Stimulation Of Poly Research Articles

The double-stranded RNA induces IL-33 expression through activation of EGFR, ERK, p38 and TBK-1 in normal human epidermal keratinocytes

The epidermis is the first line of innate host defense against external stimulus, which is mainly composed of keratinocyte. IL-33 is a member of IL-1 cytokine family that constitutively expressed in endothelial cells and epithelial cells of tissues. Viral infection is known to induce Toll-like receptor 3 (TLR3) activation through double-stranded RNA (dsRNA). We have previously reported that ultraviolet (UV) B irradiation induced IL-33 expression in normal human epidermal keratinocytes (NHEKs). We aimed to investigate whether TLR3 activation induces IL-33 expression in normal human epidermal keratinocytes and examine the mechanism of its induction. We also tried to investigate if the IL-33 which was induced by ultraviolet (UV) B irradiation can be suppressed by blocking TLR3 signal. NHEKs were stimulated with Poly I:C, the ligand for TLR3. The expression levels of mRNA for IL-33 and IFN-beta were assayed using quantitative real-time PCR. The expression of IL-33 was induced in NHEKs with the stimulation of Poly I:C and UVB which both were suppressed by the addition of TLR3 inhibitor. The induction of IL-33 by Poly I:C was dose- and time-dependent. The expression of IL-33 stimulated by Poly I:C peaked at 8 h, was suppressed at 24 through 48 h. IFN-beta were strongly induced by Poly I:C, which peaked at 4 h. We used anti-IFN-beta antibody aiming at blocking each signal, but the induction of IL-33 was not suppressed. Inhibitors for phosphorylation of extracellular signal regulated kinase (ERK), epidermal growth factor receptor (EGFR), p38 and TANK-binding kinase 1 (TBK-1) inhibited the induction of IL-33 efficiently. These results suggested that IL33 was induced through EGFR, ERK, p38, TBK-1 activation, but not through the production of IFNs by Poly I:C.

Ctenopharyngodon idella PKZ facilitates cell apoptosis through phosphorylating eIF2α.

PKZ, protein kinase containing Z-DNA binding domains, is a novel member of eIF2α kinase in fish. PKZ can be up-regulated in response to Poly I:C or heat stress, and it has a typical eIF2α kinase activity. However, the relationship between fish PKZ and apoptosis remains unclear. In the present study, effects of PKZ on apoptosis were explored. Initially, we found that PKZ and PKR were up-regulated by poly I:C in a time-dependent manner. Q-PCR analysis showed that the change of Caspase-3 mRNA was consistent with that of PKZ under the stimulation of poly I:C or transfection with PKZ siRNA in CIK cells. It suggested that PKZ might mediate the induction of apoptosis. Knockdown and overexpression assays indicated that a significant increase of apoptotic cell number was observed in the wild type PKZ (PKZ-wt) transfected PKZ-deficient cells, while mutant PKZ-K198R lost this ability. MTT also showed that overexpression of PKZ-wt in CIK cells resulted in a striking decrease of cell viability rate to about 32.5%, whereas the viability of cell with PKZ-K198R was about 88.1%. Likewise, when transfected eIF2α-wt or phosphomimetic eIF2α (S51D), CIK cells displayed higher apoptotic rate than the controls. In contrast, overexpression of phosphodeficient eIF2α (S51A) blocked induction of apoptosis. In addition, levels of eIF2α phosphorylation were significantly related to apoptosis in these CIK cells. Furthermore, when mutant eIF2α (S51A) was transiently transfected into the PKZ/PKR knockdown cells that transfected with wildtype PKZ previously, the apoptotic rates and levels of eIF2α phosphorylation were dramatically decreased. Overall, these results suggested that PKZ could facilitate apoptosis via eIF2α phosphorylation.

