Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Fragmentation”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,693 records · Page 94Linked to original sources
PubMed1991

A proteolytic fragment from human link protein is taken up and processed by monocytes and B cells.

Mild digestion of 125I-labelled human proteoglycan aggregates with trypsin or stromelysin produced specific peptides that were taken up rapidly by THP-1 monocytes. SDS/PAGE of undigested aggregate showed that the three components of molecular mass 48, 44 and 41 kDa, corresponding to isoforms of link protein originally present, had been converted into a single component of 41 kDa by trypsin treatment, and that fragments of 6-12 kDa were present in fractions containing the high-uptake peptide. Separate proteolysis of isolated proteoglycan monomer and link protein confirmed that the specific high-uptake fragment was derived from link protein. Uptake of the link fragment was rapid, reaching a maximum after 5 min, and specific, since it was blocked by metabolic or serine proteinase inhibitors and at 4 degrees C. After uptake the cleaved fragment was processed further, with 50% of the radiolabel being released as degraded peptides within 5 min. In contrast, accumulation of whole aggregate reached a maximum after 45 min and only 50% had been released after 2 h. Uptake of aggregate was less affected by inhibitors or at low temperature, suggesting that a separate mechanism existed for its turnover. The aggregate was transported to lysosomes after uptake, although the link fragment did not sediment with either lysosomes or plasma membranes, suggesting that it was present in the cytoplasm or in very labile vesicles. However, the mode of handling of the peptide by the cells remains unclear. The link fragment was taken up by several different monocytic and B cell lines, but not by mouse fibroblasts or peritoneal macrophages. These data suggest that a surface serine proteinase on monocytes and B cells enables them to process and take up a fragment of link protein derived by extracellular proteolysis.

B-Lymphocytes↗
PubMed1994

Adenosine stimulates DNA fragmentation in human thymocytes by Ca(2+)-mediated mechanisms.

Incubation of human thymocytes with an optimum concentration of adenosine and its receptor site agonist, 2-chloroadenosine, induced increases in intracellular cyclic AMP (cAMP) (from a resting 0.6 +/- 0.1 to 4.1 +/- 0.2 pmol/10(7) cells within 5 min) and Ca2+ (from the resting 85 +/- 7 nM to a peak of 210 +/- 25 nM) levels and resulted in internucleosomal DNA fragmentation and cell death (apoptosis). Other adenosine analogues were also effective at inducing DNA fragmentation, the order of potency being 2-p-(carboxyethylphenylethylamino)-5'-carboxyamidoadenosine < 5'-(N-ethylcarboxamide)adenosine < or = cyclopentyladenosine < 2-chloroadenosine (2-CA). 2-CA treatment (with an optimum concentration of 40 microM) selectively depleted a thymocyte subpopulation (15-20% of the total cells) which expressed higher levels of the CD3 molecule and which was found mainly in the CD4+CD8+ double positive immature thymocyte population. DNA fragmentation was prevented by the addition of actinomycin D or cycloheximide to the thymocyte suspension, indicating that this process required both mRNA and protein synthesis. Endonuclease activation and cell killing were dependent on an early, sustained increase in cytosolic Ca2+ concentration, most of which was of extracellular origin and was a result of an adenosine-induced inositol trisphosphate release. Other agents known to elevate intracellular cAMP levels by different mechanisms failed to induce similar DNA fragmentation, but enhanced the effect of adenosine. This suggested a supporting role for cAMP in adenosine-induced DNA fragmentation. Phorbol dibutyrate, a protein kinase. C activator, previously shown to inhibit Ca(2+)-dependent DNA fragmentation and cell killing in human thymocytes [McConkey, Hartzell, Jondal and Orrenius (1989) J. Biol. Chem. 264, 13399-13402], at 60 ng/ml concentration also prevented adenosine-induced DNA fragmentation when added prior to adenosine. This suggested a complex cross-talk between the adenosine-triggered signal transduction cascade and the activation state of protein kinase C in regulating apoptosis of human thymocytes.

