Biomedical subjects

Valerian E Kagan

Publications and source records attributed to Valerian E Kagan.

At least 19 recordsLinked to original sources

PubMed2007

Nitrosative stress inhibits the aminophospholipid translocase resulting in phosphatidylserine externalization and macrophage engulfment: implications for the resolution of inflammation.

Macrophage recognition of apoptotic cells depends on externalization of phosphatidylserine (PS), which is normally maintained within the cytosolic leaflet of the plasma membrane by aminophospholipid translocase (APLT). APLT is sensitive to redox modifications of its -SH groups. Because activated macrophages produce reactive oxygen and nitrogen species, we hypothesized that macrophages can directly participate in apoptotic cell clearance by S-nitrosylation/oxidation and inhibition of APLT causing PS externalization. Here we report that exposure of target HL-60 cells to nitrosative stress inhibited APLT, induced PS externalization, and enhanced recognition and elimination of "nitrosatively" modified cells by RAW 264.7 macrophages. Using S-nitroso-L-cysteine-ethyl ester (SNCEE) and S-nitrosoglutathione (GSNO) that cause intracellular and extracellular trans-nitrosylation of proteins, respectively, we found that SNCEE (but not GSNO) caused significant S-nitrosylation/oxidation of thiols in HL-60 cells. SNCEE also strongly inhibited APLT, activated scramblase, and caused PS externalization. However, SNCEE did not induce caspase activation or nuclear condensation/fragmentation suggesting that PS externalization was dissociated from the common apoptotic pathway. Dithiothreitol reversed SNCEE-induced S-nitrosylation, APLT inhibition, and PS externalization. SNCEE but not GSNO stimulated phagocytosis of HL-60 cells. Moreover, phagocytosis of target cells by lipopolysaccharide-stimulated macrophages was significantly suppressed by an NO. scavenger, DAF-2. Thus, macrophage-induced nitrosylation/oxidation plays an important role in cell clearance, and hence in the resolution of inflammation.

Animals↗

PubMed2006

Structural requirements for optimized delivery, inhibition of oxidative stress, and antiapoptotic activity of targeted nitroxides.

Suppression of mitochondrial production of reactive oxygen species is a promising strategy against intrinsic apoptosis typical of degenerative diseases. Stable nitroxide radicals such as 4-hydroxy-2,2,6,6-tetramethyl piperidine-1-oxyl (TEMPOL) and its analogs combine several important features, including recycleability, electron acceptance from respiratory complexes, superoxide dismutase mimicry, and radical scavenging. Although successful in antioxidant protection, their effective concentrations are too high for successful in vivo applications. Recently (J Am Chem Soc 127:12460, 2005), we reported that 4-amino 2,2,6,6-tetramethyl-1-piperidinyloxy, covalently conjugated to a five-residue segment of gramicidin S (GS), was integrated into mitochondria and blocked actinomycin D (ActD)-induced superoxide generation and apoptosis. Using a model of ActD-induced apoptosis in mouse embryonic cells, we screened a library of nitroxides to explore structure-activity relationships between their antioxidant/antiapoptotic properties and chemical composition and three-dimensional (3D) structure. High hydrophobicity and effective mitochondrial integration are necessary but not sufficient for high antiapoptotic/antioxidant activity of a nitroxide conjugate. By designing conformationally preorganized peptidyl nitroxide conjugates and characterizing their 3D structure experimentally (circular dichroism and NMR) and theoretically (molecular dynamics), we established that the presence of the beta-turn/beta-sheet secondary structure is essential for the desired activity. Monte Carlo simulations in model lipid membranes confirmed that the conservation of the d-Phe-Pro reverse turn in hemi-GS analogs ensures the specific positioning of the nitroxide moiety at the mitochondrial membrane interface and maximizes their protective effects. These new insights into the structure-activity relationships of nitroxide-peptide and -peptide isostere conjugates are instrumental for development of new mechanism-based therapeutically effective agents.

Animals↗

PubMed2006

Oxidation and cytotoxicity of 6-OHDA are mediated by reactive intermediates of COX-2 overexpressed in PC12 cells.

