Microencapsulation of linoleic acid with low- and high-molecular-weight components of soluble soybean polysaccharide and its oxidation process

Soluble soybean polysaccharide (SSPS) was fractionated into its low- (LMW) and high-molecular-weight (HMW) components to test their antioxidative and emulsifying properties. Linoleic acid was emulsified with an aqueous solution of SSPS, HMW, a mixture of LMW or HMW with maltodextrin, or maltodextrin alone. The emulsions prepared with SSPS, HWM and the mixture of HMW with maltodextrin were stable. These emulsions were spay-dried to produce microcapsules. The encapsulated linoleic acid was oxidized at 37 degrees C and at various levels of relative humidity. Linoleic acid encapsulated with the mixture of LMW with maltodextrin or HMW was stable to oxidation, and this stability increased as the weight fraction of LMW in the mixture was increased. The LMW components also had high DPPH-radical scavenging activity. These results indicate that LMW played an important role in suppressing or retarding the oxidation of linoleic acid encapsulated with SSPS. The oxidative stability of linoleic acid encapsulated with a mixture of the LMW and HMW components was high at low and high relative humidity, but not at intermediate levels of relative humidity.     

Oxidation of linoleic acid encapsulated with soluble soybean polysaccharide by spray-drying

Linoleic acid was encapsulated with a soluble soybean polysaccharide, gum arabic, or a mixture of both together with maltodextrin, and the oxidation process of the encapsulated acid was measured at 37 degrees C and at a relative humidity of 12%. The soybean polysaccharide was more effective for encapsulating the acid and suppressing the oxidation of the encapsulated acid than gum arabic. A mixture of the soybean polysaccharide and maltodextrin was also effective for this purpose when the weight fraction of the polysaccharide was equal to or greater than 0.75.     

Spray-Dried Gliadin Powders Inclusive of Linoleic Acid (Microcapsules): Their Preservability,Digestibility and Application to Bread Making

An aqueous-alcohol solution containing gliadin (or zein) and linoleic acid was pulverized by spray-drying and the powders were examined for their stability, digestibility and application. Linoleic acid in the powders was kept stable during three months’ storage in each case of low, moderate and high water activity. When the spray-dried powders were treated with such digestive enzymes as pepsin and pancreatin, linoleic acid was effectively changed into the hexane-extractable form concomitantly with the digestion of gliadin. The powders were administered to rats, instead of gluten and essential fatty acid in a semi-purified diet, and there was no abnormal excretion of fatty acid into the faeces. These facts indicate that two components of the powders could be digested and absorbed in the gut as usual. On the other hand, supplementation of bread with gliadin and linoleic acid in the form of spray-dried powders led to a considerable increase in bulk after baking, without affecting the taste and flavor. Linoleic acid in a loaf of bread seemed to be fairly resistlant to peroxidation during baking in an oven and during storage under aerobic conditions for a week.     

Nuclear activities of basic fibroblast growth factor: potentiation of low-serum growth mediated by natural or chimeric nuclear localization signals

Human basic fibroblast growth factor (FGF-2) occurs in four isoforms: a low molecular weight (LMW FGF-2, 18 kDa) and three high molecular weight (HMW FGF-2, 22, 22.5, and 24 kDa) forms. LMW FGF-2 is primarily cytoplasmic and functions in an autocrine manner, whereas HMW FGF-2s are nuclear and exert activities through an intracrine, perhaps nuclear, pathway. Selective overexpression of HMW FGF-2 forms in fibroblasts promotes growth in low serum, whereas overexpression of LMW FGF-2 does not. The HMW FGF-2 forms have two functional domains: an amino-terminal extension and a common 18-kDa amino acid sequence. To investigate the role of these regions in the intracrine signaling of HMW FGF-2, we produced stable transfectants of NIH 3T3 fibroblasts overexpressing either individual HMW FGF-2 forms or artificially nuclear-targeted LMW FGF-2. All of these forms of FGF-2 localize to the nucleus/nucleolus and induce growth in low serum. The nuclear forms of FGF-2 trigger a mitogenic stimulus under serum starvation conditions and do not specifically protect the cells from apoptosis. These data indicate the existence of a specific role for nuclear FGF-2 and suggest that LMW FGF-2 represents the biological messenger in both the autocrine/paracrine and intracrine FGF-2 pathways.     

Chlorohydrin formation from unsaturated fatty acids reacted with hypochlorous acid.