2-Chloroadenosine↗
PubMed1995

Some spectral correlates of pathological breathy and rough voice quality for different types of vowel fragments.

This study deals with the relation between listeners' ratings of pathological breathiness and roughness and certain characteristics of the voice spectrum. Two general research questions were addressed: First, which spectral parameters may serve as useful predictors of breathiness and roughness? Second, does the type of speech fragment used for analysis have an effect on the obtained regression model? Listener ratings of breathiness and roughness were obtained for three types of vowel fragments: a vowel onset segment, a mid-vowel (post-onset) segment, and a vowel segment covering the onset and the acoustically more stable post-onset parts. Results indicated that the harmonics-to-noise ratio was the best single predictor of both rated breathiness and roughness, explaining up to 54% of the true rating variance. By combining different predictors, between 75% and 80% of the breathiness variance could be explained for all three types of fragments. For roughness, a strong effect of fragment type was observed, with most variance explained in vowel onset fragments (71%), and least in post-onset fragments (52%). The effect of fragment type was also observed when regression analyses were performed with six predictors based on a factor analysis of the acoustic data.

Female↗
PubMed1999

The effect of clustered versus regular sleep fragmentation on daytime function.

Previously, we found that regular sleep fragmentation, similar to that found in patients with sleep apnoea/hypopnoea syndrome (SAHS), impairs daytime function. Apnoeas and hypopnoeas occur in groups in patients with REM or posture related SAHS. Thus, we hypothesised that clustered sleep fragmentation would have a similar impact on daytime function as regular sleep fragmentation. We studied 16 subjects over two pairs of 2 nights and 2 days. The first night of each pair was for acclimatisation. On the second night, subjects either had their sleep fragmented regularly every 90 s, or fragmented every 30 s for 30 min every 90 min, the remaining 60 min being undisturbed. We fragmented sleep with tones to produce a minimum 3 s increase in EEG frequency. During the days following each pair of nights we tested subjects daytime function. Total sleep time (TST) and microarousal frequency were similar no both study nights. We found significantly less stage 2 (55 SD 4, 62 +/- 7%; P = 0.001) and more slow wave sleep (21 SD 3, 12 +/- 6%; P < 0.001) on the clustered night. Mean sleep onset latency was similar on MSLT (clustered 10 SD 5, regular 9 +/- 4 min; P = 0.7) and MWT (clustered 32 SD 7, regular 30 +/- 7 min; P = 0.2). There was no difference in subjects mood or cognitive function after either study night. These results suggest that although there is more slow wave sleep (SWS) on the clustered night, similar numbers of sleep fragmenting events produced similar daytime function whether the events were evenly spaced or clustered.

Adult↗
PubMed1994

Low retention of white cell fragments by polyester fiber white cell-reduction platelet filters.

BACKGROUND: There is intense interest in the potential of current white cell (WBC)-reduction filters to prevent the alloimmunization of patients by the residual donor WBCs in filtered blood components transfused to them. Little attention has been paid to the capacity of current synthetic fiber filters to remove WBC membrane fragments bearing detectable leukocyte antigens. STUDY DESIGN AND METHODS: Fluorescein isothiocyanate-conjugated monoclonal antibody to a universal WBC membrane antigen (CD45) and high-speed centrifugation coupled with ficoll-hypaque differential sedimentation were used to quantitate low-density WBC fragments in single-donor platelet components before and after filtration to determine if a polyester fiber filter retained WBC fragments. RESULTS: Prefiltration measurements in 25 single-donor platelet components indicated that WBC fragments increased with length of storage up to 5 days at room temperature (p < 0.0001). When fragments in eight components were measured before and after filtration, absolute values for differences were insignificant (p = 0.15). CONCLUSION: WBC fragments were poorly retained by these polyester fiber WBC-reduction filters. The antigenicity of WBC fragments could contribute to the WBC alloimmunization of some recipients of WBC-reduced blood components.

Antibodies, Monoclonal↗
PubMed2002

Increased pollen flow counteracts fragmentation in a tropical dry forest: an example from Swietenia humilis Zuccarini.