A Janus Kinase in the JAK/STAT signaling pathway from Litopenaeus vannamei is involved in antiviral immune response

The JAK/STAT signaling pathways are conserved in evolution and mediate diversity immune responses to virus infection. In the present study, a Janus kinase (designated as LvJAK) gene was cloned and characterized from Litopenaeus vannamei. LvJAK contained the characteristic JAK homology domain (JH domain) from JH1 to JH7 and showed 19% identity (34% similarity) and 21% identity (35% similarity) to Drosophila Hopscotch protein and Human JAK2 protein, respectively. The mRNA of LvJAK was highly expressed in hepatopancreas of L. vannamei and its expression level was prominently upregulated after the stimulation of Poly (I:C) and white spot syndrome virus (WSSV) challenges. There were 10 putative STAT binding motifs in the promoter region of LvJAK, and it could be regulated by LvJAK self or (and) LvSTAT, suggesting that LvJAK is the JAK/STAT pathway target gene and could function as a positive regulator to form a positive feedback loop. In addition, the silencing of LvJAK caused higher mortality rate and virus load, suggesting that LvJAK could play an important role in defense against WSSV. This is the first report about the complete set of JAK/STAT proteins in shrimp and the results provide the evidence of the positive feedback loop mediated by JAK protein present in the JAK/STAT pathway in invertebrates.

Gene expression analysis of porcine peripheral blood mononuclear cell in response to immune stimulation of Poly I:C.

Polyinosinic-polycytidylic acid (Poly I:C) is an analogue of natural double strand RNA (dsRNA), which can simulate the viral dsRNA and stimulate the immune response. In the present study, peripheral blood mononuclear cells (PBMC) were isolated from piglets of Dapulian and Landrace with different disease resistance, and stimulated with 20 μg/mL Poly I:C for 24 hours in vitro culture. The expression of several cytokines (IL6, IL8, TNFα, IL10, IRF3, IFNα and IFNγ) and three pattern recognition receptors (TLR3, TLR4 and RIG1) was determined by qRT-PCR. The results showed that, most of the cytokines or receptors had obvious expression change compared with the control (without Poly I:C stimulation), especially the three cytokine genes IL6, IL8 and IL10, whose average expression change times were 20.71, 10.87 and 5.18, respectively. Expression comparison between breeds and among individuals of the same breed indicated that there was obvious difference not only between Dapulain and Landrace (Dapulain higher than Landrace) but also among the three individuals of the same breed. Our study simulated the infection of dsRNA to host cells using Poly I:C, and provided experimental foundation for further study on selecting the immune genes in response to Poly I:C stimulation and identifying the unique disease-resistance genes of Dapulian.

Apoptosis induction and inhibition of drug resistant tumor growth in vivo involving daunorubicin-loaded graphene–gold composites

Daunorubicin (DNR) loaded graphene-gold nanocomposites offer a novel strategy for inducing apoptosis in drug resistant leukemia cells (K562/A02; KA). In vitro and in vivo investigations on xenografted tumors in KA nude mice demonstrate that the combination of monoclonal P-glycoprotein (P-gp) antibodies and DNR anticancer drug loaded on graphene-gold nanocomposites (GGN) is an efficient drug delivery vector, with remarkable targeting and binding properties towards drug resistant KA cell lines, and induces apoptosis of KA cells and inhibits tumor growth in KA nude mice. Cellular treatment with DNR-loaded GGN remarkably reduced drug resistant-related P-gp expression and activated apoptosis-related caspase protein expression in KA cells. Cell apoptosis provoked in vitro by such nanocomposites corresponds to a rapid induction of active caspase 8,3 activities and stimulation of poly-(ADP-ribose) polymerase (PARP) proteolytic cleavage. In vivo studies indicate that DNR-loaded GGN nanocomposites effectively overcome the inhibition of drug resistant leukemia cell-induced tumor growth in KA nude mice. This nanocomposite raises the possibility of modulating apoptosis in cancer cells, and of inhibiting tumor growth, showing that nanocomposites of this kind have promising applications in efficient multifunctional therapy.

Expression patterns and association analysis of the porcine DHX58 gene

RIG-1 signalling is responsible for the detection of cytoplasmic viral RNA molecules. DEXH(Asp-Glu-X-His) box polypeptide 58 (encoded by DHX58) is a negative regulator of the RIG-1 signalling pathway. In human, the DHX58 gene can be upregulated and can inhibit the RIG-1 signalling pathway during viral infection. In this study, porcine DHX58 gene expression patterns were studied. According to our results, the porcine DHX58 gene was upregulated not only by the stimulation of Poly I:C but also by the stimulation of lipopolysaccharides (LPS). One polymorphism (g.4919G>C), detected in the ninth intron,was significantly associated with some blood parameters including the red cell distribution width of 1-day-old pigs and white blood cell counts, lymphocyte absolute counts, and platelet distribution width of 17-day-old pigs (P < 0.05). Moreover, the individuals with the genotype GG have a significantly higher mean white blood cell count than individuals with genotype CC or GC (P < 0.05). Our study indicates that DHX58 is an important gene that is associated with the immune response in swine.

