Teena Walker

Differences in DNA fragmentation following transient cerebral or decapitation ischemia in rats.

The time course of appearance of cells with DNA damage was studied in rats following transient severe forebrain ischemia. This DNA damage could be detected by in situ end-labeling on brain sections. The breaks in DNA appeared selectively by day 1 in the striatum and later in the CA1 region of the hippocampus. It was possible by double labeling to show that there was no DNA damage in astrocytes. The DNA breaks consisted of laddered DNA fragments indicative of an ordered apoptotic type of internucleosomal cleavage, which persisted without smearing for up to 7 days of reperfusion. In contrast, the DNA breaks following ischemia induced by decapitation were random and, after gel electrophoresis, consisted of smeared fragments of multiple sizes. There was some early regional cellular death, restricted to the dentate of the hippocampus, prior to the pannecrotic degeneration. It is concluded that transient forebrain ischemia leads to a type of neuronal destruction that is not random necrosis but that shares some component of the apoptotic cell death pathway.

Activation of the Rb/E2F1 Pathway by the Nonproliferative p38 MAPK during Fas (APO1/CD95)-mediated Neuronal Apoptosis

Aberrant activation of the Rb/E2F1 pathway in cycling cells, in response to mitogenic or nonmitogenic stress signals, leads to apoptosis through hyperphosphorylation of Rb. To test whether in postmitotic neurons the Rb/E2F1 pathway can be activated by the nonmitogenic stress signaling, we examined the role of the p38 stress-activated protein kinase (SAPK) in regulating Rb phosphorylation in response to Fas (CD95/APO1)-mediated apoptosis of cultured cerebellar granule neurons (CGNs). Anti-Fas antibody induced a dramatic and early activation of p38. Activated p38 was correlated with the induction of hyperphosphorylation of both endogenous and exogenous Rb. The p38-selective inhibitor, SB203580, attenuated such an increase in pRb phosphorylation and significantly protected CGNs from Fas-induced apoptosis. The cyclin-dependent kinase-mediated Rb phosphorylation played a lesser role in this neuronal death paradigm, since cyclin-dependent kinase inhibitors, such as olomoucine, roscovitine, and flavopiridol, did not significantly prevent anti-Fas antibody-evoked neuronal apoptosis. Hyperphosphorylation of Rb by p38 SAPK resulted in the release of Rb-bound E2F1. Increased E2F1 modulated neuronal apoptosis, since E2F1−/− CGNs were significantly less susceptible to Fas-mediated apoptosis in comparison with the wild-type CGNs. Taken together, these studies demonstrate that neuronal Rb/E2F1 is modulated by the nonproliferative p38 SAPK in Fas-mediated neuronal apoptosis.

The transcription factor E2F1 promotes dopamine-evoked neuronal apoptosis by a mechanism independent of transcriptional activation.

The E2F1 transcription factor plays an important role in promoting neuronal apoptosis; however, it is not clear how E2F1 does this. Here we show that E2F1 is involved in dopamine (DA)‐evoked apoptosis in cerebellar granule neurons (CGNs). E2F1 –/– CGNs and CGNs expressing an antisense E2F1 cDNA were significantly protected from DA‐toxicity relative to controls. The neuronal protection was accompanied by significantly reduced caspase 3 activity. E2F1‐mediated neuronal apoptosis did not require activation of gene transcription because: (1) ectopic expression of E2F1 or its mutants lacking the transactivation domain induced neuronal apoptosis, whereas an E2F1 mutant lacking the DNA‐binding domain did not; (2) under all of these conditions, known E2F1 target genes including cyclin A, cdc2 and p19ARF were not induced; and (3) DA‐evoked neuronal apoptosis was associated with up‐regulated E2F1, but not transcription of its target genes. Finally, E2F1‐mediated neuronal apoptosis was associated with reduced nuclear factor (NF)‐κB DNA‐binding activity. Taken together, these data suggest that E2F1 promotes DA‐evoked caspase 3‐dependent neuronal apoptosis by a mechanism independent of gene transactivation, and this may possibly occur through inhibition of anti‐apoptotic genes including NF‐κB.

Activation of DNA‐Dependent Protein Kinase May Play a Role in Apoptosis of Human Neuroblastoma Cells

Abstract : Treating SH‐SY5Y human neuroblastoma cells with 1 μM staurosporine resulted in a three‐ to fourfold higher DNA‐dependent protein kinase (DNA‐PK) activity compared with untreated cells. Time course studies revealed a biphasic effect of staurosporine on DNA‐PK activity : an initial increase that peaked by 4 h and a rapid decline that reached ~5‐10% that of untreated cells by 24 h of treatment. Staurosporine induced apoptosis in these cells as determined by the appearance of internucleosomal DNA fragmentation and punctate nuclear morphology. The maximal stimulation of DNA‐PK activity preceded significant morphological changes that occurred between 4 and 8 h (40% of total number of cells) and increased with time, reaching 70% by 48 h. Staurosporine had no effect on caspase‐1 activity but stimulated caspase‐3 activity by 10‐15‐fold in a time‐dependent manner, similar to morphological changes. Similar time‐dependent changes in DNA‐PK activity, morphology, and DNA fragmentation occurred when the cells were exposed to either 100 μM ceramide or UV radiation. In all these cases the increase in DNA‐PK activity preceded the appearance of apoptotic markers, whereas the loss in activity was coincident with cell death. A cell‐permeable inhibitor of DNA‐PK, OK‐1035, significantly reduced staurosporine‐induced punctate nuclear morphology and DNA fragmentation. Collectively, these results suggest an intriguing possibility that activation of DNA‐PK may be involved with the induction of apoptotic cell death.