Habitat destruction and the resultant fragmentation of the remaining forest are a common phenomenon in the tropics. Most investigations emphasize the potential dangers of fragmentation in isolating patches of forest and exposing populations to loss of species diversity through founder effects, genetic drift, inbreeding, and restricted gene flow. However, a limited number of studies have shown that gene flow may be extensive in tropical trees, suggesting that it may occur between forest fragments and also "isolated" remnant trees. There is an urgent need to quantify pollen flow within and between forest fragments to test the veracity of such views and determine the genetic value of such fragments for in situ conservation. Microsatellite markers are used to genotype individuals of Swietenia humilis from a highly fragmented forest mosaic to directly quantify pollen-mediated gene flow. Distances of pollen flow more than 10 times greater than previously reported were detected. Our results show that some tropical angiosperm tree species may be much more adaptable and resilient to habitat destruction and fragmentation than previously considered. The description of many remnant trees as isolated or "living dead" may be more a conditioning of human perception than a true reflection of their potential conservation value.

Ecology↗
PubMed1974

Unique double-stranded fragments of bacteriophage T5 DNA resulting from preferential shear-induced breakage at nicks.

Nicks within one strand of the bacteriophage T5 DNA molecule act as " weak points" for a novel kind mechanical breakage that can be utilized for "dissecting" the genome. The products from sheared T5(+) DNA include five unique double-stranded segments of the molecule and various combinations of adjacent segments. These specific fragments are not obtained after repair of the nicks with DNA ligase (EC 6.5.1.1). The duplex fragments and most of their single-stranded components have been separated, identified, and mapped by means of agarose gel electrophoresis. Even the complementary strands of the unique fragments separate in agarose gels; hence, there are now three useful classes of DNA fragments available from T5: the natural r-strand fragments, their complements from the normally intact l strand, and the corresponding duplex segments. By summing the apparent molecular weights of their single-stranded components, the unique duplex fragments from T5(+) DNA can be assigned molecular weights of approximately 6 x 10(6) (A), 8 x 10(6) (B), 11 x 10(6) (C), 27 x 10(6) (D), and 30 x 10(6) (E) from left to right along the genome. The most abundant overlapping fragments are segments AB (14 x 10(6)) and ABC (26 x 10(6)). Differences in the number and relative positions of nicks within two distinct groups of heatstable deletion mutants [represented by T5st(O) and T5b3] account for the large differences observed in their patterns of breakage products.

Catalysis↗
PubMed1976

Binding of triton X-100 to diphtheria toxin, crossreacting material 45, and their fragments.

Binding of the nonionic detergent [3H]Triton X-100 by diphtheria toxin, by the nontoxic serologically related protein crossreacting material (CRM) 45, and by their respective A and B fragments has been studied. If first denatured in 0.1% sodium dodecyl sulfate, all of the proteins with the exception of fragment A bind increasing amounts of Triton X-100, reaching a maximum of more than 40 mol bound per mol of protein when the detergent concentration exceeds its critical micelle concentration. No measurable amount of Triton X-100 is bound by native toxin or its A fragment of any concentration of the detergent. Undenatured CRM45 or its B45 fragment, on the other hand, readily became inserted into Triton X-100 micelles when the detergent reaches its critical micelle concentration. The results show that the toxin molecule contains a hydrophobic domain located on the portion of the B fragment that is linked to A. This region is masked in native toxin. Based on these findings, a model is proposed to describe how fragment B facilitates the transport of the enzymically active hydrophilic fragment A across the plasma membrane to reach the cytoplasm.

Binding Sites↗
PubMed1983

Rescue of transforming Epstein-Barr virus (EBV) from EBV-genome-positive epithelial hybrid cells transfected with subgenomic fragments of EBV DNA.