A Drosophila model of oculopharyngeal muscular dystrophy reveals intrinsic toxicity of PABPN1

Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset syndrome characterized by progressive degeneration of particular muscles. OPMD is caused by short GCG repeat expansions within the gene encoding the nuclear poly(A)-binding protein 1 (PABPN1) that extend an N-terminal polyalanine tract in the protein. Mutant PABPN1 aggregates as nuclear inclusions in OMPD patient muscles. We have created a Drosophila model of OPMD that recapitulates the features of the human disorder: progressive muscle degeneration, with muscle defects proportional to the number of alanines in the tract, and formation of PABPN1 nuclear inclusions. Strikingly, the polyalanine tract is not absolutely required for muscle degeneration, whereas another domain of PABPN1, the RNA-binding domain and its function in RNA binding are required. This demonstrates that OPMD does not result from polyalanine toxicity, but from an intrinsic property of PABPN1. We also identify several suppressors of the OPMD phenotype. This establishes our OPMD Drosophila model as a powerful in vivo test to understand the disease process and develop novel therapeutic strategies.

Stimulation of poly(A) synthesis by Escherichia coli poly(A)polymerase I is correlated with Hfq binding to poly(A) tails

The bacterial Lsm protein, host factor I (Hfq), is an RNA chaperone involved in many types of RNA transactions such as replication and stability, control of small RNA activity and polyadenylation. In this latter case, Hfq stimulates poly(A) synthesis and binds poly(A) tails that it protects from exonucleolytic degradation. We show here, that there is a correlation between Hfq binding to the 3' end of an RNA molecule and its ability to stimulate RNA elongation catalyzed by poly(A)polymerase I. In contrast, formation of the Hfq-RNA complex inhibits elongation of the RNA by polynucleotide phosphorylase. We demonstrate also that Hfq binding is not affected by the phosphorylation status of the RNA molecule and occurs equally well at terminal or internal stretches of poly(A).

Stimulation of poly(A) polymerase through a direct interaction with the nuclear poly(A) binding protein allosterically regulated by RNA.

During polyadenylation of mRNA precursors in metazoan cells, poly(A) polymerase is stimulated by the nuclear poly(A) binding protein PABPN1. We report that stimulation depends on binding of PABPN1 to the substrate RNA directly adjacent to poly(A) polymerase and results in an approximately 80-fold increase in the apparent affinity of poly(A) polymerase for RNA without significant effect on catalytic efficiency. PABPN1 associates directly with poly(A) polymerase either upon allosteric activation by oligo(A) or, in the absence of RNA, upon deletion of its N-terminal domain. The N-terminal domain of PABPN1 may function to inhibit undesirable interactions of the protein; the inhibition is relieved upon RNA binding. Tethering of poly(A) polymerase is mediated largely by the C-terminal domain of PABPN1 and is necessary but not sufficient for stimulation of the enzyme; an additional interaction dependent on a coiled-coil structure located within the N-terminal domain of PABPN1 is required for a productive interaction.

DETECTION OF POLY(ADP-RIBOSE) SYNTHETASE ACTIVITY IN ALVEOLAR MACROPHAGES OF RATS EXPOSED TO NITROGEN DIOXIDE AND OZONE

The toxic effects of nitrogen dioxide (NO2) and ozone (O3) are mediated through the formation of free radicals, which can cause DNA strand breaks. The present study demonstrates that exposure to NO2 and O3 causes a stimulation of poly (ADP-ribose) (poly ADPR) synthetase in alveolar macrophages of rats. Three-month-old male Sprague-Dawley rats, specific pathogen free, were exposed to either 1.2 ppm NO2 or 0.3 ppm O3 alone or a combination of these 2 oxidants continuously for 3 days. The control group was exposed to filtered room air. To evaluate whether exposure to these two oxidants (NO2 and O3) caused DNA damage to lung cells, the activity of poly ADPR synthetase was measured. Cellular DNA repair is dependent upon the formation of poly (ADP-ribose) polymerase, which is catalyzed by poly ADPR synthetase. Poly ADPR synthetase is known to be activated in response of DNA damage. The results showed that the enzyme activity was stimulated after exposure to O3 or exposure to NO2 + O3. Ozone exposure caused a 25% increase in the enzyme activity as compared to the control. Combined exposure to NO2 + O3 showed a 53% increase in the enzyme activity. These results were statistically significant as compared to the control and NO2 exposure groups. Other parameters such as total cell count, cell viability, and differential cell count were also determined. The stimulation of poly ADPR synthetase activity after O3 exposure or NO2 + O3 exposure reflects a response to lung cellular DNA repair, which may be used as an indicator for assessing DNA damage caused by oxidant injury.

