Eric P. Dixon

Global Ischemia Activates Nuclear Factor-κB in Forebrain Neurons of Rats

Background and Purpose After global ischemia, brain levels of hydrogen peroxide, oxygen radicals, and the cytokines tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) are increased. Oxygen radicals, TNF-α, and IL-1β are known to activate nuclear factor-κB (NF-κB) in vitro. The present study was performed to determine whether NF-κB was activated in vivo by global ischemia in hippocampal CA1 neurons. Methods Adult male rats were subjected to 30 minutes of four-vessel occlusion and killed 72 hours later. Levels of NF-κB p50 and p65 subunits in hippocampus were determined by immunocytochemistry, Western blot, and gel-shift analysis. Specific labeling of DNA strand breaks was demonstrated by means of an Apoptag apoptosis detection kit. Results Labeling of DNA strand breaks was present at 72 hours. Chromatin compaction and segregation, a characteristic of apoptosis, was observed in sections stained with hematoxylin and eosin. NF-κB p50 and p65 immunoreactivity localized only to nuclei of CA1 neurons at ...

Zyme, a Novel and Potentially Amyloidogenic Enzyme cDNA Isolated from Alzheimer’s Disease Brain

The deposition of the β amyloid peptide in neuritic plaques and cerebral blood vessels is a hallmark of Alzheimer’s disease (AD) pathology. The major component of the amyloid deposit is a 4.2-kDa polypeptide termed amyloid β-protein of 39–43 residues, which is derived from processing of a larger amyloid precursor protein (APP). It is hypothesized that a chymotrypsin-like enzyme is involved in the processing of APP. We have discovered a new serine protease from the AD brain by polymerase chain reaction amplification of DNA sequences representing active site homologous regions of chymotrypsin-like enzymes. A cDNA clone was identified as one out of one million that encodes Zyme, a serine protease. Messenger RNA encoding Zyme can be detected in some mammalian species but not in mice, rats, or hamster. Zyme is expressed predominantly in brain, kidney, and salivary gland. Zyme mRNA cannot be detected in fetal brain but is seen in adult brain. The Zyme gene maps to chromosome 19q13.3, a region which shows genetic linkage with late onset familial Alzheimer’s disease. When Zyme cDNA is co-expressed with the APP cDNA in 293 (human embryonic kidney) cells, amyloidogenic fragments are detected using C-terminal antibody to APP. These co-transfected cells release an abundance of truncated amyloid β-protein peptide and shows a reduction of residues 17–42 of Aβ (P3) peptide. Zyme is immunolocalized to perivascular cells in monkey cortex and the AD brain. In addition, Zyme is localized to microglial cells in our AD brain sample. The amyloidogenic potential and localization in brain may indicate a role for this protease in amyloid precursor processing and AD.

Global cerebral ischemia activates nuclear factor-κB prior to evidence of DNA fragmentation

Abstract The oxidative stress responsive transcription factor nuclear factor-κB (NF-κB) consists of a p50 (50 kDa) and p65/RelA (65 kDa) component and can be activated in vitro by TNFα, IL1β, hydrogen peroxide and oxygen radicals. All of the above factors are also known to be elevated at certain times after transient global ischemia. The present study was performed to determine if NF-κB was activated in vivo by transient global forebrain ischemia. Adult male rats were subjected to 30 min of 4-vessel occlusion (4-VO) and sacrificed at selected post-ischemic time points. Levels of NF-κB p50 and p65 subunits were determined by immunocytochemistry, Western blot and electrophoretic mobility-shift analysis. The enhancer complex was also confirmed by immuno-gel-shift analysis. Specific labeling of DNA strand breaks and DNA fragmentation was examined in situ by means of the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method. Western blot analysis of hippocampus showed induction of p50 and p65. A time course of NF-κB induction in hippocampus showed a p50-specific band at 6 h that increased in intensity over 12, 48 h and then decreased by 96 h post-ischemia. Immunocytochemistry revealed at 24 h post-ischemia that p65 and p50 immunoreactivity was present in neuronal nuclei of hippocampal CA1 neurons as well as all other hippocampal regions and several other forebrain regions which were not vulnerable to transient forebrain ischemia. At 72 h post-ischemia, nuclear NF-κB immunoreactivity had disappeared in all brain areas except in hippocampal CA1 neurons which were degenerating. No evidence for DNA fragmentation as revealed by TUNEL staining could be observed at 24 h. However, at 72 h, hippocampal CA1 neurons were heavily labeled. The results of this study demonstrate that global forebrain ischemia causes a transient activation of NF-κB in many forebrain regions. NF-κB remains persistently activated in the vulnerable hippocampal CA1 sector. Because of the persistent activation of NF-κB in these neurons, the possibility exists that NF-κB has a role in programmed cell death in hippocampal CA1 neurons.