Transfection experiments using subgenomic fragments of the B95-8 strain of Epstein-Barr virus (EBV) DNA and EBV genome (HR-1)-positive epithelial/Burkitt hybrid cells (D98/HR-1) were carried out to determine whether an interaction between the transfecting virus fragment(s) and the endogenous HR-1 EBV genome could take place. Expression of EBV-specific antigens, including early antigen and virus capsid antigen, was examined in transfected cells by immunofluorescence. Attempts were also made to recover biologically active EBV from the D98/HR-1 cells after transfection with cloned fragments of B95-8 DNA. We found that D98/HR-1 cells transfected with the BamHI H or H, F, and X fragments were positive for early antigen 3 days after transfection. Spent media from transfected D98/HR-1 cells maintained for 20-30 days in culture were pooled, filtered, concentrated, and used as a potential source of virus to inoculate human umbilical cord blood lymphocytes. No evidence of transformation was observed with such preparations. However, if spent medium from D98/HR-1 cell cultures was first treated with iododeoxyuridine (to induce EBV DNA synthesis and replicative cycle) and then transfected with the BamHI H, F, and X fragments of B95-8 DNA and used to infect cord blood lymphocytes, transformation was obtained. A lymphoblastoid cell line derived in this manner, designated HI-HFX, is an EBV nuclear antigen-positive nonproducer cell line. Similar results were obtained with preparations from iododeoxyuridine-treated D98/HR-1 cells transfected with the EB 26-36 fragment of B95-8 DNA cloned in a Charon 4A vector. The EB 26-36 fragment contains the BamHI H, F, and X regions.

Cell Transformation, Viral↗
PubMed1984

Expression of Epstein-Barr virus (EBV) DNA and cloned DNA fragments in human lymphocytes following Sendai virus envelope-mediated gene transfer.

Purified EBV DNA and cloned DNA fragments were trapped in Sendai virus (SV) envelopes during envelope reconstitution. The DNA-loaded reconstituted envelopes (RSVE/DNA) served as gene-transfer vehicles using the capability of RSVE to fuse with normal and tumor cells. The efficiency of RSVE-mediated EBV DNA transfer into lymphoid tumor cells and fresh human lymphocytes was 5-10% of the enveloped 3H-labeled EcoRI fragment B of EBV DNA. Purified intracellular EBV (B95-8 strain) DNA induced EBV nuclear antigen (EBNA) in 0.2-1% of human lymphocytes, transiently stimulated cellular DNA synthesis, but did not fully transform cells. Cloned Sal I F1 fragment [approximately equal to 9 kilobase pairs (kbp)] and a smaller BamHI K (5.2 kbp) fragment from the same region of B95-8 EBV DNA induced EBNA in 2-4% of human lymphocytes but did not stimulate DNA synthesis nor transform cells. Cloned BamHI D1 fragment (approximately equal to 9 kbp) from AG-876 virus DNA, or a combination of cloned BamHI X and H fragments (approximately equal to 2 and 7 kbp, respectively) from the similar region of B95-8 virus DNA, significantly stimulated lymphocyte DNA synthesis, but EBNA could not be detected and transformation was not achieved. Early antigen and viral capsid antigen were not observed with any of the fragments tested. Our results suggest that the induction of EBNA and stimulation of lymphocyte proliferation are not controlled by the same region of EBV DNA.

Base Sequence↗
PubMed1985

Expression of a second Epstein-Barr virus-determined nuclear antigen in mouse cells after gene transfer with a cloned fragment of the viral genome.

Large Epstein-Barr virus (EBV) DNA restriction fragments corresponding to regions transcribed in transformed, proliferating cells were cloned in a cosmid derivative of the dominant-acting selection vector pSV2-gpt. Recombinant vectors carrying the EcoRI A fragment of EBV DNA were modified in the region corresponding to the deletion of the virion DNA in the non-transforming viral substrain P3HR-1, to create a series of recombinants lacking parts of this region. The recombinant vectors were introduced into 3T3 mouse fibroblasts under selective conditions, and resistant clones shown to contain EBV DNA sequences were analyzed for the expression of EBV-related antigens detectable by direct, indirect, and anticomplement immunofluorescence techniques. Cells that contained the BamHI K fragment expressed the EBV-determined nuclear antigen (EBNA) as expected. Cells transfected with recombinant vectors containing the BamHI W, Y, and H fragment part of the EcoRI A fragment also express a nuclear antigen detectable with certain anti-EBNA-positive human sera in anticomplement immunofluorescence tests. The BamHI WYH-induced EBNA polypeptide is similar in size to the EBNA2 polypeptide in Raji cells, as shown by gel electrophoresis and immunoblotting. The antigen is not detected in cells transfected with EcoRI A-derived vectors in which the BamHI H fragment has been deleted or in cells transformed with vectors carrying the BamHI H fragment alone. Direct and indirect immunofluorescence did not reveal the presence of antigens associated with productive infection in any of the EBV DNA-transfected fibroblast clones.