Studies with poliovirus polymerase 3Dpol. Stimulation of poly(U) synthesis in vitro by purified poliovirus protein 3AB.

The synthesis in vitro of poly(U) on a poly(A) template with oligo(dT)15 primer by poliovirus RNA polymerase 3Dpol (280 ng/ml) is strongly stimulated (50-100 fold) by the addition of purified poliovirus polypeptide 3AB. The synthesis of product continues linearly with time for up to 90 min. The reaction with 3Dpol alone can be reactivated and similarly enhanced by the addition of 3AB at 30 min of incubation. Optimal stimulation is achieved under conditions where the concentration of 3Dpol and of template is low, when the molar ratio of 3AB to 3Dpol is about 100:1 and that of 3AB to poly(A) is about 25:1. In the presence of 3AB, the yield of product made by 3Dpol is much increased but its size is unchanged. From a number of basic proteins and peptides tested, a few were found which also exhibited limited enhancement of polymerase activity. The stimulatory effect of 3AB is probably related to its ability to bind both the template-primer, poly(A).oligo(dT)15, and 3Dpol (Molla, A., Harris, K. S., Paul, A. V., Shin, S. H., Mugavero, J., and Wimmer, E. J. (1994) J. Biol. Chem. 269, 27015-27020). RNA synthesis on purified poliovirus RNA with oligo(dT)15 primer is enhanced by 3AB about 5-10 fold, and this reaction is highly sensitive to detergent.

Stimulation of poly(A) tail elongation by the VP39 subunit of the vaccinia virus-encoded poly(A) polymerase.

The VP55 subunit of the vaccinia virus-encoded poly(A) polymerase can add a maximum of 35 adenylates to the 3'-end of an RNA primer in a rapid and highly processive manner, whereas the VP55-VP39 heterodimer catalyzes the formation of poly(A) tails several hundred nucleotides in length. Here, we describe the overexpression of the VP39 subunit, its purification to near homogeneity, and its ability to associate physically with VP55 and to stimulate polyadenylation. Although VP39 possessed no independent poly(A) polymerase activity, RNA primers with oligo(A) tails greater than 30 adenylates in length could be extended nearly 40-fold more rapidly in the presence of VP39. VP39 enhanced the polyadenylation rate by converting the slow, nonprocessive polyadenylation occurring after the rapid burst in the presence of monomeric VP55, to a rapid, semiprocessive reaction. The effect of VP39 was dramatic when poly(A) primers were used as, 60 mM NaCl, VP39 enhanced the polyadenylation rate 500-fold, and at 90 mM NaCl VP39 was absolutely required. Nevertheless, the VP39-containing polymerase remained selective for polyadenylation of an mRNA 3'-end in the presence of excess poly(A). These data suggest that the role of VP39 in polyadenylation is to increase the affinity of the polymerase for the growing poly(A) tail.

Benzamide potentiation of the cytotoxicity of bifunctional galactitol [correction of galacticol] in resistant P388 leukemia correlates with inhibition of DNA ligase II.

Benzamide (BA) enhances the cytotoxicity of 1,2:5,6-dianhydrogalactitol (DAG) in resistant P388 leukemia cell lines but not in the sensitive parent line. To examine the reason for this difference in response, we carried out an alkaline elution assay using proteinase K to study DNA interstrand cross-linking. At early time points, equal concentrations of DAG produced the same level of interstrand cross-linking (ICL) in the resistant and sensitive P388 leukemic cells, although marked differences were observed in their cytotoxicity toward the two cell lines. In the sensitive cells, neither the amount of DNA cross-linking nor the cytotoxicity changed during the observation period (38 h) in either the presence or the absence of BA. In contrast, the elution rate of the DNA of DAG-treated resistant cells increased with time and had reached the control levels by 38 h. However, when these cells were postincubated with BA for 38 h, the elution rate of DNA was much faster than that observed for the untreated resistant cells, indicating an accumulation of DNA single-strand breaks (SSB). The SSB accumulation caused by BA was associated with an inhibition of the activity of ligase II enzyme, which was stimulated when resistant cells were treated with DAG alone. The potentiating effect of BA on the resistant cells can thus be related to the inhibiting action of BA on the DNA-rejoining enzyme, ligase II. The lack of sensitization by BA of the DAG-treated parent cell line may be attributable to the absence of DNA-SSB formation, which is necessary for ligase II activation through the stimulation of poly(ADP-ribose) synthesis.