Bcl-Xshort is elevated following severe global ischemia in rat brains

Hippocampal CA1 neurons are highly susceptible to short periods of transient global ischemia. We have previously reported in a rat model of transient forebrain global ischemia that activation and nuclear localization of NF-kB occurs in the CA1 neurons at 24 and 72 h post reperfusion. Events following NF-kB activation would ultimately determine whether damaged cells will undergo programmed cell death. We have selected bcl-x gene expression for study because there is increasing evidence that proteins encoded by the bcl-2 gene family (bcl-2, bcl-x, bax etc) play a role in the regulation of programmed cell death. We have observed that the bcl-x gene promoter contains a putative consensus sequence for NF-kB/CS4 responsive activation. We also can show that other members of the bcl-2 multigene family contain the NF-kB/CS4 sequence in their five prime regulatory regions. In this study, we show that NF-kB p50 and NF-kB p65 act in synergy to transactivate the bcl-x promoter in co-transfected 293 cells. We also report that following ischemia and NF-kB activation, bcl-x messenger RNA levels increase in the CA1 hippocampal region. As a result of this transcriptional increase, surprisingly, it is bcl-xs, the apoptotic form of bcl-x, that is elevated. These results suggest that activation of NF-kB can lead to increased expression of bcl-x as manifested by the increase in the short form of bcl-x.

Molecular cloning of cDNA for the bovine urokinase-type plasminogen activator receptor

We isolated a full length cDNA clone for bovine u-PAR from a bovine aorta endothelial cell cDNA library and compared the structural properties of this receptor protein to the published human and murine sequences. The bovine u-PAR cDNA clone spans a nucleotide sequence stretch of 1335 bp. The open reading frame contains 330 amino acids with a 20 amino acid long putative signal peptide. The mature protein contains 6 potential N-linked glycosylation sites and a high cysteine content (9%). Bovine u-PAR revealed three homologous internal structural repeats. The NH2-terminal repeat containing the u-PA binding site showed 54% identity to the human and murine NH2-terminal domain, compared to 64% identity between human and mouse u-PAR. Southern blot analysis of genomic DNAs from 9 eukaryotic species suggests that the u-PAR gene is conserved in man, monkey, and cow.

Thrombin induces thrombomodulin mRNA expression via the proteolytically activated thrombin receptor in cultured bovine smooth muscle cells

Thrombin, an important mitogen governing smooth muscle cell proliferation, binds to cultured bovine aortic smooth muscle cells (BASMCs) via both the proteolytically activated thrombin receptor (PATR) and thrombomodulin (TM). Although TM mRNA expression and functional activity is regulated by thrombin in human endothelial cells and mouse hemangioma cells, it remains unclear in those models whether the increased TM mRNA expression observed upon thrombin stimulation is mediated through the activation of PATR or via TM occupancy. We observed in cultured BASMCs that TM mRNA is increased threefold to sixfold by either thrombin, basic fibroblast growth factor (bFGF), or platelet-derived growth factor (PDGF). The increase in TM mRNA with thrombin is time dependent (maximal at 3 hours), a consequence of increased mRNA stability, and accompanied by increases in cell surface TM functional activity. Thrombin-induced TM mRNA was reproduced by the hexameric thrombin receptor-activating peptide (TRAP6) and augmented by a TM-specific antibody. Together, these data suggest that up-regulation of TM mRNA by thrombin is mediated via the PATR. We speculate that increases in BASMC TM mRNA and activity after thrombin may contribute to the impaired thrombus formation observed after atherosclerotic vascular injury.

Mitogen crosstalk accompanying urokinase receptor expression in stimulated vascular smooth muscle cells

Thrombin and other mitogens regulate the expression of the urokinase‐type plasminogen activator receptor (uPAR) protein and mRNA levels in bovine vascular smooth muscle cells (SMC). We investigated interactions between mitogens capable of increasing uPAR mRNA levels in SMC. Up‐regulation of uPAR mRNA upon thrombin and basic fibroblast growth factor (bFGF) stimulation was preceded by a 2–3‐fold transient increase in bFGF mRNA within 1 h. TGF‐β 1 did not result in a significant change in bFGF mRNA levels. Platelet‐derived growth factor (PDGF) while substantially enhancing uPAR mRNA levels, diminished bFGF mRNA levels by 3–4‐fold. Both thrombin and bFGF induced the message for bFGF‐R 2–3‐fold. Thrombin also provoked a 3–4‐fold rise in TGF‐β 1 mRNA levels within 30 min. In summary, on the mRNA level, we demonstrated both positive as well as negative feed‐back mechanisms between different mitogens, among them bFGF revealing in addition to autoinduction also up‐regulation of the transcript concentration of its own receptor. Thus, cooperation and possible amplification of mitogenic effects might be implicated in the fine‐tuned regulation of uPAR mRNA in stimulated bovine aorta SMC.