL-1β-converting enzyme (caspase 1); inhibition of caspase 1 also attenuated the depression in contractile force after I/R (from 35% of control to 75.8% in treated atrial muscle; P < 0.01). Because caspase 1 also cleaves the precursor IL-1β, IL-1 receptor blockade was accomplished by using the IL-1 receptor antagonist. IL-1 receptor antagonist added to the perifusate also resulted in a reduction of ischemia-induced contractile dysfunction. These studies demonstrate that endogenous IL-18 and IL-1β play a significant role in I/R-induced human myocardial injury and that inhibition of caspase 1 reduces the processing of endogenous precursors of IL-18 and IL-1β and thereby prevents ischemia-induced myocardial dysfunction.

Immucillin H, a powerful transition-state analog inhibitor of purine nucleoside phosphorylase, selectively inhibits human T lymphocytes

Transition-state theory has led to the design of Immucillin-H (Imm-H), a picomolar inhibitor of purine nucleoside phosphorylase (PNP). In humans, PNP is the only route for degradation of deoxyguanosine, and genetic deficiency of this enzyme leads to profound T cell-mediated immunosuppression. This study reports the biological effects and mechanism of action of Imm-H on malignant T cell lines and on normal activated human peripheral T cells. Imm-H inhibits the growth of malignant T cell leukemia lines with the induction of apoptosis. Imm-H also inhibits activated normal human T cells after antigenic stimulation in vitro. However, Imm-H did not inhibit malignant B cells, colon cancer cell lines, or normal human nonstimulated T cells, demonstrating the selective activity of Imm-H. The effects on leukemia cells were mediated by the cellular phosphorylation of deoxyguanosine and the accumulation of dGTP, an inhibitor of ribonucleotide diphosphate reductase. Cells were protected from the toxic effects of Imm-H when deoxyguanosine was absent or when deoxycytidine was present. Guanosine incorporation into nucleic acids was selectively blocked by Imm-H with no effect on guanine, adenine, adenosine, or deoxycytidine incorporation. Imm-H may have clinical potential for treatment of human T cell leukemia and lymphoma and for other diseases characterized by abnormal activation of T lymphocytes. The design of Imm-H from an enzymatic transition-state analysis exemplifies a powerful approach for developing high-affinity enzyme inhibitors with pharmacologic activity.

RIP and FADD: two "death domain"-containing proteins can induce apoptosis by convergent, but dissociable, pathways.

With use of the yeast two-hybrid system, the proteins RIP and FADD/MORT1 have been shown to interact with the "death domain" of the Fas receptor. Both of these proteins induce apoptosis in mammalian cells. Using receptor fusion constructs, we provide evidence that the self-association of the death domain of RIP by itself is sufficient to elicit apoptosis. However, both the death domain and the adjacent alpha-helical region of RIP are required for the optimal cell killing induced by the overexpression of this gene. By contrast, FADD's ability to induce cell death does not depend on crosslinking. Furthermore, RIP and FADD appear to activate different apoptotic pathways since RIP is able to induce cell death in a cell line that is resistant to the apoptotic effects of Fas, tumor necrosis factor, and FADD. Consistent with this, a dominant negative mutant of FADD, lacking its N-terminal domain, blocks apoptosis induced by RIP but not by FADD. Since both pathways are blocked by CrmA, the interleukin 1 beta converting enzyme family protease inhibitor, these results suggest that FADD and RIP can act along separable pathways that nonetheless converge on a member of the interleukin 1 beta converting enzyme family of cysteine proteases.

Purification and cDNA cloning of a second apoptosis-related cysteine protease that cleaves and activates sterol regulatory element binding proteins.

We have purified from hamster liver a second cysteine protease that cleaves and activates sterol regulatory element binding proteins (SREBPs). cDNA cloning revealed that this enzyme is the hamster equivalent of Mch3, a human enzyme that is related to the interleukin 1beta converting enzyme. We call this enzyme Mch3/SCA-2. It is 54% identical to hamster CPP32/SCA-1, a cysteine protease that was earlier shown to cleave SREBPs at a conserved Asp between the basic helix-loop-helix leucine zipper domain and the membrane attachment domain. This cleavage liberates an NH2-terminal fragment of approximately 460 amino acids that activates transcription of genes encoding the low density lipoprotein receptor and enzymes of cholesterol synthesis. Mch3/SCA-2 and CPP32/SCA-I are synthesized as inactive 30-35 kDa precursors that are thought to be cleaved during apoptosis to generate active fragments of approximately 20 and approximately 10 kDa. The current data lend further support to the notion that SREBPs are cleaved and activated as part of the program in programmed cell death.

Suppression of apoptosis by basement membrane requires three-dimensional tissue organization and withdrawal from the cell cycle.

The basement membrane (BM) extracellular matrix induces differentiation and suppresses apoptosis in mammary epithelial cells, whereas cells lacking BM lose their differentiated phenotype and undergo apoptosis. Addition of purified BM components, which are known to induce beta-casein expression, did not prevent apoptosis, indicating that a more complex BM was necessary. A comparison of culture conditions where apoptosis would or would not occur allowed us to relate inhibition of apoptosis to a complete withdrawal from the cell cycle, which was observed only when cells acquired a three-dimensional alveolar structure in response to BM. In the absence of this morphology, both the GI cyclin kinase inhibitor p21/WAF-1 and positive proliferative signals including c-myc and cyclin DI were expressed and the retinoblastoma protein (Rb) continued to be hyperphosphorylated. When we overexpressed either c-myc in quiescent cells or p21 when cells were still cycling, apoptosis was induced. In the absence of three-dimensional alveolar structures, mammary epithelial cells secrete a number of factors including transforming growth factor alpha and tenascin, which when added exogenously to quiescent cells induced expression of c-myc and interleukin-beta1-converting enzyme (ICE) mRNA and led to apoptosis. These experiments demonstrate that a correct tissue architecture is crucial for long-range homeostasis, suppression of apoptosis, and maintenance of differentiated phenotype.