Parkinson's disease is characterized by a progressive loss of dopaminergic neurons, likely associated with dysregulation of oxidation of catechols, such as dopamine (DA) and 6-hydroxydopamine (6-OHDA), and resulting in oxidative stress. The involvement of cyclooxygenase-2 (COX-2) in pathogenesis of Parkinson's disease has been suggested. However, specific COX-2 triggered mechanisms participating in catalysis of DA oxidation and enhanced catechol-induced cytotoxicity remain poorly characterized. Here, we demonstrate that in a model biochemical system, recombinant heme-reconstituted COX-2 induced oxidation of 6-OHDA in the course of its peroxidase (H(2)O(2)-dependent) and cyclooxygenase (arachidonic acid (AA)-dependent) catalytic half-cycles. Similarly, COX-2 was able to stimulate 6-OHDA oxidation during its peroxidase- and cyclooxygenase half-cycles and caused oxidative stress in homogenates of PC12 cells stably overexpressing the enzyme (but not in mock-transfected cells). In addition, the increased levels of COX-2 were associated with enhanced cytotoxicity of 6-OHDA in stably transfected PC12 cells. Finally, co-oxidation of 6-OHDA by COX-2 triggered production of superoxide radicals critical for both propagation of 6-OHDA oxidation and induction of oxidative stress in COX-2 overexpressing cells. Thus, we conclude that both peroxidase and cyclooxygenase half-cycles of COX-2-catalyzed reactions are essential for COX-2-dependent activation of 6-OHDA oxidation, oxygen radical production, oxidative stress, and cytotoxicity.

Animals↗

PubMed2006

Mechanisms of cardiolipin oxidation by cytochrome c: relevance to pro- and antiapoptotic functions of etoposide.

Execution of apoptotic program in mitochondria is associated with accumulation of cardiolipin peroxidation products required for the release of proapoptotic factors into the cytosol. This suggests that lipid antioxidants capable of inhibiting cardiolipin peroxidation may act as antiapoptotic agents. Etoposide, a widely used antitumor drug and a topoisomerase II inhibitor, is a prototypical inducer of apoptosis and, at the same time, an effective lipid radical scavenger and lipid antioxidant. Here, we demonstrate that cardiolipin oxidation during apoptosis is realized not via a random cardiolipin peroxidation mechanism but rather proceeds as a result of peroxidase reaction in a tight cytochrome c/cardiolipin complex that restrains interactions of etoposide with radical intermediates generated in the course of the reaction. Using low-temperature and ambient-temperature electron paramagnetic resonance spectroscopy of H(2)O(2)-induced protein-derived (tyrosyl) radicals and etoposide phenoxyl radicals, respectively, we established that cardiolipin peroxidation and etoposide oxidation by cytochrome c/cardiolipin complex takes place predominantly on protein-derived radicals of cytochrome c. We further show that etoposide can inhibit cytochrome c-catalyzed oxidation of cardiolipin competing with it as a peroxidase substrate. Peroxidase reaction of cytochrome c/cardiolipin complexes causes cross-linking and oligomerization of cytochrome c. With nonoxidizable tetraoleoyl-cardiolipin, the cross-linking occurs via dityrosine formation, whereas bifunctional lipid oxidation products generated from tetralinoleoyl-cardiolipin participate in the production of high molecular weight protein aggregates. Protein aggregation is effectively inhibited by etoposide. The inhibition of cardiolipin peroxidation by etoposide, however, is realized at far higher concentrations than those at which it induces apoptotic cell death. Thus, oxidation of cardiolipin by the cytochrome c/cardiolipin peroxidase complex, which is essential for apoptosis, is not inhibited by proapoptotic concentrations of the drug.

Antineoplastic Agents, Phytogenic↗

PubMed2006

Peroxidase activity and structural transitions of cytochrome c bound to cardiolipin-containing membranes.

