Mechanisms of cell death in primary cortical neurons and PC12 cells.

Increasing evidence suggests that the regulation of neuronal cell death is complex. In this study we compared the neurotoxic effects of tumor necrosis factor-alpha (TNFalpha), nitric oxide, and thrombin on primary rat cortical cell cultures and the neuronal PC12 cell line. Release of lactate dehydrogenase (LDH) and the intracellular accumulation of nucleosomes were used as indicators of necrosis and apoptosis, respectively. There was significant LDH release in both neuronal cell types, however, the pattern of LDH release was variable and agonist-dependent. In response to the nitric oxide generator, sodium nitroprusside (SNP), cortical cells exhibited pronounced LDH release and dramatic morphologic changes, whereas in differentiated PC12 cells, TNFalpha evoked release of LDH with no associated morphologic changes. Both neuronal cell types, but not undifferentiated PC12 cells, responded to TNFalpha and thrombin with increased apoptosis. Caspase inhibition, but not antioxidant treatment, reduced nucleosome accumulation in primary cortical cells, but not in differentiated PC12 cells. In the differentiated PC12 cells, caspase inhibition reduced TNFalpha-mediated LDH release, but not nucleosome accumulation. These data suggest mechanisms involved in neuronal cell death utilize multiple pathways that vary depending on the neurotoxic insult and are also influenced by subtle differences among neuronal cell phenotypes.

Simultaneous monitoring of slow cell motility and calcium signals of the guinea pig outer hair cells.

'Slow' motility (shape changes over seconds to minutes) of the mammalian cochlear outer hair cell (OHC) could play a protection role from intense sound pressure and is associated with elevation of the cytosolic free Ca(2+) concentration ([Ca(2+)](i)). In the present work, a new approach was elaborated using fluorescent imaging for continuous monitoring of both [Ca(2+)](i) changes and slow motility of OHCs employing the Ca(2+) fluorescent indicator Fura-2. Whole OHC fluorescence and that of cell segments were analyzed to discriminate between fluorescence changes caused by [Ca(2+)](i) rise and those related to change of the cell shape. The reliability of the method was examined by simultaneous monitoring of [Ca(2+)](i) and OHC length changes induced by change of buffer osmolarity or by increase of KCl concentration. The method revealed that the time course of [Ca(2+)](i) increase and rate of cell shortening often do not coincide. It was also observed that [Ca(2+)](i) increased in 70 mM KCl more slowly than the rate of KCl delivery to OHCs. The comparison of the time courses of [Ca(2+)](i) elevation, induced by increase of K(+)/Na(+) ratio and by substitution of Na(+) with N-methyl-D-glucamine(+), indicated that the relatively slow kinetics of [Ca(2+)](i) increase in the OHC is partially attributed to regulation of Ca(2+) homeostasis by the Na(+)/Ca(2+) exchanger.

Cerebrovasculature-mediated neuronal cell death.

The presence of significant vascular disease in patients with Alzheimer's disease (AD) and the recognition of the ApoE genotype as a risk factor for both coronary disease and AD support an association between AD and vascular disease. It is our hypothesis that brain microvessels contribute to the pathogenesis of AD by producing soluble factors that injure or kill neurons. In this study we report that AD microvessels produce factors that are noxious to neurons and that these vessels can evoke neuronal cell death in vitro. In these experiments, microvessels are isolated from the cerebral cortices of AD patients and non-demented elderly and young controls. Microvessels isolated from AD brains produce high levels of a known neurotoxin nitric oxide, compared to vessels from aged-matched controls. In addition, we demonstrate a direct neurotoxic effect of AD microvessels when co-cultured with primary rat cerebral cortical neurons. In contrast, vessels from elderly non-demented donors are less lethal, and brain vessels from younger donors are not neurotoxic. Similarly, AD vessels exhibit a dose-dependent toxicity in co-culture with the human neurons. Finally, treatment of AD microvessels with the protein synthesis inhibitor cycloheximide reduces AD vessel neurotoxicity, suggesting that the neurotoxic factor is a protein. These findings suggest that the cerebral microvasculature is a source of factors that can injure neurons and implicate a novel mechanism of vascular-mediated neuronal cell death in AD.

Mechanisms of resistance. Expression of coat protein.

Expression of viral CP genes in transgenic plants can lead to virus resistance by interference of either the transcript or the protein with virus infection. Dependence of resistance on CP accumulation can be most convincingly shown by comparison of plants that accumulate CP with plants that accumulate a nontranslatable CP transcript. Even in cases in which CP accumulation is required, the degree of resistance does not always correlate with CP levels in transgenic plants. In cases in which CPMR can be overcome by inoculation with viral RNA instead of virions, interference with virion disassembly is the likely cause of resistance. Classical crossprotection can also sometimes be overcome by RNA inoculation, and, in this case, appears to work by a similar mechanism. There is no evidence yet that CPMR is caused by a nonspecific plant defense response that might be triggered by the accumulating CP. Measurement of virus accumulation in protoplasts prepared from transgenic plants was used to show interference with early events of virus infection. There is no clear evidence yet for inhibition of local virus spread in transgenic plants. A reduced rate of virus accumulation in inoculated leaves can usually also be explained with reduced rate of replication. However, in the case of CPMR to CMV, it appears that early events, as well as systemic spread, are affected. Reduced vector transmission of virus infection from inoculated transgenic plants to nontransgenic plants has been observed. It is not known whether this is just a consequence of lower virus levels in the transgenic plants or whether direct interference with acquisition and transmission by the vector is also involved. In addition to virion formation, CP can function in different ways in plant virus infections. Replication, long-distance spread, and vector transmission can also depend on the presence of CP. Expression of genes encoding nonfunctional CPs in transgenic plants can be tried in order to interfere with normal CP function. Knowledge of CP function(s) in a particular plant-virus interaction will be useful to design gene constructs. Since CP accumulation levels in transgenic plants do not always correlate with resistance, newly generated transgenic plant lines are now frequently tested for virus resistance before further characterization. However, if the objective of a transformation experiment is also to study the mechanism of CPMR, it is necessary to determine transcript and protein levels in the transgenic plants. Gene constructs encoding nontranslatable and antisense CP transcripts should be included in the experiment. If possible, transgenic plants should be inoculated with virions and viral nucleic acid, and replication in isolated protoplasts should be determined.

