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Cells exposed to extreme physicochemical or mechanical stimuli die in an uncontrollable manner, as a result of their immediate structural breakdown. Such an unavoidable variant of cellular demise is generally referred to as 'accidental cell death' (ACD). In most settings, however, cell death is initiated by a genetically encoded apparatus, correlating with the fact that its course can be altered by pharmacologic or genetic interventions. 'Regulated cell death' (RCD) can occur as part of physiologic programs or can be activated once adaptive responses to perturbations of the extracellular or intracellular microenvironment fail. The biochemical phenomena that accompany RCD may be harnessed to classify it into a few subtypes, which often (but not always) exhibit stereotyped morphologic features. Nonetheless, efficiently inhibiting the processes that are commonly thought to cause RCD, such as the activation of executioner caspases in the course of apoptosis, does not exert true cytoprotective effects in the mammalian system, but simply alters the kinetics of cellular demise as it shifts its morphologic and biochemical correlates. Conversely, bona fide cytoprotection can be achieved by inhibiting the transduction of lethal signals in the early phases of the process, when adaptive responses are still operational. Thus, the mechanisms that truly execute RCD may be less understood, less inhibitable and perhaps more homogeneous than previously thought. Here, the Nomenclature Committee on Cell Death formulates a set of recommendations to help scientists and researchers to discriminate between essential and accessory aspects of cell death. [ABSTRACT FROM AUTHOR]

Human and rabbit corneas were stored at 4 degrees C in K-Sol with and without antioxidants (ascorbic acid, reduced glutathione, alpha-tocopherol, and retinol acetate) for two to three weeks. All the corneas were then examined visually and by scanning electron microscopy. They appeared clear and slightly oedematous. Scanning electron micrographs were used to grade corneal endothelial cell morphology in a masked manner in terms of cell shape, cell borders, cell swelling, and apical holes. Corneas stored in K-Sol without antioxidants showed changes in cell shape, cell borders, and apical holes. Human corneas showed more morphological changes than rabbit corneas. The results suggest that antioxidants in K-Sol have an important role in the preservation of endothelial cell morphology. [ABSTRACT FROM PUBLISHER]

Arachidonoyldiacylglycerol (20:4-DAG) is a second messenger derived from phosphatidylinositol 4,5-bisphosphate and generated by stimulation of glutamate metabotropic receptors linked to G proteins and activation of phospholipase C. 20:4-DAG signaling is terminated by its phosphorylation to phosphatidic acid, catalyzed by diacylglycerol kinase (DGK). We have cloned the murine DGKepsilon gene that showed, when expressed in COS-7 cells, selectivity for 20:4-DAG. The significance of DGKepsilon in synaptic function was investigated in mice with targeted disruption of the DGKepsilon. DGKepsilon(-/-) mice showed a higher resistance to electroconvulsive shock with shorter tonic seizures and faster recovery than DGKepsilon(+/+) mice. The phosphatidylinositol 4,5-bisphosphate-signaling pathway in cerebral cortex was greatly affected, leading to lower accumulation of 20:4-DAG and free 20:4. Also, long-term potentiation was attenuated in perforant path-dentate granular cell synapses. We propose that DGKepsilon contributes to modulate neuronal signaling pathways linked to synaptic activity, neuronal plasticity, and epileptogenesis.

Platelet-activating factor (PAF), a bioactive lipid (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) derived from phospholipase A(2) and other pathways, has been implicated in neural plasticity and memory formation. Long-term potentiation (LTP) can be induced by the application of PAF and blocked by a PAF receptor (PAF-R) inhibitor in the hippocampal CA1 and dentate gyrus. To further investigate the role of PAF in synaptic plasticity, we compared LTP in dentate granule cells from hippocampal slices of adult mice deficient in the PAF-R and their age-matched wild-type littermates. Whole cell patch-clamp recordings were made in the current-clamp mode. LTP in the perforant path was induced by a high-frequency stimulation (HFS) and defined as >20% increase above baseline of the amplitude of excitatory postsynaptic potentials (EPSPs) from 26 to 30 min after HFS. HFS-induced enhancement of the EPSP amplitude was attenuated in cells from the PAF-R-deficient mice (163 +/- 14%, mean +/- SE; n = 32) when compared with that in wild-type mice (219 +/- 17%, n = 32). The incidence of LTP induction was also lower in the cells from the deficient mice (72%, 23 of 32 cells) than in the wild-type mice (91%, 29 of 32 cells). Using paired-pulse facilitation as a synaptic pathway discrimination, it appeared that there were differences in LTP magnitudes in the lateral perforant path but not in the medial perforant path between the two groups. BN52021 (5 microM), a PAF synaptosomal receptor antagonist, reduced LTP in the lateral path in the wild-type mice. However, neither BN52021, nor BN50730 (5 microM), a microsomal PAF-R antagonist, reduced LTP in the lateral perforant path in the receptor-deficient mice. These data provide evidence that PAF-R-deficient mice are a useful model to study LTP in the dentate gyrus and support the notion that PAF actively participates in hippocampal synaptic plasticity.