During apoptosis, cytochrome c (cyt c) is released from intermembrane space of mitochondria into the cytosol where it triggers the caspase-dependent machinery. We discovered that cyt c plays another critical role in early apoptosis as a cardiolipin (CL)-specific oxygenase to produce CL hydroperoxides required for release of pro-apoptotic factors [Kagan, V. E., et al. (2005) Nat. Chem. Biol. 1, 223-232]. We quantitatively characterized the activation of peroxidase activity of cyt c by CL and hydrogen peroxide. At low ionic strength and high CL/cyt c ratios, peroxidase activity of the CL/cyt c complex was increased >50 times. This catalytic activity correlated with partial unfolding of cyt c monitored by Trp(59) fluorescence and absorbance at 695 nm (Fe-S(Met(80)) band). The peroxidase activity increase preceded the loss of protein tertiary structure. Monounsaturated tetraoleoyl-CL (TOCL) induced peroxidase activity and unfolding of cyt c more effectively than saturated tetramyristoyl-CL (TMCL). TOCL/cyt c complex was found more resistant to dissociation by high salt concentration. These findings suggest that electrostatic CL/cyt c interactions are central to the initiation of the peroxidase activity, while hydrophobic interactions are involved when cyt c's tertiary structure is lost. In the presence of CL, cyt c peroxidase activity is activated at lower H(2)O(2) concentrations than for isolated cyt c molecules. This suggests that redistribution of CL in the mitochondrial membranes combined with increased production of H(2)O(2) can switch on the peroxidase activity of cyt c and CL oxidation in mitochondria-a required step in execution of apoptosis.

Acridine Orange↗

PubMed2006

Nitric oxide inhibits peroxidase activity of cytochrome c.cardiolipin complex and blocks cardiolipin oxidation.

The increased production of NO during the early stages of apoptosis indicates its potential involvement in the regulation of programmed cell death through yet to be identified mechanisms. Recently, an important role for catalytically competent peroxidase form of pentacoordinate cytochrome c (cyt c) in a complex with a mitochondria-specific phospholipid, cardiolipin (CL), has been demonstrated during execution of the apoptotic program. Because the cyt c.CL complex acts as CL oxygenase and selectively oxidizes CL in apoptotic cells in a reaction dependent on the generation of protein-derived (tyrosyl) radicals, we hypothesized that binding and nitrosylation of cyt c regulates CL oxidation. Here we demonstrate by low temperature electron paramagnetic resonance spectroscopy that CL facilitated interactions of ferro- and ferri-states of cyt c with NO and NO(-), respectively, to yield a mixture of penta- and hexa-coordinate nitrosylated cyt c. In the nitrosylated cyt c.CL complex, NO chemically reacted with H(2)O(2)-activated peroxidase intermediates resulting in their reduction. A dose-dependent quenching of H(2)O(2)-induced protein-derived radicals by NO donors was shown using direct electron paramagnetic resonance measurements as well as immuno-spin trapping with antibodies against protein 5,5-dimethyl-1-pyrroline N-oxide-nitrone adducts. In the presence of NO donors, H(2)O(2)-induced oligomeric forms of cyt c positively stained for 3-nitrotyrosine confirming the reactivity of NO toward tyrosyl radicals of cyt c. Interaction of NO with the cyt c.CL complex inhibited its peroxidase activity with three different substrates: CL, etoposide, and 3,3'-diaminobenzidine. Given the importance of CL oxidation in apoptosis, mass spectrometry analysis was utilized to assess the effects of NO on oxidation of 1,1'2,2'-tertalinoleoyl cardiolipin. NO effectively inhibited 1,1'2,2'-tertalinoleoyl cardiolipin oxidation catalyzed by the peroxidase activity of cyt c. Thus, NO can act as a regulator of peroxidase activity of cyt c.CL complexes.

Animals↗

PubMed2006

Moderate ascorbate deficiency increases myogenic tone of arteries from pregnant but not virgin ascorbate-dependent rats.

Plasma ascorbic acid is decreased in women with the pregnancy disorder preeclampsia. We used a mutant strain of rats (Osteogenic Disorder Shionogi), dependent on dietary sources of vitamin C, to investigate whether reduced intake of the vitamin would differentially affect vascular function in late-pregnant (day 19) and age-matched virgin rats. The animals were given either 1 mg/mL of ascorbic acid ad libitum in drinking water [fully supplemented (FS)] or 0.25 mg/mL [marginally supplemented (MS)]. Fetal weights were 21% lower in MS than FS rats, whereas mean maternal weights and pup numbers did not differ. Small mesenteric arteries (diameter, 268+/-7 microm) were mounted in a pressurized arteriograph. Myogenic reactivity (contractile response to step increases in intraluminal pressure) was increased in arteries from MS pregnant compared with FS pregnant rats to levels observed in virgin rats. Ascorbic acid intake did not affect myogenic responses of arteries from virgin rats. Hence, the normal pregnancy-induced reduction in myogenic reactivity was abrogated in MS pregnant animals. Inhibition of nitric oxide synthase had no effect on the myogenicity of arteries from virgin or MS pregnant rats but increased myogenicity of FS pregnant rats to the level of MS pregnant rats. Free radical scavengers reversed the accentuated myogenicity of MS pregnant rats without affecting FS pregnant or virgin rat arteries. These data indicate that moderate ascorbate deprivation increases mesenteric artery myogenic responsiveness during pregnancy. This increase may result from a decrease in nitric oxide-mediated modulation of the myogenic contractile response.

