Cellular Poly Research Articles

Inverse electron demand Diels–Alder (iEDDA) functionalisation of macroporous poly(dicyclopentadiene) foams

Inverse electron demand Diels-Alder reactions performed on the double bonds in open cellular macroporous poly(dicyclopentadiene) monoliths yield a high degree of functionalisation (up to 2 mmol pyridazines per g or 8 mmol N per g) with grafted di(pyridyl)pyridazines in a single step.

Induction of Stress Granule-Like Structures in Vesicular Stomatitis Virus-Infected Cells

Previous studies from our laboratory revealed that cellular poly(C) binding protein 2 (PCBP2) downregulates vesicular stomatitis virus (VSV) gene expression. We show here that VSV infection induces the formation of granular structures in the cytoplasm containing cellular RNA-binding proteins, including PCBP2, T-cell-restricted intracellular antigen 1 (TIA1), and TIA1-related protein (TIAR). Depletion of TIA1 via small interfering RNAs (siRNAs), but not depletion of TIAR, results in enhanced VSV growth and gene expression. The VSV-induced granules appear to be similar to the stress granules (SGs) generated in cells triggered by heat shock or oxidative stress but do not contain some of the bona fide SG markers, such as eukaryotic initiation factor 3 (eIF3) or eIF4A, or the processing body (PB) markers, such as mRNA-decapping enzyme 1A (DCP1a), and thus may not represent canonical SGs or PBs. Our results revealed that the VSV-induced granules, called SG-like structures here, contain the viral replicative proteins and RNAs. The formation and maintenance of the SG-like structures required viral replication and ongoing protein synthesis, but an intact cytoskeletal network was not necessary. These results suggest that cells respond to VSV infection by aggregating the antiviral proteins, such as PCBP2 and TIA1, to form SG-like structures. The functional significance of these SG-like structures in VSV-infected cells is currently under investigation.

Human RECQL5 participates in the removal of endogenous DNA damage

Human RECQL5 is a member of the RecQ helicase family, which maintains genome stability via participation in many DNA metabolic processes, including DNA repair. Human cells lacking RECQL5 display chromosomal instability. We find that cells depleted of RECQL5 are sensitive to oxidative stress, accumulate endogenous DNA damage, and increase the cellular poly(ADP-ribosyl)ate response. In contrast to the RECQ helicase family members WRN, BLM, and RECQL4, RECQL5 accumulates at laser-induced single-strand breaks in normal human cells. RECQL5 depletion affects the levels of PARP-1 and XRCC1, and our collective results suggest that RECQL5 modulates and/or directly participates in base excision repair of endogenous DNA damage, thereby promoting chromosome stability in normal human cells.

Interaction of cellular poly(C)-binding protein 2 with nonstructural protein 1β is beneficial to Chinese highly pathogenic porcine reproductive and respiratory syndrome virus replication

Non-structural protein1β (Nsp1β) of porcine reproductive and respiratory syndrome virus (PRRSV) has been recognized to be involved in suppressing the host innate immune response and mediating viral subgenomic mRNA transcription. In the present study, we have analyzed the interaction of Nsp1β of Chinese highly pathogenic PRRSV (HP-PRRSV) with cellular poly(C)-binding 2 (PCBP2) by means of the yeast two-hybrid screening in a pulmonary alveolar macrophages (PAMs) cDNA library and co-immunoprecipitation (Co-IP) assay. Our results indicated that the Nsp1β of the HP-PRRSV is able to bind and interact with cellular PCBP2 strongly in both the infected cells and plasmid transfected cells. Their minimal binding regions were identified to be the residues 85–203 aa (PCPβ and CTE domains) for the Nsp1β and the residues 96–168 aa (KH2 domain) for PCBP2, respectively. Next, we used confocal immunofluorescence analysis and discovered that, during PRRSV infection in MARC-145 cells and/or pasmid-transfected cells, the Nsp1β and PCBP2 mainly colocalized in the cytoplasm and perinuclear pattern. Moreover, the siRNA-mediated silencing of PCBP2 gene in the MARC-145 cells resulted in significant reduction of the virus titer in supernatants as well as viral proteins, while no significant effects on the expression of the type I interferon α and interferon β, suggesting that the interaction of the Nsp1β with cellular PCBP2 is beneficial to Chinese HP-PRRSV replication in MARC-145 cells.