Coat protein-mediated resistance in transgenic tobacco expressing the tobacco mosaic virus coat protein from tissue-specific promoters.

Coat protein-mediated resistance (CP-MR) was studied in transgenic Nicotiana tabacum 'Xanthi nn' and 'Xanthi NN' that express chimeric tobacco mosaic virus (TMV) coat protein (CP) gene constructs using two different tissue-specific promoters. The Phaseolus vulgaris pal2 promoter leads to gene expression in the upper leaf epidermis and the xylem, while the rolC promoter from Agrobacterium rhizogenes leads to gene expression in pholem and leaf hair tip cells. Tissue-specific gene expression was verified using the gusA(uidA) reporter gene, while accumulation of TMV CP was verified by Western blot analysis. Transgenic Xanthi nn plants harboring the pal2-CP gene construct were partially resistant to TMV infection. On Xanthi NN plants that expressed the pal2-CP gene construct, fewer necrotic lesions were formed after TMV inoculation compared to nontransformed control plants. The level of resistance, however, was substantially less than in plant lines that expressed TMV CP from the cauliflower mosaic virus 35S promoter. By contrast, expression of the rolC-CP construct did not confer resistance in either Xanthi nn or Xanthi NN. The results provide further evidence that CP-MR to systemic TMV infection in tobacco is probably due to inhibition of infection rather than to effects on long-distance spread through the phloem.

Plant Resistance to Virus Diseases through Genetic Engineering: Can a Similar Approach Control Plant-parasitic Nematodes?

Genetically engineered resistance against plant virus diseases has been achieved by transforming plants with gene constructs that encode viral sequences. Several successful field trials of virus-resistant transgenic plants have been carried out. Specific features of virus infection make it possible to interfere with different steps of the infection and disease cycle by accumulating products of chimeric genes introduced into transgenic plants. In this paper we describe the most common methods of producing virus-resistant transgenic plants and discuss the possibility of applying the concept of pathogen-derived resistance to non-viral pathogens.

The effect of light on the biosynthesis of leaf-specific thionins in barley, Hordeum vulgare.

In barley seedlings grown in the dark large amounts of thionin-specific mRNAs are present, the concentration of which rapidly declines once the seedling is exposed to light. This rapid light effect is mediated by a complex interaction of possibly two photoreceptors, phytochrome and a blue-light-absorbing photoreceptor. Parallel to the decline in mRNA content, the de novo synthesis of leaf-specific thionins ceases rapidly upon illumination of etiolated seedlings. However, thionins which have accumulated before the onset of illumination remain stable within the seedling at high concentrations. In younger leaves of mature, nonstressed barley plants grown under a 16-h-light/8-h-dark cycle thionins are still present, although at much lower concentrations. In these plants, synthesis and accumulation of thionins occur predominantly in the meristematic zone at the leaf basis, which is shielded from light through the sheath of the preceding leaf. In mature light-adapted barley plants, mRNA encoding leaf-specific thionins may reaccumulate if these plants are exposed to pathogens or other stresses. Thus, the inhibitory effect of light on the biosynthesis of thionins may be overruled by stress- and pathogen-induced signals.

Intracellular thionins of barley. A second group of leaf thionins closely related to but distinct from cell wall-bound thionins.

Leaf thionins of barley have been identified as a novel class of cell wall proteins, toxic to plant pathogenic fungi, and possibly involved in the defense mechanism of plants (Bohlmann, H., Clausen, S., Behnke, S., Giese, H., Hiller, C., Reimann-Philipp, U., Schrader, G., Barkholt, V., and Apel, K., (1988) EMBO J. 7, 1559-1565). In the present work a second subfraction of thionins has been detected within the leaf cell, mainly in the vacuole. Thionins of both groups are closely related to each other. They are toxic to phytopathogenic fungi as well as to plant protoplasts, they share similar amino acid sequences, and their synthesis in etiolated seedlings of barley is down-regulated by light. Despite these similarities each of the two subfractions of thionins could be clearly distinguished by its subcellular distribution. In ultrathin sections of embedded etiolated leaf material, cell wall thionins could be immunogold labeled specifically by an antiserum raised against a fusion protein of Escherichia coli beta-galactosidase and the 15,000 Mr precursor polypeptide of thionins. This antiserum did not react with intracellular thionins. Inversely, intracellular thionins were recognized specifically by an anti-serum raised against soluble leaf thionins. The possible function of intracellular thionins as part of a defense mechanism has been discussed.

Leaf-specific thionins of barley-a novel class of cell wall proteins toxic to plant-pathogenic fungi and possibly involved in the defence mechanism of plants.

A novel class of highly abundant polypeptides with antifungal activity has been detected in cell walls of barley leaves. Similar polypeptides known as thionins occur not only in monocotyledonous but also in various dictoyledonous plants. The leaf-specific thionins of barley are encoded by a complex multigene family, which consists of at least 50-100 members per haploid genome. All of these genes are confined to chromosome 6. The toxicity of these thionins for plant pathogenic fungi and the fact that their synthesis can also be triggered by pathogens strongly suggest that thionins are a naturally occurring, inducible plant protein possibly involved in the mechanism of plant defence against microbial infections.