Administration, Oral↗

PubMed2006

Oxidative stress in immature brain after traumatic brain injury.

High oxygen demand along with the abundance of readily oxidizable substrates yielding productive oxidative metabolism are required for the normal function of the brain. This necessitates the existence of the complex and multicomponent antioxidant system in the brain for protection against oxidative damage. However, during development, individual components of the antioxidant system are not equally expressed and not always sufficient to fulfill their tasks in a coordinated way. As a result, the developing brain may be more vulnerable to oxidative insults than the adult brain. Traumatic brain injury is one of the damaging acute impacts that challenge the brain antioxidant reserves by exposing them to a number of decompartmentalized prooxidant molecules. This review focuses on the sources and assessment of oxidative stress and the link between oxidative stress and cell death pathways in the immature brain after experimental and clinical traumatic brain injury.

Animals↗

PubMed2006

Cell-surface protein disulfide isomerase is required for transnitrosation of metallothionein by S-nitroso-albumin in intact rat pulmonary vascular endothelial cells.

S-nitrosation of the metal binding protein, metallothionein (MT) appears to be a critical link in affecting endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS)-derived nitric oxide (NO)-induced changes in cytoplasmic and nuclear labile zinc, respectively. Although low molecular weight S-nitrosothiols also appear to affect this signaling system, less is known about the ability of extracellular protein nitrosothiols to transnitrosate MT. Accordingly, we synthesized fluorescently labeled S-nitroso-albumin (SNO-albumin, a major protein S-nitrosothiol in plasma) and determined, via confocal microscopy in fixed tissue, that it is transported into cultured rat pulmonary vascular endothelial cells in a temperature sensitive fashion. The cells were transfected with an expression vector that encodes human MT-IIa cDNA sandwiched between enhanced cyan (donor) and yellow (acceptor) fluorescent proteins (FRET-MT) that can detect conformational changes in MT through fluorescence resonance energy transfer (FRET). SNO-albumin and the membrane-permeant low molecular weight S-nitroso-l-cysteine ethyl ester (l-SNCEE) caused a conformational change in FRET-MT as ascertained by full spectral laser scanning confocal microscopy in live rat pulmonary vascular endothelial cells, a result which is consistent with transnitrosation of the reporter molecule. Transnitrosation of FRET-MT by SNO-albumin, but not l-SNCEE, was sensitive to antisense oligonucleotide-mediated inhibition of the expression of cell surface protein disulfide isomerase (csPDI). These results extend the original observations of Ramachandran et al. (Ramachandran N, Root P, Jiang XM, Hogg PJ, Mutus B. Proc Natl Acad Sci U S A 98: 9539-9544, 2001) and suggest that csPDI-mediated denitrosation helps to regulate the ability of the major plasma NO carrier (SNO-albumin) to transnitrosate endothelial cell molecular targets (e.g. MT).

Animals↗

PubMed2005

Thioredoxin and lipoic acid catalyze the denitrosation of low molecular weight and protein S-nitrosothiols.