Stimulated neutrophils produce hypochlorous acid (HOCl) via the myeloperoxidase-catalyzed reaction of hydrogen peroxide with chloride. The reactions of HOCl with oleic, linoleic, and arachidonic acids both as free fatty acids or bound in phosphatidylcholine have been studied. The products were identified by gas chromatography-mass spectrometry of the methylated and trimethylsilylated derivatives. Oleic acid was converted to the two 9,10-chlorohydrin isomers in near stoichiometric yield. Linoleic acid, at low HOCl:fatty acid ratios, yielded predominantly a mixture of the four possible monochlorohydrin isomers. Bischlorohydrins were also formed, in increasing amounts at higher HOCl concentrations. Arachidonic acid gave a complex mixture of mono- and bischlorohydrins, the relative proportions depending on the amount of HOCl added. Linoleic acid appears to be slightly more reactive than oleic acid with HOCl. Reactions of oleic and linoleic acids with myeloperoxidase, hydrogen peroxide, and chloride gave chlorohydrin products identical to those with HOCl. Lipid chlorohydrins have received little attention as products of reactions of neutrophil oxidants. They are more polar than the parent fatty acids, and if formed in cell membranes could cause disruption to membrane structure. Since cellular targets for HOCl appear to be membrane constituents, chlorohydrin formation from unsaturated lipids could be significant in neutrophil-mediated cytotoxicity.     

Properties of wilt-toxins produced by Verticillium albo-atrum Reinke & Berth.

Cultures of Verticillium albo-atrum grown in a liquid, mineral salts/sucrose medium, were harvested at daily intervals and subjected to gel-filtration on Sephadex G-25 columns eluted with demineralized water. Ultraviolet scanning of the eluate indicated a broad division into high and low molecular weight fractions (HMW and LMW). The HMW material was found to be a variable mixture of a protein component (M.W. c. 100,000) possessing mild cellulase activity, and fructosan (M.W. 5,000–10,000). Both components produced wilting in a lucerne detached-leaf bioassay. Heat denaturation resulted only in the loss of the protein component with a corresponding reduction in wilt-toxicity. A number of LMW components were found to produce leaf necrosis, but only in high concentrations and long-duration assays. These were considered to be of doubtful significance in the wilt syndrome. The possible action mechanisms and the basis of resistance are discussed.     

Adiponectin Oligomers and Ectopic Fat in Liver and Skeletal Muscle in Humans

We aimed at determining which circulating forms of the adipokine adiponectin that increases lipid oxidation in liver and skeletal muscle are related to ectopic fat in these depots in humans. Plasma total‐, high‐molecular weight (HMW)‐, middle‐molecular weight (MMW)‐, and low‐molecular weight (LMW) adiponectin were quantified by an enzyme‐linked immunosorbent assay. Their relationships with liver‐ and intramyocellular fat, measured using ¹H magnetic resonance spectroscopy, were investigated in 54 whites without type 2 diabetes. Liver fat, adjusted for gender, age, and total body fat, was associated only with HMW adiponectin (r = ?0.35, P = 0.012), but not with total‐, MMW‐, or LMW adiponectin. In addition, subjects with fatty liver (liver fat ≥5.56%, n = 15) had significantly lower HMW‐ (P = 0.04), but not total‐, MMW‐, or LMW adiponectin levels, compared to controls (n = 39). Similarly, intramyocellular fat correlated only with HMW (r = ?0.32, P = 0.039), but not with the other circulating forms of adiponectin. These data indicate that, among circulating forms of adiponectin, HMW is strongly related to ectopic fat, thus possibly representing the form of adiponectin regulating lipid oxidation in liver and skeletal muscle.     

Preparation of a Water-in-oil-in-water (W/O/W) Type Microcapsules by a Single-droplet-drying Method and Change in Encapsulation Efficiency of a Hydrophilic Substance during Storage

