Lipid peroxidation generates reactive aldehydes, most notably hydroxynonenal (HNE), which covalently bind amino acid residue side chains leading to protein inactivation and insolubility. Specific adducts of lipid peroxidation have been demonstrated in intimate association with the pathological lesions of Alzheimer disease (AD), suggesting that oxidative stress is a major component of AD pathogenesis. Some HNE-protein products result in protein crosslinking through a fluorescent compound similar to lipofuscin, linking lipid peroxidation and the lipofuscin accumulation that commonly occurs in post-mitotic cells such as neurons. In this study, brain tissue from AD and control patients was examined by immunocytochemistry and immunoelectron microscopy for evidence of HNE-crosslinking modifications of the type that should accumulate in the lipofuscin pathway. Strong labeling of granulovacuolar degeneration (GVD) and Hirano bodies was noted but lipofuscin did not contain this specific HNE-fluorophore. These findings directly implicate lipid crosslinking peroxidation products as accumulating not in the lesions or the lipofuscin pathways, but instead in a distinct pathway, GVD, that accumulates cytosolic proteins.
fluorophore; granulovacuolar degeneration; Hirano body; hydroxynonenal; lipid peroxidation; lipofuscin; oxidative stress; protein cross-linking
Heart mitochondria, which, depending on their location within cardiomyofibers, are classified as either subsarcolemmal or interfibrillar, are the major sources of the high-energy compound, adenosine triphosphate. Physiological differences between these two populations are reflected by differences in the morphology of their cristae, with those of subsarcolemmal mitochondria being mostly lamelliform, and those of interfibrillar mitochondria being mostly tubular. What determines the configuration of cristae, not only in cardiac mitochondria but in mitochondria in general, is unclear. The morphology of cardiac mitochondria, as well as their physiology, is responsive to the exigencies posed by a large variety of pathological situations. Giant cardiac mitochondria make an appearance in certain types of cardiomyopathy and as a result of dietary, pharmacological, and toxicological manipulation; such megamitochondria probably arise by a combination of fusion and true growth. Some of these enlarged organelles occasionally contain a membrane-bound deposit of β-glycogen. Those giant mitochondria induced by experimental treatment usually can be restored to normal dimensions simply by supplying the missing nutrient or by deleting the noxious substance. In some conditions, such as endurance training and ischemia, the mitochondrial matrices become pale. Dense rods or plates are present in the outer compartment of mitochondria under certain conditions. Biochemical alterations in cardiac mitochondria appear to be important in heart failure. In aging, only interfibrillar mitochondria exhibit such changes, with the subsarcolemmal mitochondria unaffected. In certain heart afflictions, biochemical defects are not accompanied by obvious morphological transformations. Mitochondria clearly play a cardinal role in homeostasis of the heart.
We measured the loss of cardiac mitochondrial function related to aging in males of three rat strains presenting with different longevity and aging phenotypes: the Fischer 344 (F344), the Brown Norway (BN), and the hybrid F344×BN. The F344 rat has a short life span and a ∼45% decrease in coupled mitochondrial oxidation in the cardiac permeabilized fibers from the old rats compared with the young rats. Citrate synthase activity in the permeabilized fibers (mitochondrial content) did not change significantly with aging. The BN live longer compared with the F344 and have a 15%–18% loss of mitochondrial respiration in the aged rats compared with the young rats. The differences are not significant. In hybrids, more resistant to aging than are the BN and the F344, mitochondrial function is preserved during aging. The difference in longevity of the different strains is correlated with mitochondrial dysfunction in the heart, suggesting the importance of mitochondria in cardiac aging.
Aging; Mitochondrial; Oxidative phosphorylation; Rat; Strains
A distinct conformational transition from the α-helix-rich cellular prion protein (PrPC) into its β-sheet-rich pathological isoform (PrPSc) is the hallmark of prion diseases, a group of fatal transmissible encephalopathies that includes spontaneous and acquired forms. Recently, a PrPSc-like intermediate form characterized by the formation of insoluble aggregates and protease-resistant PrP species termed insoluble PrPC (iPrPC) has been identified in uninfected mammalian brains and cultured neuronal cells, providing new insights into the molecular mechanism(s) of these diseases. Here, we explore the molecular characteristics of the spontaneously formed iPrPC in cultured neuroblastoma cells expressing wild-type or mutant human PrP linked to two familial prion diseases. We observed that although PrP mutation at either residue 183 from Thr to Ala (PrPT183A) or at residue 198 from Phe to Ser (PrPF198S) affects glycosylation at both N-linked glycosylation sites, the T183A mutation that results in intracellular retention significantly increased the formation of iPrPC. Moreover, while autophagy is increased in F198S cells, it was significantly decreased in T183A cells. Our results indicate that iPrPC may be formed more readily in an intracellular compartment and that a significant increase in PrPT183A aggregation may be attributable to the inhibition of autophagy.