Poly(A) binding protein abundance regulates eukaryotic translation initiation factor 4F assembly in human cytomegalovirus-infected cells

By commandeering cellular translation initiation factors, or destroying those dispensable for viral mRNA translation, viruses often suppress host protein synthesis. In contrast, cellular protein synthesis proceeds in human cytomegalovirus (HCMV)-infected cells, forcing viral and cellular mRNAs to compete for limiting translation initiation factors. Curiously, inactivating the host translational repressor 4E-BP1 in HCMV-infected cells stimulates synthesis of the cellular poly(A) binding protein (PABP), significantly increasing PABP abundance. Here, we establish that new PABP synthesis is translationally controlled by the HCMV-encoded UL38 mammalian target of rapamycin complex 1-activator. The 5' UTR within the mRNA encoding PABP contains a terminal oligopyrimidine (TOP) element found in mRNAs, the translation of which is stimulated in response to mitogenic, growth, and nutritional stimuli, and proteins encoded by TOP-containing mRNAs accumulated in HCMV-infected cells. Furthermore, UL38 expression was necessary and sufficient to regulate expression of a PABP TOP-containing reporter. Remarkably, preventing the rise in PABP abundance by RNAi impaired eIF4E binding to eIF4G, thereby reducing assembly of the multisubunit initiation factor eIF4F, viral protein production, and replication. This finding demonstrates that viruses can increase host translation initiation factor concentration to foster their replication and defines a unique mechanism whereby control of PABP abundance regulates eIF4F assembly.

The 3′ Untranslated Region of the Rabies Virus Glycoprotein mRNA Specifically Interacts with Cellular PCBP2 Protein and Promotes Transcript Stability

Viral polymerase entry and pausing at intergenic junctions is predicted to lead to a defined polarity in the levels of rhabdovirus gene expression. Interestingly, we observed that the rabies virus glycoprotein mRNA is differentially over-expressed based on this model relative to other transcripts during infection of 293T cells. During infection, the rabies virus glycoprotein mRNA also selectively interacts with the cellular poly(rC)-binding protein 2 (PCBP2), a factor known to influence mRNA stability. Reporter assays performed both in electroporated cells and in a cell-free RNA decay system indicate that the conserved portion of the 3′ UTR of the rabies virus glycoprotein mRNA contains an RNA stability element. PCBP2 specifically interacts with reporter transcripts containing this 72 base 3′ UTR sequence. Furthermore, the PCBP2 interaction is directly associated with the stability of reporter transcripts. Therefore, we conclude that PCBP2 specifically and selectively interacts with the rabies virus glycoprotein mRNA and that this interaction may contribute to the post-transcriptional regulation of glycoprotein expression.

Expression of antioxidant molecules after peripheral nerve injury and regeneration

Oxidative stress is considered to be one of the main causes of neural damage after injury. However, little is known about the changes in mRNA expression of antioxidant molecules that occur after injury and regeneration of the peripheral nerve. In the present study, the rat median nerve was transected, and transcriptional changes were studied at day 6 and day 12 after injury in both the proximal and the distal stumps, in the absence or presence of microsurgical repair. The expression profiles of the following genes were investigated: three metallothionein isoforms (MT-1, MT-2, and MT-3), the main antioxidant enzymes (catalase, superoxide dismutase, and glutathione-S-transferase), and the marker of cellular damage poly(ADP-ribose) polymerase-1 (PARP-1). The results showed that, in the proximal nerve stump, MT-3 mRNA expression was significantly and markedly up-regulated in the absence of surgical repair, whereas MT-1 and MT-2 showed significant down-regulation. In the distal nerve portion, mRNA expression of all MT isoforms decreased significantly in the absence of microsurgical reconstruction, whereas, after repair, MT-3 mRNA expression alone was up-regulated. Expression of all the antioxidant enzymes decreased significantly after repair in the proximal nerve portion, but a significant general increase in their mRNA expression was revealed in the distal nerve stump. PARP-1 expression was significantly up-regulated in the proximal nerve portion without repair but dramatically reduced after reconstruction. In contrast, PARP-1 expression increased markedly in the distal stump after surgical repair. Taken together, these findings indicate that antioxidant molecules are differentially modulated and might, therefore, play an important role in peripheral nerve injury and regeneration.