Microcapsules of a water-in-oil-in-water (W/O/W) emulsion, which contained a hydrophilic substance, 1,3,6,8-pyrenetetrasulfonic acid tetrasodium salt (PTSA), in its inner aqueous phase, was prepared by hot-air-drying or freeze-drying the emulsion using a single-droplet-drying method. Pullulan, maltodextrin, or gum arabic was used as a wall material, and the oily phase was tricaprylin, oleic acid, olive oil, or a mixture of tricaprylin and olive oil. An encapsulation efficiency higher than 0.95 was reached except for the microcapsules prepared using gum arabic and oleic acid. The hot-air-dried microcapsules were generally more stable than the freeze-dried microcapsules at 37°C and various relative humidities. The stability was higher for the microcapsules with tricaprylin as the oily phase than for the microcapsules with oleic acid. The higher stability of the microcapsules with tricaprylin would be ascribed to the lower partition coefficient of PTSA to the oily phase. There was a tendency for the stability to be higher at lower relative humidity for both the hot-air- and freeze-dried microcapsules. The volumetric fraction of olive oil in its mixture with tricaprylin did not significantly affect either the encapsulation efficiency or the stability of the hot-air-dried microcapsules.     

Preparation of a water-in-oil-in-water (W/O/W) type microcapsules by a single-droplet-drying method and change in encapsulation efficiency of a hydrophilic substance during storage

Microcapsules of a water-in-oil-in-water (W/O/W) emulsion, which contained a hydrophilic substance, 1,3,6,8-pyrenetetrasulfonic acid tetrasodium salt (PTSA), in its inner aqueous phase, was prepared by hot-air-drying or freeze-drying the emulsion using a single-droplet-drying method. Pullulan, maltodextrin, or gum arabic was used as a wall material, and the oily phase was tricaprylin, oleic acid, olive oil, or a mixture of tricaprylin and olive oil. An encapsulation efficiency higher than 0.95 was reached except for the microcapsules prepared using gum arabic and oleic acid. The hot-air-dried microcapsules were generally more stable than the freeze-dried microcapsules at 37 degrees C and various relative humidities. The stability was higher for the microcapsules with tricaprylin as the oily phase than for the microcapsules with oleic acid. The higher stability of the microcapsules with tricaprylin would be ascribed to the lower partition coefficient of PTSA to the oily phase. There was a tendency for the stability to be higher at lower relative humidity for both the hot-air- and freeze-dried microcapsules. The volumetric fraction of olive oil in its mixture with tricaprylin did not significantly affect either the encapsulation efficiency or the stability of the hot-air-dried microcapsules.     

A dual, concentration-dependent absorption mechanism of linoleic acid by rat jejunum in vitro.

Linoleic acid absorption was studied using everted rat jejunal sacs. At low concentrations (42-1260 microM), the relationship between linoleic acid concentration and its absorption rate fitted best to a rectangular hyperbola. At high concentrations (2.5-4.2 mM) the relationship between the two parameters was linear. The separate additions of 2,4-dinitrophenol, cyanide, or azide, or decrease in the incubation temperature from 37 to 20 degrees C did not change the absorption rate of linoleic acid. Absorption rate of linoleic acid at low concentrations increased as the hydrogen ion and taurocholate concentrations were increased or as the unstirred water layer thickness was decreased. Linoleic acid absorption rate was decreased after the additions of lecithin, oleic, linolenic, and arachidonic acids or the substitution of taurocholate with the nonionic surfactant Pluronic F 68. These observations indicate that a concentration-dependent, dual mechanism of transport is operative in linoleic acid absorption. Facilitated diffusion is the predominant mechanism of absorption at low concentrations, while at high concentrations, simple diffusion is predominant. At low concentrations, the absorption rate of linoleic acid is influenced by the pH, surfactant type and concentration, the simultaneous presence of other polyunsaturated fatty acids, and the thickness of the unstirred water layer.     

Selective channelling of arachidonic and linoleic acids into glycerolipids of rat hepatocytes in primary culture.

Rat hepatocytes in primary culture were incubated with a mixture of linoleic and arachidonic acid at various total fatty acid/serum albumin molar ratios. Mixed fatty acids were taken up at the same rate and distributed with the same pattern as fatty acids added separately. The rates of total uptake, incorporation into hepatocyte and secreted triacylglycerols and beta-oxidation were linearly related to the fatty acid/albumin ratios, whereas the rate of incorporation into phospholipids was saturable. Neither the uptake rate nor the distribution of both fatty acids considered together varied with the arachidonic acid/linoleic acid molar ratio. Changes in this ratio and in the uptake rate led to significant variations in the respective fate of the fatty acids. The preferential channelling of arachidonic acid versus linoleic acid into beta-oxidation and phosphatidylinositol was greatest at a low uptake rate and then decreased as the uptake rose. Conversely, the preferential channelling of arachidonic acid versus linoleic acid into phosphatidylcholine, but not phosphatidylethanolamine, increased with the uptake rate. Moreover, both arachidonic acid and linoleic acid were preferentially incorporated into the 1-palmitoyl molecular species of phosphatidylcholine and phosphatidylethanolamine at a low uptake rate, and of phosphatidylcholine at a high uptake rate. This could be related to the synthesis of biliary phosphatidylcholine, of which 1-palmitoyl-2-linoleoyl and 1-palmitoyl-2-arachidonoyl are the main molecular species. Linoleic and arachidonic acid were selectively distributed into distinct metabolic pools of triacylglycerol, the intrahepatocyte pool which preferentially incorporated linoleic acid at a low uptake rate and the secreted pool in which the relative enrichment of arachidonic acid increased with the uptake rate. This strengthens the central role of hepatic secretion in the supply of arachidonic acid to peripheral tissues.     