To further understand the molecular mechanism of glucocorticoid action on gene expression, DNA-binding activities of the cis-acting transcription factors activator protein 1 (AP1), AP2, Egr1 (zif268), NF-kappaB, the signal transducers and activators of transcription proteins gamma interferon activation site (GAS), Sis-inducible element, and the TATA binding protein transcription factor II D (TFIID) were examined in human epidermal keratinocytes. The cytokine interleukin 1beta (IL-1beta) and platelet-activating factor (PAF), both potent mediators of inflammation, were used as triggers for gene expression. Budesonide epimer R (BUDeR) and dexamethasone (DEX) were studied as potential antagonists. BUDeR or DEX before IL-1beta- or PAF-mediated gene induction elicited strong inhibition of AP1-, GAS-, and in particular NF-kappaB-DNA binding (P < 0.001, ANOVA). Only small effects were noted on AP2, Egr1 (zif268), and Sis-inducible element-DNA binding (P > 0.05). No significant effect was noted on the basal transcription factor TFIID recognition of TATA-containing core promoter sequences (P > 0.68). To test the hypothesis that changing cis-acting transcription factor binding activity may be involved in inflammatory-response related gene transcription, RNA message abundance for human cyclooxygenase (COX)-1 and -2 (E.C.1.14.99.1) was assessed in parallel by using reverse transcription-PCR. Although the COX-1 gene was found to be expressed at constitutively low levels, the TATA-containing COX-2 gene, which contains AP1-like, GAS, and NF-kappaB DNA-binding sites in its immediate promoter, was found to be strongly induced by IL-1beta or PAF (P < 0.001). BUDeR and DEX both suppressed COX-2 RNA message generation; however, no correlation was associated with TFIID-DNA binding. These results suggest that on stimulation by mediators of inflammation, although the basal transcription machinery remains intact, modulation of cis-activating transcription factor AP1, GAS, and NF-kappaB-DNA binding by the glucocorticoids BUDeR and DEX play important regulatory roles in the extent of specific promoter activation and hence the expression of key genes involved in the inflammatory response.

Purpose: Prostaglandin (PG) D synthase is present in neural tissues and cerebrospinal fluid (beta-trace). This enzyme belongs to the lipocalin family which consists of transporter proteins for lipophilic substances in the extracellular space. PGD synthase is found in retinal pigment epithelium, from where it is secreted into the interphotoreceptor matrix. The authors have undertaken the localization of this unique enzyme within the tissues and spaces of the anterior segment of the eye., Methods: Iris, ciliary body, lens, and aqueous and vitreous humors were collected from adult rats and mice. PGD synthase activity was determined, and the protein was quantified by Western blot analysis and localized immunohistochemically. Finally, in situ hybridization was performed to localize PGD synthase mRNA., Results: PGD synthase was most abundant in the aqueous and vitreous humors. It was less abundant in tissue cytosolic fractions; these fractions had almost 10-fold as much as their corresponding membrane-bound fractions. Lens tissue had the lowest amount observed. PGD synthase was localized to the epithelial cells of the iris and the ciliary body and to the adjacent extracellular chambers, but PGD synthase mRNA was found only within the epithelial cells. Several glycosylated forms of PGD synthase were also detected., Conclusions: PGD synthase was synthesized within the epithelial cells of the iris and the ciliary body and was then secreted into the aqueous and vitreous humors, where it accumulated as an active enzyme.