Involvement of the transcription factor E2F1/Rb in kainic acid-induced death of murine cerebellar granule cells.

The full mechanisms underlying neuronal death following excitotoxic insult remain unclear, despite many in vivo and in vitro studies. Recent work has focused on various signaling molecules and pathways, normally strictly regulated, that can trigger death if perturbed. The transcription factor, E2F1 is pivotal in controlling cell death under stress situations. The current study aimed to investigate the role of this transcription factor in modulating neuronal death following kainic acid (KA) treatment of cultured mouse cerebellar granule cells (CGCs). KA-induced death of CGCs was attenuated by the selective KA/AMPA receptor antagonist CNQX, but not MK-801. Such neuronal death was caspase-3-independent and did not activate many known death genes, such as Fas receptor, caspase-8 and p38. However, hyperphosphorylation of Rb showed a transient increase which may lead to activation of E2F1. Indeed E2F1 +/+ and -/- CGCs showed a differential response to KA-mediated toxicity, in that E2F1 -/- neurons were significantly less susceptible to KA compared to E2F1 +/+ neurons, albeit both E2F1 +/+ and -/- neurons expressed similar levels of KA receptors and responded similarly to kainate antagonist, CNQX. Using selective inhibitors to CDKs, such as olomoucine, roscovitine and flavopiridol, and the inhibitor SB203580 to p38 MAPK, we ruled out the possibility that Rb inactivation through hyperphosphorylation was due to either upstream kinases. Therefore activation of Rb/E2F1 pathway appears to involve novel interactions yet to be elucidated.

Plaque assay and replication of Tipula iridescent virus in Spodoptera frugiperda ovarian cells

A plaque assay was developed for the study of Tipula iridescent virus (TIV) replication using a cell line derived from the fall army worm Spodoptera frugiperda (Sf9). Infection and plaque formation were monitored with time by phase contrast microscopy, video and fluorescent light microscopy. Structure of virions, viroplasmic centres and organelles of infected cells were examined by transmission electron microscopy (TEM). After 4 h postinfection, plaques were visibly detected within the cell monolayer by the presence of localized cell damage and production of numerous vesicular-like cytoplasmic structures. Quantitation of virions present per A260 unit of TIV preparation was determined by TEM. The number of visible plaques corresponded to virus concentration and 1 A260 produced approximately 10(5) plaques. DNA hybridization analysis revealed no gross differences in genomic DNA from TIV propagated in either Sf9 cells or wax moth Galleria mellonella larvae. These findings indicate that Sf9 is permissive for replication of TIV and superior by some parameters to other cell lines currently in use for the study of host cell/TIV interactions.

Medium-induced fragility of Schwanniomyces*

Schwanniomyces occidentalis has attracted interest because of its ability to metabolize starch and similar complex carbohydrates. Studies have been undertaken, mostly using defined media, to ascertain conditions for optimal production and secretion of hydrolytic enzymes. Here we demonstrate the fragility ofSchw. occidentalis in many defined media. We especially examined viability in YNB (Yeast Nitrogen Base) plus 1% glucose. Without phosphate supplementation, viability was routinely very low at stationary phase (usually less than 37%), whereas viability of stationary-phase cultures in phosphate-supplemented YNB usually exceeded 97%. The negative implications of having many, presumably permeabilized, dead cells present in assays for secretion of enzymes by living yeast cells are discussed.

Induction of Amylases in Schwanniomyces occidentalis

Total depolymerization of starch, made up of non-branched amylose and branched amylopectin (Fig.1), into its component glucose units is catalyzed by a consortium of at least four enzymes: a-amylase (Moranelli et al. 1987), b-amylase, glucoamylase (g), and debranching enzyme (R) (Sills et al. 1987). The versatile yeast Schwanniomyces alluvius produces all four in liberal quantities and all are extracellular; thus its ability to grow well on starch as sole carbon source (Calleja et al. 1984, 1987; Lusena et al. 1984). These different enzymic activities can be separated electrophoretically and isolated, purified, and characterized as different molecular species; however,all of them will be treated here as if they were one amylolytic activChapter 44 Protocol

Subjective Analysis of Medium-Induced Fragility of Schwanniomyces occidentalis

To ascertain the proportion of non-viable cells in various stationary-phase cultures of Schwanniomyces occidentalis by comparison of plate counts with hemocytometer counts. The experimental cultures are grown in defined media supplemented with various buffers.

Ethanol Production from Starch by Schwanniomyces occidentalis

Fermentation of starch to ethanol involves liquefaction and saccharification first, essentally a hydrolysis, before the resultant simple sugars are converted to ethanol. The saccharification may be accomplished by acid or enzymatic hydrolysis before fermentation by some organism, commonly a yeast (Fig. 1). Alternatively, a co-culture or a sequential culture of an amylolytic organism and a homofermentative yeast may accomplish the conversion. Or again, an organism that is both amylolytic and homofermentative may be used. The most commonly used, Saccharomyces cerevisiae, is not naturally amylolytic. Efforts have been made in various laboratories to make it so. Chapter 45 Protocol