Control of antibody high and low molecular weight species by depth filtration-based cell culture harvesting

Depth filtration-based harvesting is widely used in mAb manufacturing to remove cell and process-related impurities. However, it has not been studied on control of product-related impurities, which are very critical for product quality. In this article, we studied the interactions of depth filter with high and low molecular weight species (HMWs and LMWs) for their direct removal from cell culture. The process parameters (filter, loading, temperature, and flux) were evaluated for adsorption of HMWs and LMWs by depth filters. The adsorption is significantly dependent on filter media and loading capacity and is mainly on the basis of hydrophobic interaction during harvesting. The HMW and LMW species were characterized as HMW1, HMW2, LMW1, and LMW2. The increasing binding from LMW2 to LMW1, HMW1, and HMW2 is correlated with their increasing hydrophobicity score. Adsorption using enriched HMW sample demonstrated similar total protein binding capacity (36–40 g/m²) between depth filters D0HC and X0HC. However, X0HC has stronger HMW binding than D0HC (71% vs 43% of bound protein), indicating more hydrophobic interaction in X0HC. HMW2 DBC on X0HC reached 12 g/m², similar to protein binding on hydrophobic interaction membrane adsorbers. Further study showed LMW can induce HMW formation. This study provides a critical understanding of HMW and LMW interaction with depth filters. The strategy of HMW and LMW control by depth filtration-based harvesting was implemented successfully in mAb manufacturing.     

Purification and characterization of rat low molecular weight kininogen

Low molecular weight (LMW) kininogen was isolated from pooled rat plasma by chromatography on DEAE-Sephadex A-50, CM-Sephadex C-50, Blue-Sepharose CL-6B, and Sephadex G-100. It was shown to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoelectrophoresis. The molecular weight of rat LMW kininogen was determined to be 72,000 by SDS-PAGE. The LMW kininogen contained 83.5% protein, 4.0% hexose, 5.5% hexosamine, and 2.7% sialic acid. Kinin liberated from LMW kininogen by trypsin treatment was identified as an Ile-Ser-bradykinin(T-kinin) by analysis involving ion exchange column chromatography on CM-Sephadex C-25 and high performance liquid chromatography on a reverse-phase column (ODS-120T). LMW kininogen formed kinin with rat submaxillary gland kallikrein, but the kinin liberated was only 14% of the total kinin content, that is, that released by trypsin. In order to determine the immunochemical properties of LMW kininogen, specific antiserum was prepared in rabbits. The antiserum cross-reacted with high molecular weight (HMW) kininogen, but spur formation was observed between the LMW and HMW kininogens. The kininogen level in rat plasma was estimated to be 433 microgram/ml by a quantitative single radial immunodiffusion test.     

Mapping of functional domains of human high molecular weight and low molecular weight kininogens using murine monoclonal antibodies