Cellular Poly(C) Binding Proteins 1 and 2 Interact with Porcine Reproductive and Respiratory Syndrome Virus Nonstructural Protein 1β and Support Viral Replication

Porcine reproductive and respiratory syndrome virus (PRRSV) infection of swine results in substantial economic losses to the swine industry worldwide. Identification of cellular factors involved in PRRSV life cycle not only will enable a better understanding of virus biology but also has the potential for the development of antiviral therapeutics. The PRRSV nonstructural protein 1 (nsp1) has been shown to be involved in at least two important functions in the infected hosts: (i) mediation of viral subgenomic (sg) mRNA transcription and (ii) suppression of the host's innate immune response mechanisms. To further our understanding of the role of the viral nsp1 in these processes, using nsp1β, a proteolytically processed functional product of nsp1 as bait, we have identified the cellular poly(C)-binding proteins 1 and 2 (PCBP1 and PCBP2) as two of its interaction partners. The interactions of PCBP1 and PCBP2 with nsp1β were confirmed both by coimmunoprecipitation in infected cells and/or in plasmid-transfected cells and also by in vitro binding assays. During PRRSV infection of MARC-145 cells, the cytoplasmic PCBP1 and PCBP2 partially colocalize to the viral replication-transcription complexes. Furthermore, recombinant purified PCBP1 and PCBP2 were found to bind the viral 5' untranslated region (5'UTR). Small interfering RNA (siRNA)-mediated silencing of PCBP1 and PCBP2 in cells resulted in significantly reduced PRRSV genome replication and transcription without adverse effect on initial polyprotein synthesis. Overall, the results presented here point toward an important role for PCBP1 and PCBP2 in regulating PRRSV RNA synthesis.

Interdependent genotoxic mechanisms of monomethylarsonous acid: Role of ROS-induced DNA damage and poly(ADP-ribose) polymerase-1 inhibition in the malignant transformation of urothelial cells

Exposure of human bladder urothelial cells (UROtsa) to 50 nM of the arsenic metabolite, monomethylarsonous acid (MMA III), for 12 weeks results in irreversible malignant transformation. The ability of continuous, low-level MMA III exposure to cause an increase in genotoxic potential by inhibiting repair processes necessary to maintain genomic stability is unknown. Following genomic insult within cellular systems poly(ADP-ribose) polymerase-1 (PARP-1), a zinc finger protein, is rapidly activated and recruited to sites of DNA strand breaks. When UROtsa cells are continuously exposed to 50 nM MMA III, PARP-1 activity does not increase despite the increase in MMA III-induced DNA single-strand breaks through 12 weeks of exposure. When UROtsa cells are removed from continuous MMA III exposure (2 weeks), PARP-1 activity increases coinciding with a subsequent decrease in DNA damage levels. Paradoxically, PARP-1 mRNA expression and protein levels are elevated in the presence of continuous MMA III indicating a possible mechanism to compensate for the inhibition of PARP-1 activity in the presence of MMA III. The zinc finger domains of PARP-1 contain vicinal sulfhydryl groups which may act as a potential site for MMA III to bind, displace zinc ion, and render PARP-1 inactive. Mass spectrometry analysis demonstrates the ability of MMA III to bind a synthetic peptide representing the zinc-finger domain of PARP-1, and displace zinc from the peptide in a dose-dependent manner. In the presence of continuous MMA III exposure, continuous 4-week zinc supplementation restored PARP-1 activity levels and reduced the genotoxicity associated with MMA III. Zinc supplementation did not produce an overall increase in PARP-1 protein levels, decrease the levels of MMA III-induced reactive oxygen species, or alter Cu–Zn superoxide dismutase levels. Overall, these results present two potential interdependent mechanisms in which MMA III may increase the susceptibility of UROtsa cells to genotoxic insult and/or malignant transformation: elevated levels of MMA III-induced DNA damage through the production of reactive oxygen species, and the direct MMA III-induced inhibition of PARP-1.

Zinc-finger antiviral protein inhibits HIV-1 infection by selectively targeting multiply spliced viral mRNAs for degradation