The nitrosation of cellular thiols has attracted much interest as a regulatory mechanism that mediates some of the pathophysiological effects of nitric oxide (NO). In cells, virtually all enzymes contain cysteine residues that can be subjected to S-nitrosation, whereby this process often acts as an activity switch. Nitrosation of biological thiols is believed to be mediated by N2O3, metal-nitrosyl complexes, and peroxynitrite. To date, however, enzymatic pathways for S-denitrosation of proteins have not been identified. Herein, we present experimental evidence that two ubiquitous cellular dithiols, thioredoxin and dihydrolipoic acid, catalyze the denitrosation of S-nitrosoglutathione, S-nitrosocaspase 3, S-nitrosoalbumin, and S-nitrosometallothionenin to their reduced state with concomitant generation of nitroxyl (HNO), the one-electron reduction product of NO. In these reactions, formation of NO and HNO was assessed by ESR spectrometry, potentiometric measurements, and quantification of hydroxylamine and sodium nitrite as end reaction products. Nitrosation and denitrosation of caspase 3 was correlated with its proteolytic activity. We also report that thioredoxin-deficient HeLa cells with mutated thioredoxin reductase denitrosate S-nitrosothiols less efficiently. We conclude that both thioredoxin and dihydrolipoic acid may be involved in the regulation of cellular S-nitrosothiols.

Caspase 3↗

PubMed2005

Neuroprotective effects of TEMPOL in central and peripheral nervous system models of Parkinson's disease.

TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) is a stable nitroxyl antioxidant. Previous studies have suggested that TEMPOL is protective in acute disorders thought to involve reactive oxygen species (ROS), such as ischemic stroke and cardiac reperfusion injury. Oxidized TEMPOL can be recycled to its redox-active reducing form by co-administration with polynitroxylated albumin, making it a candidate as a pharmacological "reservoir" for reducing potential of use in chronic disorders involving ROS. The present studies examine the efficacy of TEMPOL in cell culture and animal models of the central and peripheral dysfunction associated with Parkinson's disease, a disorder in the pathogenesis of which ROS generated from dopamine have been implicated. Antioxidants have been proposed as both preventive and symptomatic therapy for Parkinson's disease. TEMPOL protects MN9D dopaminergic mesencephalic cells in culture from 6-hydroxydopamine (6-OHDA)-induced apoptosis. Translocation of the p65 component of NF-kappaB to the nucleus accompanies protection by TEMPOL. In vivo, intraperitoneal TEMPOL protects mice from intrastriatal 6-OHDA-induced cell and dopamine metabolite loss in the striatum. TEMPOL also protects mice against the 6-OHDA-induced rotational behavior elicited by intrastriatal administration of d-amphetamine. In addition, TEMPOL protects mice from the ptosis, activity level decrement, and mortality induced by intraperitoneal administration of 6-OHDA, a model of autonomic dysfunction in Parkinson's disease. Adjunctive use of polynitroxylated albumin enhances the in vitro and in vivo effects of TEMPOL.

Active Transport, Cell Nucleus↗

PubMed2005

Orphan nuclear receptor pregnane X receptor sensitizes oxidative stress responses in transgenic mice and cancerous cells.

Efficient handling of oxidative stress is critical for the survival of organisms. The orphan nuclear receptor pregnane X receptor (PXR) is important in xenobiotic detoxification through its regulation of phase I and phase II drug-metabolizing/detoxifying enzymes and transporters. In this study we unexpectedly found that the expression of an activated human PXR in transgenic female mice resulted in a heightened sensitivity to paraquat, an oxidative xenobiotic toxicant. Heightened paraquat sensitivity was also seen in wild-type mice treated with the mouse PXR agonist pregnenolone-16alpha-carbonitrile. The PXR-induced paraquat sensitivity was associated with decreased activities of superoxide dismutase and catalase, enzymes that scavenge superoxide and hydrogen peroxide, respectively. Paradoxically, the general expression and activity of glutathione S-transferases, a family of phase II enzymes that detoxify electrophilic and cytotoxic substrates, was also induced in the transgenic mice. PXR regulates glutathione S-transferase expression in an isozyme-, tissue-, and sex-specific manner, and this regulation is independent of the nuclear factor-erythroid 2 p45-related factor 2/Kelch-like Ech-associated protein 1 pathway. In cell cultures, expression of activated human PXR sensitizes the cancerous colon and liver cells to the cytotoxic effect of paraquat, which is associated with an increased production of the reactive oxygen species. The current study reveals a novel function of PXR in the mammalian oxidative stress response, and this regulatory pathway may be implicated in carcinogenesis by sensitizing normal and cancerous tissues to oxidative cellular damage.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Mitochondrial targeting of selective electron scavengers: synthesis and biological analysis of hemigramicidin-TEMPO conjugates.