Stimulation of poly(ADP-ribose) synthesis by free radicals in C3H10T1/2 cells: relationship with NAD metabolism and DNA breakage

We have studied the effect of H2O2 and O2- produced by xanthine and xanthine oxidase on NAD catabolism, poly(ADP-ribose) synthesis, and production of DNA single-strand breaks in C3H10T1/2 cells. The results show a correlation between the induction of DNA single-strand breaks, the decrease of NAD pool, and the accumulation of polymer. New techniques, based on affinity chromatography and reversed-phase high pressure liquid chromatography, have allowed an accurate determination of polymer contents and showed a 20-fold stimulation of polymer biosynthesis induced by active oxygen species. Inhibition experiments performed with 3-aminobenzamide have shown that the decrease in NAD levels after exposure of cells to active oxygen species was caused by stimulation of poly(ADP-ribosyl)ation and of another cellular process.

Acetaldehyde activation of poly(ADP-ribose) polymerase in hepatocytes of mice treated in vivo

The activities of poly(ADP-ribose) polymerase and of DNA polymerases α and β and the level of cytochrome P450 were determined in mouse parenchymal liver cells 5 h after treatment with 0.1, 0.3, 1.0, and 3.0 μmole of acetaldehyde. Injection with 1.0 and 3.0 μmole of acetaldehyde induced an increase in poly(ADP-ribose) polymerase activity and in the p450 level, but had no effect on DNA polymerases. The stimulation of poly(ADP-ribose) polymerase activity can be used as an index of induced DNA damage. The possibility of using this experimental approach with other cells derived from mice treated in vivo with different xenobiotics is discussed

Poly(ADP-ribose) synthesis and inhibition of DNA synthesis in Chinese hamster cells treated with methylating agents and thymidine

A possible role of poly(ADP-ribose) synthesis in modulating the response of V79 cells to DNA damage induced by N-methyl- N′-nitrosoguanidine (MNNG) and methyl methanesulfonate (MMS) was investigated. Inhibition of [ 3H]thymidine (dThd) incorporation into DNA and lowering of NAD + levels in intact cells were employed as parameters of DNA-synthesis inhibition and poly(ADP-ribose) synthesis, respectively. Dose responses of these parameters were studied in cells 2 and 24 h after treatment with the methylating agents in medium with or without dThd. The initial inhibition of DNA synthesis was uniformly associated with stimulation of poly(ADP-ribose) synthesis whether the cells were treated with MNNG or MMS, incubated with or without 20 μM dThd which did not inhibit poly(ADP-ribose) synthesis, or incubated with 3 mM dThd which did inhibit the latter synthesis. By contrast, the DNA-synthesis inhibition detected 24 h after treatment with MNNG was not associated with poly(ADP-ribose) synthesis. These data suggest that (i) the mechanism of this later inhibition of DNA synthesis is different from that of the initial inhibition, (ii) DNA-synthesis inhibition does not stimulate poly(ADP-ribose synthesis, and (iii) single-strand breaks, resulting from N-methylation of the DNA, stimulate poly(ADP-ribose) synthesis, which may produce the initial inhibition of DNA synthesis. The initial inhibition of DNA synthesis was not uniformly associated with mutagenesis and dThd facilitation of MNNG-induced cytotoxicity and mutagenesis. This indicates that O-methylation of DNA does not stimulate poly(ADP-ribose) synthesis. Our data suggest that, in V79 cells treated with methylating agents, poly(ADP-ribose) synthesis is stimulated by single-strand breaks, inhibits DNA synthesis, and thereby serves to allow time for repair of the DNA prior to replication.