The zinc-finger antiviral protein (ZAP) was originally identified as a host factor that inhibits the replication of Moloney murine leukemia virus. Here we report that ZAP inhibits HIV-1 infection by promoting the degradation of specific viral mRNAs. Overexpression of ZAP rendered cells resistant to HIV-1 infection in a ZAP expression level-dependent manner, whereas depletion of endogenous ZAP enhanced HIV-1 infection. Both human and rat ZAP inhibited the propagation of replication-competent HIV-1. ZAP specifically targeted the multiply spliced but not unspliced or singly spliced HIV-1 mRNAs for degradation. We provide evidence indicating that ZAP selectively recruits cellular poly(A)-specific ribonuclease (PARN) to shorten the poly(A) tail of target viral mRNA and recruits the RNA exosome to degrade the RNA body from the 3' end. In addition, ZAP recruits cellular decapping complex through its cofactor RNA helicase p72 to initiate degradation of the target viral mRNA from the 5' end. Depletion of each of these mRNA degradation enzymes reduced ZAP's activity. Our results indicate that ZAP inhibits HIV-1 by recruiting both the 5' and 3' mRNA degradation machinery to specifically promote the degradation of multiply spliced HIV-1 mRNAs.

In the absence of cellular poly (A) binding protein, the glycolytic enzyme GAPDH translocated to the cell nucleus and activated the GAPDH mediated apoptotic pathway by enhancing acetylation and serine 46 phosphorylation of p53

The cytoplasmic poly (A) binding protein (PABP) interacts with 3′ poly (A) tract of eukaryotic mRNA and is important for both translation and stability of mRNA. Previously, we have shown that depletion of PABP by siRNA prevents protein synthesis and consequently leads to cell death through apoptosis. In the present investigation, we studied the mechanism of cell apoptosis. We show that in the absence of PABP, the glycolytic enzyme GAPDH translocated to the cell nucleus and activated the GAPDH mediated apoptotic pathway by enhancing acetylation and serine 46 phosphorylation of p53. As a result, p53 translocated to the mitochondria to initiate Bax mediated apoptosis.

Depletion of cellular poly (A) binding protein prevents protein synthesis and leads to apoptosis in HeLa cells

The cytoplasmic poly (A) binding protein (PABP) is important in mRNA translation and stability. In yeast, depletion of PABP leads to translation arrest. Similarly, the PABP gene in Drosophila is important for proper development. It is however uncertain, whether mammalian PABP is essential for mRNA translation. Here we showed the effect of PABP depletion on mRNA metabolism in HeLa cells by using a small interfering RNA. Our results suggest that depletion of PABP prevents protein synthesis and consequently leads to cell death through apoptosis. Interestingly, no detectable effect of PABP depletion on transcription, transport and stability of mRNA was observed.

Cellular cap-binding proteins associate with influenza virus mRNAs

The influenza virus RNA polymerase synthesizes three types of RNA: genomic vRNA, anti-genomic cRNA and mRNA. Both vRNA and cRNA are bound by the viral RNA polymerase and nucleoprotein to form ribonucleoprotein complexes. Viral mRNAs are also proposed to be bound by the RNA polymerase to prevent their endonucleolytic cleavage, regulate the splicing of M1 mRNA, and facilitate translation. Here, we used standard immunoprecipitation, biochemical purification and RNA immunoprecipitation assays to investigate the association of viral and host factors with viral mRNA. We found that viral mRNA associates with the viral non-structural protein 1 (NS1), cellular poly(A)-binding protein 1 (PABP1), the 20 kDa subunit NCBP1 of the nuclear cap-binding complex (CBC), the RNA and export factor-binding protein REF/Aly and the translation initiation factor eIF4E. However, our data suggest that the RNA polymerase might not form part of the viral messenger ribonucleoprotein (mRNP) complex. We propose a model in which viral mRNAs, by associating with cellular cap-binding proteins, follow the pathways normally used by cellular mRNAs for splicing, nuclear export and translation.

Minocycline Protects Cardiac Myocytes Against Simulated Ischemia–Reperfusion Injury by Inhibiting Poly(ADP-ribose) Polymerase-1

There is an increase in reactive oxygen and nitrogen species in cardiomyocytes during myocardial ischemia/reperfusion injury. This leads to oxidative DNA damage and activation of nuclear repair enzymes such as poly(ADP-ribose) polymerase-1 (PARP-1). PARP-1 activation promotes DNA repair under normal conditions. However, excessive activation of PARP-1 leads to cell death. We report that PARP-1 enzymatic activity is directly inhibited by minocycline, and we propose that one mechanism of minocycline cardioprotection is the result of PARP-1 inhibition. Using cultured adult rat cardiac myocytes, we evaluated the mechanism of minocycline protection in which PARP-1 activation was induced by simulated ischemia/reperfusion injury using oxygen–glucose deprivation.We found an increase in reactive oxygen species production, PARP-1 activation, and PARP-1-mediated cell death after simulated ischemia/reperfusion. Cell death was significantly reduced by the PARP inhibitors 3, 4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone (10 μM) and PJ-34 (500 nM) or by minocycline (500 nM). Cellular NAD(+) depletion and poly(ADP-ribose) formation, which are biochemical markers of PARP-1 activation, were also blocked by minocycline. Finally, simulated ischemia/reperfusion led to induction of the mitochondrial permeability transition, which was prevented by minocycline. Therefore, we propose that the protective effect of minocycline on cardiac myocyte survival is the result of inhibition of PARP-1 activity.