Animals↗
PubMed1991

Structurally intact (78-kDa) forms of maternal lactoferrin purified from urine of preterm infants fed human milk: identification of a trypsin-like proteolytic cleavage event in vivo that does not result in fragment dissociation.

Two forms of lactoferrin, an intact lactoferrin and a "nicked" but apparently intact (i.e., 78-kDa) form, have been isolated from the urine of preterm infants fed human milk. These two forms of lactoferrin, demonstrated to be entirely of maternal origin, were copurified using affinity columns of immobilized single-stranded DNA-agarose. The relative concentrations of the intact lactoferrin and the "nicked" lactoferrin were determined after denaturation and separation by reverse-phase HPLC. N-terminal sequence analyses showed that the intact 78-kDa form had lost two residues from its N terminus. The nicked 78-kDa form was composed of only two fragments; one fragment was identified as the N terminus of the N-lobe (residues 3-283). The other fragment started with Ser-284 and included the alpha-helical structures at the C terminus of the N-lobe, as well as the entire C-lobe. Although no disulfide bonds connect these two fragments, they were tightly associated in vivo and were not separated in vitro except under denaturing conditions. Limited in vitro digestion of human milk lactoferrin with trypsin produced a nicked, but stable (78-kDa), form of DNA-binding lactoferrin nearly indistinguishable from the isolated urinary lactoferrin, except for the absence of one additional arginine residue at the N terminus of the N-lobe. Residues involved in the stable molecular interaction between fragments were evaluated using data obtained from the high-resolution crystal structure of hololactoferrin. Two features, entirely within the N-lobe, account for the lack of fragment dissociation after cleavage at residue 283 in vivo: an extensive interface at the hinge region behind the iron-binding cleft and an "anchor" sequence traversing the remainder of the N-lobe at 90 degrees relative to the fragment interface. These results document the remarkably limited degradation of absorbed lactoferrin in vivo and suggest that iron-binding activity, receptor-binding properties, and postulated immune cell regulatory functions remain intact.

Amino Acid Sequence↗
PubMed1995

Repair of x-ray-induced DNA double-strand breaks in specific Not I restriction fragments in human fibroblasts: joining of correct and incorrect ends.

An assay that allows measurement of absolute induction frequencies for DNA double-strand breaks (dsbs) in defined regions of the genome and that quantitates rejoining of correct DNA ends has been used to study repair of dsbs in normal human fibroblasts after x-irradiation. The approach involves hybridization of single-copy DNA probes to Not I restriction fragments separated according to size by pulsed-field gel electrophoresis. Induction of dsbs is quantitated from the decrease in the intensity of the hybridizing restriction fragment and an accumulation of a smear below the band. Rejoining of dsbs results in reconstitution of the intact restriction fragment only if correct DNA ends are joined. By comparing results from this technique with results from a conventional electrophoresis assay that detects all rejoining events, it is possible to quantitate the misrejoining frequency. Three Not I fragments on the long arm of chromosome 21 were investigated with regard to dsb induction, yielding an identical induction rate of 5.8 X 10(-3) break per megabase pair per Gy. Correct dsb rejoining was measured for two of these Not I fragments after initial doses of 80 and 160 Gy. The misrejoining frequency was about 25% for both fragments and was independent of dose. This result appears to be representative for the whole genome as shown by analysis of the entire Not I fragment distribution. The correct rejoining events primarily occurred within the first 2 h, while the misrejoining kinetics included a much slower component, with about half of the events occurring between 2 and 24 h. These misrejoining kinetics are similar to those previously reported for production of exchange aberrations in interphase chromosomes.