Thirty-four monoclonal antibodies directed against human high molecular weight (HMW) and low molecular weight (LMW) kininogens and their derivatives were obtained, and the specificities of the antibodies were assayed by enzyme-linked immunosorbent assay (ELISA). By use of HMW kininogen, kinin-free HMW kininogen, kinin-free and fragment 1.2 (fr 1.2) free HMW kininogen, fr 1.2-light chain of HMW kininogen, LMW kininogen, kinin-free LMW kininogen, heavy chain of LMW kininogen, and light chain of LMW kininogen, the monoclonal antibodies were characterized and classified into four groups: (A) 20 monoclonal antibodies reacting with only the heavy chain, a common region of HMW and LMW kininogens; each of these monoclonal antibodies possessed the specificity to domain 1 (2 monoclonal antibodies), domain 2 (2 monoclonal antibodies), domain 3 (7 monoclonal antibodies), and both domains 2 and 3 (7 monoclonal antibodies) of the heavy chain; (B) 7 monoclonal antibodies reacting with fr 1.2, a unique histidine-rich region; (C) 5 monoclonal antibodies reacting with the light chain of HMW kininogen; (D) 2 monoclonal antibodies reacting with the light chain of LMW kininogen. Two monoclonal antibodies in the first group (group A), designated HKG H7 and H12, effectively suppressed the thiol proteinase inhibitor activity of HMW kininogen to papain and calpains and of LMW kininogen to papain, but the others did not affect it. Further, all the monoclonal antibodies which recognized the fr 1.2 or light chain of HMW kininogen (groups B and C) suppressed the clotting activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Preatherosclerosis and adiponectin subfractions in obese adolescents

We evaluated total adiponectin, high-molecular weight (HMW), medium-molecular weight (MMW), low-molecular weight (LMW) adiponectin subfractions, clinical parameters, routine lab parameters, lipids, metabolic, inflammatory biomarkers, and intima-media thickness (IMT) of common carotid arteries in 70 obese juveniles and adolescents with preatherosclerosis and 55 normal weight controls of similar age and gender distribution. Compared with the controls, the obese probands had a significantly increased IMT (P < 0.001) and elevated ultra-sensitive C-reactive protein (P < 0.001) indicating early vascular burden. Total and HMW adiponectin were significantly decreased in the obese cohort. The ratio between HMW and total adiponectin was significantly decreased in obese probands whereas the LMW/total adiponectin ratio was increased. Overall, total-, HMW, and MMW adiponectin were significantly negatively correlated with carotid IMT. The HMW/total adiponectin ratio correlated significantly negatively, and the LMW/total adiponectin ratio significantly positively with the IMT. Furthermore, HMW adiponectin was significantly positively correlated with high-density lipoprotein (HDL)-cholesterol and serum apolipoprotein A1, and negatively with BMI, triglycerides, homeostatic model assessment (HOMA)-index, leptin, liver transaminases, and uric acid. This remained stable after controlling for gender. Multiple regression analysis of body measures and all other lab parameters showed the strongest correlation between HMW adiponectin and carotid IMT (beta = -0.35, P < 0.001). Taken together, our study provides the first evidence that preatherosclerosis in obese juveniles and adolescents is associated with altered subfractions of adiponectin, whereas after multiple testing the HMW subfraction showed a better correlation to IMT compared with total adiponectin.     

Mitoxantrone and ametantrone inhibit hydroperoxide-dependent initiation and propagation reactions in fatty acid peroxidation

The anthracenedione antineoplastic agents mitoxantrone and ametantrone are potent inhibitors of basal and drug-stimulated lipid peroxidation in a variety of subcellular systems (Kharasch, E. D., and Novak, R. F. (1983) J. Pharmacol. Exp. Ther. 226, 500-506). The mechanism by which these compounds function as antioxidants has been investigated using enzymic and chemical systems. Mitoxantrone and ametantrone inhibited NADPH-cytochrome P-450 reductase- and xanthine oxidase-catalyzed conjugated diene formation from linoleic acid in a concentration-dependent manner with half-maximal inhibition achieved at approximately 0.5 microM anthracenedione. Inhibition of linoleic acid peroxidation was not attributable to a decrease in P-450 reductase activity, hydroxyl radical scavenging, or iron chelation by the anthracenediones. Nonenzymic fatty acid peroxidation was also inhibited by the anthracenediones. Linoleic acid oxidation initiated by superoxide (ferrous iron autoxidation) or by hydroxyl radicals (Fenton's reagent) was diminished by mitoxantrone and ametantrone after a brief delay, suggesting an effect subsequent to activated oxygen-dependent initiation. In contrast, linoleic acid oxidation initiated by iron-dependent hydroperoxide decomposition was inhibited immediately. Reinitiation of linoleic acid oxidation in an anthracenedione-inhibited system was accomplished only by superoxide generation, but not by fatty acid hydroperoxide decomposition. These results suggest the anthracenediones diminished neither oxygen radical formation nor oxygen radical-dependent initiation of peroxidation. Rather, inhibition of fatty acid peroxidation by mitoxantrone and ametantrone results from the inhibition of hydroperoxide-dependent initiation and propagation reactions.     