Prion protein; prion disease; insoluble prion protein; neuroblastoma cells; mutation; autophagy
Singlet oxygen is produced by absorption of red light by the phthalocyanine dye, Pc 4, followed by energy transfer to dissolved triplet oxygen. Mitochondria pre-incubated with Pc 4 were illuminated by red light and the damage to mitochondrial structure and function by the generated singlet oxygen was studied. At early illumination times (3–5 min. of red light exposure), state 3 respiration was inhibited (50%) while state 4 activity increased, resulting in effectively complete uncoupling. Individual complex activities were measured and only complex IV activity was significantly reduced and exhibited a dose response while the activities of electron transport complexes I, II and III were not significantly affected. Cyt c release was an increasing function of irradiation time with 30% being released following 5 min. of illumination. Mitochondrial expansion along with changes in the structure of the cristae were observed by transmission electron microscopy following 5 min. of irradiation with an increase of large vacuoles and membrane rupture occurring following more extensive exposures.
Singlet oxygen; Mitochondria; Cytochrome c oxidase; Electron transport complexes; Cytochrome c release; Photodynamic therapy
In Africa, infant susceptibility to Plasmodium falciparum malaria increases substantially as fetal hemoglobin (HbF) and maternal immune IgG disappear from circulation. During the first few months of life, however, resistance to malaria is evidenced by extremely low parasitemias, the absence of fever, and the almost complete lack of severe disease. This resistance has previously been attributed in part to poor parasite growth in HbF-containing red blood cells (RBCs). A specific role for maternal immune IgG in infant resistance to malaria has been hypothesized but not yet identified.
Methods and Findings
We found that P. falciparum parasites invade and develop normally in fetal (cord blood, CB) RBCs, which contain up to 95% HbF. However, these parasitized CB RBCs are impaired in their binding to human microvascular endothelial cells (MVECs), monocytes, and nonparasitized RBCs – cytoadherence interactions that have been implicated in the development of high parasite densities and the symptoms of malaria. Abnormal display of the parasite's cytoadherence antigen P. falciparum erythrocyte membrane protein-1 (PfEMP-1) on CB RBCs accounts for these findings and is reminiscent of that on HbC and HbS RBCs. IgG purified from the plasma of immune Malian adults almost completely abolishes the adherence of parasitized CB RBCs to MVECs.
Our data suggest a model of malaria protection in which HbF and maternal IgG act cooperatively to impair the cytoadherence of parasitized RBCs in the first few months of life. In highly malarious areas of Africa, an infant's contemporaneous expression of HbC or HbS and development of an immune IgG repertoire may effectively reconstitute the waning protective effects of HbF and maternal immune IgG, thereby extending the malaria resistance of infancy into early childhood.
The autosomal recessive kidney disease nephronophthisis (NPHP) constitutes the most frequent genetic cause of terminal renal failure in the first 3 decades of life. Ten causative genes (NPHP1–NPHP9 and NPHP11), whose products localize to the primary cilia-centrosome complex, support the unifying concept that cystic kidney diseases are “ciliopathies”. Using genome-wide homozygosity mapping, we report here what we believe to be a new locus (NPHP-like 1 [NPHPL1]) for an NPHP-like nephropathy. In 2 families with an NPHP-like phenotype, we detected homozygous frameshift and splice-site mutations, respectively, in the X-prolyl aminopeptidase 3 (XPNPEP3) gene. In contrast to all known NPHP proteins, XPNPEP3 localizes to mitochondria of renal cells. However, in vivo analyses also revealed a likely cilia-related function; suppression of zebrafish xpnpep3 phenocopied the developmental phenotypes of ciliopathy morphants, and this effect was rescued by human XPNPEP3 that was devoid of a mitochondrial localization signal. Consistent with a role for XPNPEP3 in ciliary function, several ciliary cystogenic proteins were found to be XPNPEP3 substrates, for which resistance to N-terminal proline cleavage resulted in attenuated protein function in vivo in zebrafish. Our data highlight an emerging link between mitochondria and ciliary dysfunction, and suggest that further understanding the enzymatic activity and substrates of XPNPEP3 will illuminate novel cystogenic pathways.