Transcribed DNA is preferentially located in the perichromatin region of mammalian cell nuclei

The precise localization of transcribed DNA and resulting RNA is an important aspect of the functional architecture of the nucleus. To this end we have developed a novel in situ hybridization approach in combination with immunoelectron microscopy, using sense and anti-sense RNA probes that are derived from total cellular or cytoplasmic poly(A+) RNA. This new technology is much more gentle than classical in situ hybridization using DNA probes and shows excellent preservation of nuclear structure. Carried out on ultrathin sections of fixed and resin-embedded COS-7 cells, it revealed at high resolution the localization of the genes that code for the cellular mRNAs. Quantitative analysis shows that most transcribed DNA is concentrated in the perichromatin region, i.e. the interface between subchromosomal compact chromatin domains and the interchromatin space essentially devoid of DNA. The RNA that is produced is found mainly in the perichromatin region and the interchromatin space. These results imply that in the mammalian nucleus the chromatin fiber is folded so that active genes are predominantly present in the perichromatin region, which is the most prominent site of transcription.

Ex vivo supplementation with nicotinic acid enhances cellular poly(ADP-ribosyl)ation and improves cell viability in human peripheral blood mononuclear cells

Poly(ADP-ribosyl)ation is a posttranslational modification of proteins, which is mainly catalyzed by poly(ADP-ribose) polymerase-1 (PARP-1) by using NAD+ as substrate and is directly triggered by DNA strand breaks. Under mild genotoxic stress poly(ADP-ribose) (PAR) formation plays an important role in DNA repair whereas severe genotoxic stress and the ensuing overactivation of PARP-1 induce cellular NAD+ depletion, energy failure and ultimately cell death. We are interested in studying the consequences of moderately enhanced enzymatic activity under conditions of DNA damage. Here we chose supplementation of cells with the NAD+ precursor nicotinic acid (NA) as a strategy. In order to reliably assess PAR accumulation in living cells we first developed a novel, sensitive flow-cytometric method for the rapid analysis of poly(ADP-ribose) accumulation (RAPARA). Our data showed that ex vivo supplementation of human peripheral blood mononuclear cells (PBMC) with low concentrations of NA significantly raised cellular NAD+ levels by 2.1-fold. Upon X-irradiation or exposure to hydrogen peroxide or N-methyl-N′-nitro-N-nitrosoguanidine, PAR accumulation was significantly increased and sustained in NA-supplemented cells. Furthermore, NA-supplemented PBMC displayed significantly higher cell viability due to a lower rate of necrotic cell death. In summary, ex vivo supplementation of human PBMC with NA increases cellular NAD+ levels, boosts the cellular poly(ADP-ribosyl)ation response to genotoxic treatment, and protects from DNA-damage-induced cell death.

Enzyme characteristics of recombinant poly(ADP-ribose) polymerases-1 of rat and human origin mirror the correlation between cellular poly(ADP-ribosyl)ation capacity and species-specific life span

Poly(ADP-ribosyl)ation is a posttranslational modification, which is involved in many cellular functions, including DNA repair and maintenance of genomic stability, and has also been implicated in cellular and organismal ageing. We have previously reported that maximum poly(ADP-ribosyl)ation capacity in mononuclear blood cells is correlated with mammalian life span. Here we show that the difference between a long-lived and a short-lived species tested (i.e. man and rat) is directly mirrored by the enzymatic parameters of recombinant poly(ADP-ribose) polymerase-1 (PARP-1), i.e. substrate affinity and reaction velocity. In addition, we have characterized two human PARP-1 alleles and assign their activity difference to their respective initial velocity and not substrate affinity.