Stimulation of poly(ADP-ribose) synthetase activity in the lungs of mice exposed to a low level of ozone

Toxic effects of O 3 are mediated through the formation of free radicals, which can cause DNA strand breaks. Cellular DNA repair is dependent upon the formation of poly(ADP-ribose) (polyADPR) catalyzed by polyADPR synthetase. In order to evaluate whether O 3 exposure inflicted DNA damage in lung tissue, we measured the activity of polyADPR synthetase (known to be activated in response to DNA damage) in mouse lungs after exposure to 0.45 ppm (882 μg/m 3) O 3 for up to 7 days. The enzyme activity was stimulated with O 3 exposure relative to unexposed controls, showing a 20% ( P < 0.05) increase at Day 5 and 42% ( P < 0.001) at Day 7 of O 3 exposure. In addition, the activity of superoxide dismutase (SOD), known to be stimulated in response to production of superoxide anion (.O 2 −), was measured as an indicator of free radical involvement. Relative to unexposed controls, the SOD activity in exposed animal lungs increased to the peak level at Day 5 (48%, P < 0.001) and then declined at Day 7 of O 3 exposure but was still higher than controls (17%, P < 0.05). When animals, after 5 days of O 3 exposure, were allowed to recover in filtered room air, the activities of both enzymes declined to their respective control values in 6 days. These results suggest a possible temporal relationship between O 3 injury and the activities of polyADPR synthetase and a free radical scavenging enzyme, SOD. The stimulation of polyADPR synthetase activity with O 3 exposure, reflecting a response to lung cellular DNA repair, may be a sensitive indicator for assessing DNA damage in oxidant injury.

Androgen regulation of poly(A) polymerase in the ventral prostate of rat

The effect of various hormones on the levels of poly(A) polymerase in the ventral prostate of castrated rats was investigated. It was observed that this enzyme is specifically induced by androgens; progresterone and estradiol-17β did not cause stimulation of poly(A) polymerase activity. Dihydro-testosterone-induced poly(A) polymerase was inhibited by cordycepin, actinomycin-D and cycloheximide, which indicates that the genetic transcription leading to the enzyme poly(A) polymerase is regulated by androgens in the ventral prostate.

Interferon and sodium butyrate inhibit the stimulation of poly(ADP-ribose) synthetase in mouse cells stimulated to divide

The activity of poly(ADP-ribose) synthetase, a chromatin-bound enzyme, increases when quiescent 3T3 cells are stimulated to proliferate. The elevation of enzymatic activity requires de novo RNA and protein synthesis. Interferon (IFN) or sodium butyrate, when added to quiescent cells at the time of stimulation, suppressed the rise of enzymatic activity as well as initiation of DNA synthesis in cells. However, other DNA synthesis inhibitors like methotrexate, FudR and hydroxyurea had little effect on the elevation of poly(ADP-ribose) synthetase in quiescent cells.

Stimulation of poly(ADP-ribosyl)ation during Ehrlich ascites tumor cell "starvation" and suppression of concomitant DNA fragmentation by benzamide.

Incubation of Ehrlich ascites tumor cells in their own ascites fluid induced a reversible metabolic adaptation to these "starvation" conditions which was associated with a fragmentation of DNA. Endogenous poly(ADP-ribose) residues also increased, reaching within 1-3 h values 6-10 times higher than in cells taken directly from the mouse peritoneum. The NAD content changed only slightly while dimethyl sulfate-induced accumulation of poly(ADP-ribose) (10-fold within 30 min) was associated with a rapid depletion of NAD (85% lost at 30 min). Nevertheless, turnover of poly(ADP-ribose) as measured by the decay rate of the polymer upon addition of benzamide was dramatically stimulated in both situations, reaching apparently identical half-lives (t 1/2 approximately equal to 1 min) in "starved" and in alkylated cells. However, since penetration of benzamide into the nucleus may be the rate-limiting factor in these studies, turnover of poly(ADP-ribose) in dimethyl sulfate-treated cells may still be much higher than that in "starved" cells. In cells treated with dimethyl sulfate, suppression of poly(ADP-ribose) synthesis by benzamide did not interfere with DNA fragmentation or with DNA resealing as determined by the nucleoid procedure. By contrast, starvation induced a type of DNA incision that was prevented by benzamide. It is proposed that starvation-induced scission of DNA occurs at specific ("regulatory?") sites requiring poly(ADP-ribose) formation to take place, while fragmentation of DNA at random as seen with alkylating agents is associated with, but not dependent on, increased poly(ADP-ribosyl)ation.