Ingestion of a blood meal by the female mosquito Anopheles gambiae (L., Diptera: Culicidae), results in a dramatic distention of the midgut epithelium. Here we report that these events correlate with a transient increase of actin mRNA and protein abundance. The newly synthesized actin may provide a pool of actin protein needed to remodel epithelial cell cytoarchitecture. We also document changes in midgut epithelial cell morphology. Upon blood ingestion, the columnar cells flatten accompanied by the loss of microvilli on the lumenal side and the unfolding of the labyrinth on the basal side. These changes correlate with the large increase of epithelial surface area needed to accommodate the blood meal. Actin gene expression, actin synthesis and cell morphology all return to the pre-feeding state by 24 h after blood intake.
actin; Anopheles gambiae; midgut; microvilli; blood feeding
While considerable evidence supports the causal relationship between increases in c-Myc (Myc) and cardiomyopathy as a part of a “fetal re-expression” pattern, the functional role of Myc in mechanisms of cardiomyopathy remains unclear. To address this, we developed a bitransgenic mouse that inducibly expresses Myc under the control of the cardiomyocyte-specific MHC promoter. In adult mice the induction of Myc expression in cardiomyocytes in the heart led to the development of severe hypertrophic cardiomyopathy followed by ventricular dysfunction and ultimately death from congestive heart failure. Mechanistically, following Myc activation, cell cycle markers and other indices of DNA replication were significantly increased suggesting that cell cycle-related events might be a primary mechanism of cardiac dysfunction. Furthermore, pathological alterations at the cellular level included alterations in mitochondrial function with dysregulation of mitochondrial biogenesis and defects in electron transport chain complexes I and III. These data are consistent with the known role of Myc in several different pathways including cell cycle activation, mitochondrial proliferation, and apoptosis, and indicate that Myc activation in cardiomyocytes is an important regulator of downstream pathological sequelae. Moreover, our findings indicate that the induction of Myc in cardiomyocytes is sufficient to cause cardiomyopathy and heart failure, and that sustained induction of Myc, leading to cell cycle re-entry in adult cardiomyocytes, represents a maladaptive response for the mature heart.
The C-transmembrane form of prion protein (CtmPrP) has been implicated in prion disease pathogenesis, but the factors underlying its biogenesis and cyotoxic potential remain unclear. Here we show that CtmPrP interferes with cytokinesis in cell lines where it is transported to the plasma membrane. These cells fail to separate following cell division, assume a variety of shapes and sizes, and contain multiple nuclei, some of which are pyknotic. Furthermore, the synthesis and transport of CtmPrP to the plasma membrane are modulated through a complex interaction between cis- and trans-acting factors and the endoplasmic reticulum translocation machinery. Thus, insertion of eight amino acids before or within the N region of the N signal peptide (N-SP) of PrP results in the exclusive synthesis of CtmPrP regardless of the charge conferred to the N region. Subsequent processing and transport of CtmPrP are modulated by specific amino acids in the N region of the N-SP and by the cell line of expression. Although the trigger for CtmPrP upregulation in naturally occurring prion disorders remains elusive, these data highlight the underlying mechanisms of CtmPrP biogenesis and neurotoxicity and reinforce the idea that CtmPrP may serve as the proximate cause of neuronal death in certain prion disorders.
Plasmodium sporozoites develop within oocysts residing in the mosquito midgut. Mature sporozoites exit the oocysts, enter the hemolymph, and invade the salivary glands. The circumsporozoite (CS) protein is the major surface protein of salivary gland and oocyst sporozoites. It is also found on the oocyst plasma membrane and on the inner surface of the oocyst capsule. CS protein contains a conserved motif of positively charged amino acids: region II-plus, which has been implicated in the initial stages of sporozoite invasion of hepatocytes. We investigated the function of region II-plus by generating mutant parasites in which the region had been substituted with alanines. Mutant parasites produced normal numbers of sporozoites in the oocysts, but the sporozoites were unable to exit the oocysts. In in vitro as well, there was a profound delay, upon trypsin treatment, in the release of mutant sporozoites from oocysts. We conclude that the exit of sporozoites from oocysts is an active process that involves the region II-plus of CS protein. In addition, the mutant sporozoites were not infective to young rats. These findings provide a new target for developing reagents that interfere with the transmission of malaria.