Synthetic hemigramicidin S-peptidyl TEMPO conjugates are effectively delivered into cells and mitochondria, where they act as electron scavengers and exert protection against apoptosis. Our delivery approach is based on the use of specific structural signaling features recognizable by cells as mitochondria targeting sequences and offers considerable therapeutic anti-apoptotic potential.

Animals↗

PubMed2005

Novel predictors of overt nephropathy in subjects with type 1 diabetes. A nested case control study from the Pittsburgh Epidemiology of Diabetes Complications cohort.

BACKGROUND: Predictors of diabetic nephropathy are only partly known and traditional risk factors do not adequately explain disease risk. We thus examined novel risk factors for overt nephropathy (ON) in type 1 diabetes. METHODS: The EDC is a prospective study of childhood-onset type 1 diabetes. When first seen (1986-1988), mean age was 28 and diabetes duration 19 years. In the subsequent 10 years, 56 of 485 subjects without ON in 1986-88 developed ON. An age, duration (+/-3 years), and sex-matched control was identified for 47 cases. Forty-two matched pairs had available stored plasma samples obtained prior to ON onset in cases, and complete standard risk factor data. RESULTS: Cases had a higher baseline albumin excretion rate (AER), HbA1, pulse rate, non-HDL cholesterol, fibrinogen, small LDL and lower eGDR and LDL particle size compared to controls (all P values<0.05). Multiple measures of immune complexes were increased in cases (P<0.05), whereas borderline elevations were seen for total antioxidant reserve (P=0.06) and retinol (P=0.08). Multivariably, other than AER, LDL particle size and IgG-IC were predictive beyond the standard predictors. CONCLUSION: Besides AER, the immunecomplexes and lipoprotein subclasses may provide additional information in the assessment of ON risk in type 1 diabetes.

Adolescent↗

PubMed2005

Cytochrome c acts as a cardiolipin oxygenase required for release of proapoptotic factors.

Programmed death (apoptosis) is turned on in damaged or unwanted cells to secure their clean and safe self-elimination. The initial apoptotic events are coordinated in mitochondria, whereby several proapoptotic factors, including cytochrome c, are released into the cytosol to trigger caspase cascades. The release mechanisms include interactions of B-cell/lymphoma 2 family proteins with a mitochondria-specific phospholipid, cardiolipin, to cause permeabilization of the outer mitochondrial membrane. Using oxidative lipidomics, we showed that cardiolipin is the only phospholipid in mitochondria that undergoes early oxidation during apoptosis. The oxidation is catalyzed by a cardiolipin-specific peroxidase activity of cardiolipin-bound cytochrome c. In a previously undescribed step in apoptosis, we showed that oxidized cardiolipin is required for the release of proapoptotic factors. These results provide insight into the role of reactive oxygen species in triggering the cell-death pathway and describe an early role for cytochrome c before caspase activation.

Animals↗

PubMed2005

Bcl-2-mediated potentiation of neocarzinostatin-induced apoptosis: requirement for caspase-3, sulfhydryl groups, and cleavable Bcl-2.

Overexpression of antiapoptotic Bcl-2 family members is thought to contribute to chemotherapeutic resistance of neural crest tumors. Paradoxical potentiation by Bcl-2 of apoptosis induced by the antineoplastic prodrug, neocarzinostatin (NCS), has been observed in PC12 pheochromocytoma cells. Prior studies have indicated that the cleavage of Bcl-2 to its proapoptotic counterpart mediated by caspase-3 is responsible for this potentiation of apoptosis. This has led to the hypothesis that induction of caspase-3 expression in bcl-2-transfected, caspase-3-deficient MCF-7 cells, will result in Bcl-2 cleavage and Bcl-2-dependent potentiation of NCS-induced apoptosis. These studies have further led to the hypothesis that both cleavable Bcl-2 and sulfhydryl groups are required for the activity of caspase-3 in this regard. As hypothesized, co-transfection of bcl-2-transfected MCF-7 cells with a caspase-3 expression construct results in cleavage of Bcl-2 and potentiation of dose-dependent, NCS-mediated cell death. Furthermore, PC12 cells transfected with an expression construct for cleavage-resistant Bcl-2 demonstrated attenuated potentiation of apoptosis relative to their counterparts transfected with wild-type bcl-2. Finally, irreversible oxidative titration of sulfhydryl groups resulted in concentration-dependent attenuation of apoptosis in PC12 cells, along with prevention of caspase-3 activation and Bcl-2 cleavage. These results definitively demonstrate the requirement for caspase-3, cleavable Bcl-2, and available sulfhydryl groups (separate from those required for NCS activation) in potentiation of NCS-induced apoptosis by Bcl-2.