Calcium-translocating and cardiotonic properties of oxidation products of linoleic acid

Calcium-translocating activity of linoleic acid and its lipoxygenase (linoleate: oxygen oxidoreductase; EC 1.13.11.12) metabolites or autoxidation products was determined in vitro by estimation of 45Ca transport from a bulk aqueous to a bulk organic phase. Fresh commercial linoleic acid, tested immediately after removal from a sealed vial, stimulated calcium translocation only at concentrations greater than 1 mM. In contrast, 45Ca translocation by linoleic acid exposed to air was detectable at 10 microM. Oxidation products of linoleic acid obtained either by incubation with lipoxygenase or by autoxidation were much less potent than the calcium ionophore A23187. The products obtained by enzymic oxidation of linoleic acid enhanced contractility in the Langendorff-perfused guinea pig heart up to 45% over control (at 3 X 10(-8) M). The inotropic response was transient with rapid onset and not affected by the beta-adrenergic antagonist, propranolol. The autoxidation products of linoleic acid increased cardiac contractility up to 43% at 10(-6) M. In contrast, fresh linoleic acid caused only a negative inotropic effect at 10(-8) to 3 X 10(-7) M, progressing to contracture at 10(-6) M. These findings suggest that conflicting reports on the cardiostimulant effect of linoleic acid may be due to varying levels of the autoxidation products. Linoleic acid metabolites in vivo may have a physiological role in myocardial function related to their Ca2+-ionophoric activity.     

Water-soluble inhibitor(s) of tumor respiration formed from ultraviolet-induced oxidation of linoleic and linolenic acids

Inhibition of Ehrlich ascites carcinoma respiration by aqueous extracts of oxidized linoleic or linolenic acid (aqueous emulsions UV-irradiated, 90 min) was associated entirely with relatively involatile compounds which were both thiobarbituric acid (TBA)-reactive and peroxidase-reactive. Inhibitory compounds were heat stable and migrated in thin-layer chromatography with aldehydes, "hydroperoxides," and TBA-reactive compounds. Peroxidase-catalyzed reduction of the "hydroperoxide" diminished the inhibition. At 4.7 x 10(-5) M "hydroperoxide" concentration, the residues from both linoleic and linolenic acid inhibited tumor oxygen consumption to a similar degree. However, at this concentration of "hydroperoxide" only the dried extract from linolenic acid was able to produce inhibition (100%) of aerobic glucose utilization by tumor cells. No glycolytic inhibition by the dried residue of oxidized linoleic acid was observed. At least 12 compounds (approximate chain length, 7C-13C) containing alpha,Beta-unsaturated carbonyl groups were isolated by gas-liquid chromatography (GLC) of dried extracts of oxidized linolenic acid. No single fraction inhibited tumor respiration, but the recombined mixture of all compounds caused complete respiratory inhibition of ascites tumor cells. Less material was required to inhibit oxygen consumption before than after GLC presumably because the more highly inhibitory components of the extract (along with "hydroperoxides" and TBA-reactive compounds) were lost during GLC. Extracts from oxidized linolenic acid were found to produce in all tumor cells cytoplasmic evaginations which were readily detected by phase microscopy.     

The contribution of apoptosis-inducing factor, caspase-activated DNase, and inhibitor of caspase-activated DNase to the nuclear phenotype and DNA degradation during apoptosis

We have assessed the contribution of apoptosis-inducing factor (AIF) and inhibitor of caspase-activated DNase (ICAD) to the nuclear morphology and DNA degradation pattern in staurosporine-induced apoptosis. Expression of D117E ICAD, a mutant that is resistant to caspase cleavage at residue 117, prevented low molecular weight (LMW) DNA fragmentation, stage II nuclear morphology, and detection of terminal deoxynucleotidyl transferase staining. However, high molecular weight (HMW) DNA fragmentation and stage I nuclear morphology remained unaffected. On the other hand, expression of either D224E or wild type ICAD had no effect on DNA fragmentation or nuclear morphology. In addition, both HMW and LMW DNA degradation required functional executor caspases. Interestingly, silencing of endogenous AIF abolished type I nuclear morphology without any effect on HMW or LMW DNA fragmentation. Together, these results demonstrate that AIF is responsible for stage I nuclear morphology and suggest that HMW DNA degradation is a caspase-activated DNase and AIF-independent process.