Abstract 1032: Impact of HOXC9 recapitulation on cancer biology and clinical outcome in gastric cancer

Abstract The HOX genes encode transcription factors and may be implicated in the growth and the progression of many types of tumors. There are growing lines of evidence that dysregulation of HOX gene expression plays important roles in cancer. The aim of this study is to determine the level of HOXC9 expression during gastric cancer progression and the impact of its alteration on cancer biology and clinical outcome. HOXC9 expression was evaluated in normal mucosa, malignant legions, gastric cancer tissues by RT-PCR, western blot and immunohistochemistry. We established AGS gastric cancer cell in which HOXC9 was knock-down by shRNA and conditionally knock-down the HOXC9 expression by tetracycline-regulated gene expression (Tet-Off) system. The effect of HOXC9 knock-down on cell proliferation and apoptosis in AGS gastric cancer line were analyzed by tetrazolium salt-based proliferation assay (WST assay) and FACScan. Knockdown of HOXC9 by specific shRNA (67.3%) increased apoptosis, compared to scambled (7.3%) after 48 hours treatment of doxycycline, and increased active form of cellular apoptosis-related enzyme capsae-9, caspase-3 and poly (ADP-ribose) polymerase, whereas decreased expression of Bcl-xL, a mitochondria dependent anti-apoptosis factor. There was a gain or substantial increase of HOXC9 protein expression according to gastric cancer progression. Kaplan-Meier survival curves showed that HOXC9 overexpression is correlated with inferior disease-specific survival (P<0.001). multivariate Cox repression analysis revealed HOXC9 expression was an independent poor prognostic factor (Hazard ratio, 1.74; 95% CI, 1.01 to 2.99; P=0.047). We then demonstrated that HOXC9 directly regulated Bcl-xL transcription by binding to a HOXC9-responsive element in the Bcl-xL promoter by reporter gene assay, chromatin immunoprecipitation (ChIP), and electrophoretic mobility shift assay (EMSA). In conclusion, these results suggest that HOXC9 overexpressed in advanced gastric cancer setting and its expression should be useful as a prognostic marker in gastric cancer patients. HOXC9 dependent Bcl-xL overexpresion may be an important mechanism by which HOXC9 protects gastric cancer cells from apoptosis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1032.

Inorganic polyphosphate differentiates human mesenchymal stem cells into osteoblastic cells

The existence of inorganic polyphosphates [poly(P)] in human cells has been demonstrated. In osteoblasts, it is suggested that the concentration of cellular poly(P) is relatively high. In this study, we examined whether poly(P) accelerates the differentiation of human mesenchymal stem cells (hMSCs) from patients with osteoarthritis (OA) and rheumatoid arthritis (RA) into osteoblastic cells. Alkaline phosphatase (ALP) activity was induced by poly(P) in hMSCs from both OA and RA. In Alizarin Red S and osteocalcin EIA, there was a significant difference between the control and poly(P) group. In real-time PCR, there was a significant difference in ALP, collagen type 1A, osteocalcin, and bone sialoprotein between the control and poly(P) group. Our findings suggest that poly(P) have the potent role of differentiating hMSCs into osteoblastic cells at the early and later stages of osteoblastic differentiation.

Postnatal Age Influences Hypoglycemia-Induced Poly(ADP-ribose) Polymerase-1 Activation in the Brain Regions of Rats

Poly(ADP-ribose) polymerase-1 (PARP-1) overactivation plays a significant role in hypoglycemia-induced brain injury in adult rats. To determine the influence of postnatal age on PARP-1 activation, developing and adult male rats were subjected to acute hypoglycemia of equivalent severity and duration. The expression of PARP-1 and its downstream effectors, apoptosis-inducing factor (Aifm1), caspase 3 (Casp3), NF-kappaB (Nfkb1) and bcl-2 (Bcl2), and cellular poly(ADP-ribose) (PAR) polymer expression were assessed in the cerebral cortex, hippocampus, striatum, and hypothalamus at 0 h and 24 h posthypoglycemia. Compared with the control group, PARP-1 expression increased in the cerebral cortex of adult rats 24 h posthypoglycemia, but not at 0 h, and it was accompanied by increased number of PAR-positive cells. The expression was not altered in other brain regions. Aifm1, Nfkb1, Casp3, and Bcl2 expressions also increased in the cerebral cortex of adult rats 24 h posthypoglycemia. Conversely, hypoglycemia did not alter PARP-1 expression and its downstream effectors in any brain region in developing rats. These data parallel the previously demonstrated pattern of hypoglycemia-induced brain injury and suggest that PARP-1 overactivation may determine age- and region-specific vulnerability during hypoglycemia.