Animals↗

PubMed2005

Unusual inflammatory and fibrogenic pulmonary responses to single-walled carbon nanotubes in mice.

Single-walled carbon nanotubes (SWCNT) are new materials of emerging technological importance. As SWCNT are introduced into the life cycle of commercial products, their effects on human health and environment should be addressed. We demonstrated that pharyngeal aspiration of SWCNT elicited unusual pulmonary effects in C57BL/6 mice that combined a robust but acute inflammation with early onset yet progressive fibrosis and granulomas. A dose-dependent increase in the protein, LDH, and gamma-glutamyl transferase activities in bronchoalveolar lavage were found along with accumulation of 4-hydroxynonenal (oxidative biomarker) and depletion of glutathione in lungs. An early neutrophils accumulation (day 1), followed by lymphocyte (day 3) and macrophage (day 7) influx, was accompanied by early elevation of proinflammatory cytokines (TNF-alpha, IL-1beta; day 1) followed by fibrogenic transforming growth factor (TGF)-beta1 (peaked on day 7). A rapid progressive fibrosis found in mice exhibited two distinct morphologies: 1) SWCNT-induced granulomas mainly associated with hypertrophied epithelial cells surrounding SWCNT aggregates and 2) diffuse interstitial fibrosis and alveolar wall thickening likely associated with dispersed SWCNT. In vitro exposure of murine RAW 264.7 macrophages to SWCNT triggered TGF-beta1 production similarly to zymosan but generated less TNF-alpha and IL-1beta. SWCNT did not cause superoxide or NO.production, active SWCNT engulfment, or apoptosis in RAW 264.7 macrophages. Functional respiratory deficiencies and decreased bacterial clearance (Listeria monocytogenes) were found in mice treated with SWCNT. Equal doses of ultrafine carbon black particles or fine crystalline silica (SiO2) did not induce granulomas or alveolar wall thickening and caused a significantly weaker pulmonary inflammation and damage.

Animals↗

PubMed2005

An epigrammatic (abridged) recounting of the myriad tales of astonishing deeds and dire consequences pertaining to nitric oxide and reactive oxygen species in mitochondria with an ancillary missive concerning the origins of apoptosis.

Mitochondria play a central role in the life and death of cells. These organelles serve as the major energy-producing power-house, whereby the generation of ATP is associated with the utilization of molecular oxygen. A significant fraction (2-3%) of molecular oxygen consumed by mitochondria may be reduced in a one-electron fashion to yield a series of reactive oxygen species (ROS) such as superoxide anion radical, hydrogen peroxide, and hydroxyl radical. ROS are capable of damaging components of the electron transport apparatus and can, in turn, disrupt mitochondrial functioning, limiting cellular ATP levels and ultimately resulting in cell death. ROS-induced disruption of electron transport can perpetuate production of deleterious ROS and propagate mitochondrial damage. Consequently, mitochondria are highly enriched with water-soluble and lipid-soluble antioxidants (glutathione, ascorbate, Vitamin E, and coenzyme Q) and antioxidant enzymes, such as superoxide dismutase, glutathione peroxidase, catalase, thioredoxins, and peroxiredoxin. Another important antioxidant acting as a very effective scavenger of reactive lipid (peroxyl, alkoxyl) radicals is nitric oxide (NO) generated by mitochondrial nitric oxide synthase. However, NO can also be very disruptive to mitochondria function, a process facilitated by its high reactivity with superoxide. This interaction results in the formation of peroxynitrite, an oxidant capable of causing oxidative/nitrosative stress, further aggravating mitochondrial dysfunction, causing ATP depletion and damage to cells. Thus, in the most general sense, the effects of NO in mitochondria may be either protective or deleterious depending on specific conditions of local redox environment (redox potential, ratio of oxidized to reduced glutathione, transition metals, and the presence of other oxygen- and nitrogen-centered radicals).

Animals↗