Cell Line↗
PubMed1995

The functional role of the domains of troponin-C investigated with thrombin fragments of troponin-C reconstituted into skinned muscle fibers.

Proteolysis of rabbit fast skeletal troponin-C (RSTnC) with thrombin produces four separate fragments containing the following Ca2+-binding site(s): TH1 (residues 1-120) sites I-III; TH2 (121-159) site IV; TH3 (1-100) sites I and II; and TH4 (101-120) site III. We studied the ability of these fragments to restore the steady state isometric force in TnC-depleted skinned skeletal muscle fibers. Interestingly, we found that all investigated fragments of RSTnC possessed some of the properties of native RSTnC, but none of them could fully regulate contraction in the fibers like intact RSTnC. TH1 was the most effective in the force restoration (65%) whereas the smaller fragments developed about 50% (TH3 and TH4) or 20% (TH2) of the initial force of unextracted fibers. Additionally, much higher concentrations of TH2, TH3, and TH4 compared to RSTnC OR TH1 were necessary for force development suggesting a decreased affinity of these fragments to their binding site(s) in the fibers. Like intact RSTnC, TH1 was able to interact with the fibers in a Ca(2+)-independent (Mg(2+)-dependent) manner, indicating that at a minimum, Ca(2+)-binding site III is required for this type of binding. The initial binding of the other fragments to the TnC-depleted fibers occurred only in the presence of Ca2+. TH2 and TH4 appeared to bind to two different binding sites in the fibers. The binding to one of the sites caused partial force restoration. This binding of TH2 and TH4 was abolished when Ca2+ was removed. TH2 and TH4 binding to the second site required Ca2+ initially but was maintained in the presence of Mg2+. This interaction of TH2 and TH4 partially blocked the rebinding of RSTnC to the fibers. The latter results suggest that site III and IV in these small fragments, when removed from the constraints of the parent protein, may assume conformations that allow them to function, to a certain extent, like both the regulatory sites (I and II) and the Ca(2+)-Mg2+ sites(III and IV) of TnC.

Animals↗
PubMed1995

Electrophoresis of chromatin on nondenaturing agarose gels containing Mg2+. Self-assembly of small chromatin fragments and folding of the 30-nm fiber.

We show that nondenaturing agarose gels can be used for the study of the structure and dynamic properties of native (uncross-linked) chromatin. In gels containing 1.7 mM Mg2+, chicken erythrocyte chromatin fragments having from about 6 to 50 nucleosomes produce well defined bands. These bands have an electrophoretic mobility that decreases only slightly with molecular weight. This surprising behavior is not observed in low ionic strength gels. Fragments with less than 6 nucleosomes and low content of histones H1-H5 give rise to broad bands in gels with Mg2+. In contrast, fragments containing only 3-4 nucleosomes but with the normal H1-H5 content are able to form associated structures with a mobility similar to that observed for high molecular weight chromatin. Electron microscopy results indicate that the associated fragments and the fragments of higher molecular weight show similar electrophoretic properties because they become very compact in the presence of Mg2+ and form cylindrical structures with a diameter of approximately 33 nm. Our results suggest that the interactions involved in the self-assembly of small fragments are the same that direct the folding of larger fragments; in both cases, the resulting compact chromatin structure is formed from a basic element containing 5-7 nucleosomes.

Animals↗
PubMed1999

Fragmentation of the large subunit of ribulose-1,5-bisphosphate carboxylase by reactive oxygen species occurs near Gly-329.