Purpose: To investigate the effect of the inflammatory mediator platelet-activating factor (PAF) in the induction of the inducible prostaglandin H synthase-cyclooxygenase-2 (COX-2) gene expression in corneal epithelium., Methods: Rabbit corneas were incubated in organ culture with or without carbamyl PAF (cPAF, 100 nM). The effects of PAF antagonist BN50730 (10 microM), protein synthesis inhibitor cycloheximide (CHX; 30 micrograms/ml), RNA synthesis inhibitor actinomycin D (50 micrograms/ml), and tumor promoter phorbol ester (TPA); (100 nM) were tested. Total RNA for corneal epithelium was analyzed by Northern blot analysis using mouse COX-2 cDNA fragments labeled with 32P as probes. Western blots were performed using mouse monoclonal antibodies. Primary cultures of rabbit corneal epithelium were loaded with the fluorescent dye fluo-3 AM and changes in intracellular calcium concentration [Ca2+]i were analyzed by laser scanning confocal microscopy., Results: Platelet-activating factor induction of COX-2 expression was detectable by Northern blot analysis at 2 hours, peaked at 4 hours, and remained increased for as long as 8 hours. At 16 hours, there was a marked increase in COX-2 expression. The effect was abolished by the PAF antagonist. TPA also induced COX-2 gene expression. Neither PAF-nor TPA-induced expression was inhibited by CHX. In a Ca(2+)-free medium, there was a 50% inhibition of COX-2 gene induction by PAF. The calcium ionophore A23187 also caused an increase in expression of COX-2 messenger RNA; this did not occur in Ca(2+)-free medium. Confocal microscopy imaging showed that after the addition of PAF, there was a transient increase in [Ca2+]i in corneal epithelial cells that peaked between 30 and 60 seconds. The increase was inhibited in the presence of BN50730 or in a Ca(2+)-free medium. A23187 also caused a transient increase in [Ca2+]i that was not altered in cells previously treated with PAF or BN50730., Conclusions: PAF may enhance prostaglandin synthesis in the corneal epithelium by increasing COX-2 gene expression. This increase is by means of transcriptional activation of the gene and results in increased COX-2 protein formation. Influx of Ca2+ due to PAF stimulation is required to induce the COX-2 gene. A PAF antagonist abolishes all PAF effects and could be of therapeutic value by modulating ocular inflammation at the level of COX-2 gene expression.

Glutathione-Independent prostaglandin D synthase, identical to beta-trace, (a major CSF protein), is localized in the CNS. This enzyme, lipocalin-type prostaglandin D synthase, is a member of the lipocalin family of secretory proteins that transport small lipophilic substances. This enzyme's activity in adult rat retina was enriched sixfold in retinal pigment epithelium (RPE) and even more in interphotoreceptor matrix (IPM), all higher than brain. Western blots with anti-lipocalin-type prostaglandin D synthase showed three distinct immunoreactive bands. In the retinal cytosolic fraction, only one band was observed (M(r) 25,000); in IPM, the larger component occurred (M(r), 26,000). The RPE membrane-bound fraction showed two bands (M(r) 20,000 and 23,000), indicating synthesis, and the cytosolic fraction contained two bands (M(r) 23,000 and 26,000), indicating modification for release into IPM. At least two glycosylation sites occurred on the prostaglandin D synthase moiety, explaining the three immunoreactive bands in Western blots. Immunohistochemistry with polyclonal antibodies against this lipocalin-type enzyme showed intense localization in RPE, but less in photoreceptor outer and inner segments. In situ hybridization showed mRNA specifically expressed in RPE. Thus, lipocalin-type prostaglandin D synthase is predominantly expressed in RPE and actively accumulated in IPM. This may demonstrate gene sharing because, while catalyzing prostaglandin D2 synthesis, it may perform an additional, unrelated role in IPM. This enzyme is secreted from the RPE into IPM from which it is then taken up by photoreceptors. However, the nature of its ligand(s) is not known; they may be retinoids and/or docosahexanoic acid.

Purpose: To determine whether platelet-activating factor (PAF), a lipid mediator that is accumulated in the cornea after alkali burn, induces the gene expression of urokinase-type plasminogen activator (uPA) in the corneal epithelium. Possible signaling mechanisms of uPA gene induction by PAF also were examined., Methods: Rabbit corneas were cultured with or without PAF. One hour before stimulation, PAF antagonists or other modulators were added to PAF. In some experiments, the corneas were permeabilized to introduce guanosine triphosphate analogs into the corneal epithelial cells. Corneal epithelia were then harvested for Northern blot analysis, nuclear runoff transcription assay, and zymography., Results: Platelet-activating factor induced uPA mRNA expression in the corneal epithelium. New protein synthesis was not required for the induction of uPA mRNA. The induction was at the level of transcription as shown by nuclear runoff assays. Additionally, both actinomycin D and alpha-amanitin inhibited the increase in uPA mRNA by PAF. The message was translated into protein, which was secreted into the conditioned medium. An antagonist with high affinity for intracellular PAF binding sites (BN 50730) inhibited uPA gene expression and cellular secretion of the protein. The effect of PAF was not mediated by G proteins and was independent of protein kinase C- and cyclic adenosine monophosphate-dependent signal transduction pathways. Okadaic acid increased the expression of uPA and, at longer times, augmented the effect of PAF, suggesting that a signaling pathway that requires phosphorylation is involved in activated uPA mRNA synthesis., Conclusions: After corneal injury and inflammation, PAF may be an important initiator of the proteolytic cascade, leading to epithelial defects and corneal ulceration. Antagonists of PAF could be useful in the prevention of these diseases.