Malaria affects hundreds of millions of people, and kills at least 1 million children per year. The infective stages of the malaria parasites, named “sporozoites,” are found in the salivary gland of Anopheles mosquitoes, and are injected along with the saliva during blood feeding. From the skin, sporozoites enter the blood circulation and invade liver cells where the parasites multiply. When they exit the liver, these parasites infect blood cells and can cause severe symptoms. If ingested by mosquitoes, the blood-stage parasites continue their lifecycle in the insect stomach. Thousands of sporozoites are formed within a cyst-like structure (oocyst). The sporozoites come out of the oocyst and infect the salivary gland, where they remain until injected back into humans. Malaria parasites are increasingly resistant to drugs, mosquitoes are difficult to eliminate, and effective vaccines are not yet available. New tools to combat malaria are urgently needed. One exciting approach, although the application is in the distant future, is to release in endemic areas genetically modified mosquitoes that are resistant to parasite growth. This paper provides a new target for generating these “transmission-block” mosquitoes and shows that the exit of sporozoites from the oocysts is an active process that requires the enzymatic digestion of components of the oocyst wall. If these enzymes are inhibited in transgenic mosquitoes, sporozoites will never reach the salivary gland.
Most Apicomplexan parasites, including the human pathogens Plasmodium, Toxoplasma, and Cryptosporidium, actively invade host cells and display gliding motility, both actions powered by parasite microfilaments. In Plasmodium sporozoites, thrombospondin-related anonymous protein (TRAP), a member of a group of Apicomplexan transmembrane proteins that have common adhesion domains, is necessary for gliding motility and infection of the vertebrate host. Here, we provide genetic evidence that TRAP is directly involved in a capping process that drives both sporozoite gliding and cell invasion. We also demonstrate that TRAP-related proteins in other Apicomplexa fulfill the same function and that their cytoplasmic tails interact with homologous partners in the respective parasite. Therefore, a mechanism of surface redistribution of TRAP-related proteins driving gliding locomotion and cell invasion is conserved among Apicomplexan parasites.
gliding motility; cell invasion; Apicomplexan parasites; thrombospondin-related anonymous protein; micronemal protein 2
Immunoglobulin (Ig)A provides the initial immune barrier to viruses at mucosal surfaces. Specific IgA interrupts viral replication in polarized epithelium during receptor-mediated transport, probably by binding to newly synthesized viral proteins. Here, we demonstrate by immunoelectron microscopy that specific IgA monoclonal antibodies (mAbs) accumulate within Sendai virus–infected polarized cell monolayers and colocalize with the hemagglutinin– neuraminidase (HN) viral protein in a novel intracellular structure. Neither IgG specific for HN nor irrelevant IgA mAbs colocalize with viral protein. Treatment of cultures with viral-specific IgA but not with viral-specific IgG or irrelevant IgA decreases viral titers. These observations provide definitive ultrastructural evidence of a subcellular compartment in which specific IgA and viral envelope proteins interact, further strengthening our hypothesis of intracellular neutralization of virus by specific IgA antibodies. Our results have important implications for intracellular protein trafficking, viral replication, and viral vaccine development.
immunoglobulin A; Sendai virus; mucosal immunity; colocalization; hemagglutinin–neuraminidase protein
In addition to its potent efficacy in animal models against Candida sp., Aspergillus fumigatus, and Histoplasma capsulatum, the clinical candidate pneumocandin MK-991 (formerly L-743,872) was also extremely potent against Pneumocystis carinii in models of immune-compromised animals. MK-991 was approximately 14 times more potent than the original natural product lead, pneumocandin B0. The 90% effective dose (ED90) of MK-991 for cyst clearance in the rat model for pneumocystis was 0.011 mg/kg of body weight when delivered parenterally for 4 days twice a day (b.i.d.). In a mouse model, under the same experimental parameters, the ED90 was 0.02 mg/kg. MK-991 was also effective orally, with an ED90 for cyst clearance of 2.2 mg/kg against acute infection in rats (b.i.d. for 4 days). Complete prevention of P. carinii development was achieved in immunocompromised mice at a daily oral dose of 2.25 mg/kg. As reported previously for other pneumocandins and echinocandins, MK-991 selectively prevented the development of P. carinii cysts. When used as a prophylactic agent, neither stage of the organism appeared in the lungs of animals. In response to an acute infection, cysts were eliminated rapidly, while trophozoite forms persisted. Despite good efficacy as an oral agent in murine models, the low oral absorption of this class may limit the use of MK-991 to parenteral therapy.