The large subunit (LSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) in the illuminated lysates of wheat (Triticum aestivum L.) chloroplasts is broken down by reactive oxygen radicals into 37- and 16-kDa polypeptides. Analysis of the terminal amino acid residues of both fragments revealed that the C terminus of the 37-kDa fragment was Ser-328 and the N terminus of the 16-kDa fragment was Thr-330. Gly-329, which links the two fragments, was missing, suggesting that the fragmentation of the LSU in the lysates driven by oxygen-free radicals occurs at Gly-329. Purified rubisco, exposed to a hydroxyl radical-generating system, was also cleaved at the same site of the LSU. The cleavage site was positioned at the N-terminal end of the flexible loop (loop 6) within the beta/alpha-barrel domain, constituting the catalytic site of rubisco. The binding of a reaction intermediate analogue, 2-carboxyarabinitol 1,5-bisphosphate, to the active form of rubisco completely protected the enzyme from the fragmentation. The fragmentation was differentially affected by CO2, Mg2+, ribulose 1, 5-bisphosphate, or 2-carboxyarabinitol 1,5-bisphosphate. All these results indicate that the conformation of the catalytic site of the enzyme is involved as an important factor determining the breakdown of rubisco by reactive oxygen species. Reactive oxygen species generated at its catalytic site by a Fenton-type reaction may trigger the site-specific degradation of the LSU in the lysates of chloroplasts in the light.

Amino Acid Sequence↗
PubMed2001

Roles of DNA fragmentation factor and poly(ADP-ribose) polymerase in an amplification phase of tumor necrosis factor-induced apoptosis.

During apoptosis, endonucleases cleave DNA into 50-300-kb fragments and subsequently into internucleosomal fragments. DNA fragmentation factor (DFF) is implicated in apoptotic DNA cleavage; this factor comprises DFF45 and DFF40 subunits, the former of which acts as a chaperone and inhibitor of the catalytic subunit and whose cleavage by caspase-3 results in DFF activation. Disruption of the DFF45 gene blocks internucleosomal DNA fragmentation and confers resistance to apoptosis in primary thymocytes. The role of DFF-mediated DNA fragmentation in apoptosis was investigated in primary fibroblasts from DFF45(-/-) and control (DFF45(+/+)) mice. DFF45 deficiency rendered fibroblasts resistant to apoptosis induced by tumor necrosis factor (TNF). TNF induced rapid cleavage of DNA into approximately 50-kb fragments in DFF45(+/+) fibroblasts but not in DFF45(-/-) cells, indicating that DFF mediates this initial step in DNA processing. The TNF-induced activation of poly(ADP-ribose) polymerase (PARP), which requires PARP binding to DNA strand breaks, and the consequent depletion of the PARP substrate NAD were markedly delayed in DFF45(-/-) cells, suggesting a role for DFF in PARP activation. The activation of caspase-3 and mitochondrial events important in apoptotic signaling, including the loss of mitochondrial membrane potential and the release of cytochrome c, induced by TNF were similarly delayed in DFF45(-/-) fibroblasts. DFF45(-/-) and DFF45(+/+) cells were equally sensitive to the DNA-damaging agent and PARP activator N-methyl-N'-nitro-N-nitrosoguanidine. Inhibition of PARP by 3-aminobenzamide partially protected DFF45(+/+) cells against TNF-induced death and inhibited the associated release of cytochrome c and activation of caspase-3. These results suggest that the generation of 50-kb DNA fragments by DFF, together with the activation of PARP, mitochondrial dysfunction, and caspase-3 activation, contributes to an amplification loop in the death process.

Animals↗
PubMed2002

The RasGAP N-terminal fragment generated by caspase cleavage protects cells in a Ras/PI3K/Akt-dependent manner that does not rely on NFkappa B activation.

RasGAP, a regulator of Ras GTPase family members, is cleaved at low levels of caspase activity into an N-terminal fragment (fragment N) that generates potent anti-apoptotic signals. At higher levels of caspase activity, fragment N is further cleaved into two fragments that strongly potentiate apoptosis. RasGAP could thus function as a sensor of caspase activity to determine whether a cell should survive or not. Here we show that fragment N protects cells by activating the Ras-PI3K-Akt pathway. Surprisingly, even though nuclear factor kappaB (NFkappaB) can be activated by Akt, it plays no role in the anti-apoptotic functions of fragment N. This indicates that Akt effectors are differentially regulated when fragment N is generated.

Apoptosis↗