Thiol protecting agents and antioxidants inhibit the mitochondrial permeability transition promoted by etoposide: implications in the prevention of etoposide-induced apoptosis

Etoposide (VP-16) is known to promote cell apoptosis either in cancer or in normal cells as a side effect. This fact is preceded by the induction of several mitochondrial events, including increase in Bax/Bcl-2 ratio followed by cytochrome c release and consequent activation of caspase-9 and -3, reduction of ATP levels, depolarization of membrane potential (DeltaPsi) and rupture of the outer membrane. These events are apoptotic factors essentially associated with the induction of the mitochondrial permeability transition (MPT). VP-16 has been shown to stimulate the Ca2+-dependent MPT induction similarly to prooxidants and to promote apoptosis by oxidative stress mechanisms, which is prevented by glutathione (GSH) and N-acetylcysteine (NAC). Therefore, the aim of this work was to study the effects of antioxidants and thiol protecting agents on MPT promoted by VP-16, attempting to identify the underlying mechanisms on VP-16-induced apoptosis. The increased sensitivity of isolated mitochondria to Ca2+-induced swelling, Ca2+ release, depolarization of DeltaPsi and uncoupling of respiration promoted by VP-16, which are prevented by cyclosporine A proving that VP-16 induces the MPT, are also efficiently prevented by ascorbate, the primary reductant of the phenoxyl radicals produced by VP-16. The thiol reagents GSH, dithiothreitol and N-ethylmaleimide, which have been reported to prevent the MPT induction, also protect this event promoted by VP-16. The inhibition of the VP-16-induced MPT by antioxidants agrees with the prevention of etoposide-induced apoptosis by GSH and NAC and suggests the generation of oxidant species as a potential mechanism underlying the MPT that may trigger the release of mitochondrial apoptogenic factors responsible for apoptotic cascade activation.     

Glycosylphosphatidylinositol-anchored mucin-like glycoproteins from Trypanosoma cruzi bind to CD1d but do not elicit dominant innate or adaptive immune responses via the CD1d/NKT cell pathway

It has been proposed that self and protozoan-derived GPI anchors are natural ligands of CD1d. In this study, we investigated the ability of GPI anchors from Trypanosoma cruzi to bind to CD1d and mediate activation of NKT cells. We observed that GPI-anchored mucin-like glycoproteins (GPI mucins), glycoinositolphospholipids (GIPLs), and their phosphatidylinositol moieties bind to rCD1d and inhibit the stimulation of a NKT hybridoma by the alpha-galactosylceramide-CD1 complex. However, these GPI anchors and related structures were unable to activate NKT cells in vitro or in vivo. We found that high titers of Ab anti-GPI mucins, but not anti-GIPLs, were detected in sera from wild-type as well as in TAP1(-/-), CD1d(-/-), and MHC class II(-/-) mice after immunization. However, T-dependent anti-GPI mucin Ab isotypes, such as IgG1, IgG2a, IgG2b, and IgG3, were absent on MHC class II(-/-), but were conserved in CD1d(-/-) and TAP1(-/-) mice. Furthermore, we found that CD1d(-/-) mice presented a robust cytokine as well as anti-GPI mucins and anti-GIPL Ab responses, upon infection with T. cruzi parasites. These results indicate that, despite binding to CD1d, GPI mucins and related structures expressed by T. cruzi appear not to evoke dominant CD1d-restricted immune responses in vivo. In contrast, MHC class II is critical for the production of the major Ig G isotypes against GPI mucins from T. cruzi parasites.     

DNA polymerase clamp shows little turnover at established replication sites but sequential de novo assembly at adjacent origin clusters

The spatial and temporal organization of DNA replication was investigated in living cells with a green fluorescent protein fusion to the DNA polymerase clamp PCNA. In situ extractions and photobleaching experiments revealed that PCNA, unlike RPA34, shows little if any turnover at replication sites, suggesting that it remains associated with the replication machinery through multiple rounds of Okazaki fragment synthesis. Photobleaching analyses further showed that the transition from earlier to later replicons occurs by disassembly into a nucleoplasmic pool of rapidly diffusing subcomponents and reassembly at newly activated sites. The fact that these replication sites were de novo assembled in close proximity to earlier ones suggests that activation of neighboring origins may occur by a domino effect possibly involving local changes in chromatin structure and accessibility.     

Protein transduction: a novel tool for tissue regeneration

Tissue regeneration in humans is limited and excludes vitals organs like heart and brain. Transformation experiments with oncogenes like T antigen have shown that retrodifferentiation of the respective cells is possible but hard to control. To bypass the risk of cancer formation a protein therapy approach has been developed. The transient delivery of proteins rather than genes could still induce terminally-differentiated cells to reenter the cell cycle. This approach takes advantage of protein-transducing domains that mediate the transfer of cargo proteins into cells. The goal of this brief review is to outline the basics of protein transduction and to discuss potential applications for tissue regeneration.     

Determinants of human immunodeficiency virus (HIV) prevalence in homosexual and bisexual men screened for admission to a cohort study of HIV negatives in Belo Horizonte,Brazil: Project Horizonte

Project Horizonte, an open cohort of homosexual and bisexual human immunodeficiency virus (HIV-1) negative men, is a component of the AIDS Vaccine Program, in Belo Horizonte, Minas Gerais, Brazil. The objective of this study was to compare volunteers testing HIV positive at cohort entry with a sample of those who tested HIV negative in order to identify risk factors for prevalent HIV infection, in a population being screened for enrollment at Project Horizonte. A nested case-control study was conducted. HIV positive volunteers at entry (cases) were matched by age and admission date to three HIV negative controls each. Selected variables used for the current analysis included demographic factors, sexual behavior and other risk factors for HIV infection. During the study period (1994-2001), among the 621 volunteers screened, 61 tested positive for HIV. Cases were matched to 183 HIV negative control subjects. After adjustments, the main risk factors associated with HIV infection were unprotected sex with an occasional partners, OR = 3.7 (CI 95% 1.3-10.6), receptive anal intercourse with an occasional partner, OR = 2.8 (95% CI 0.9-8.9) and belonging to the negro racial group, OR = 3.4 (CI 95% 1.1-11.9). These variables were associated with an increase in the risk of HIV infection among men who have sex with men at the screening for admission to an open HIV negative cohort.     

Glutathione cycle impairment mediates A beta-induced cell toxicity

Alzheimer's disease is widely held to be associated with oxidative stress due, in part, to the action of amyloid beta-peptide (A beta). We observed that A beta 25-35 induced an increase in reactive oxygen species (ROS) in NT2 rho+ cells, leading to protein and lipid oxidation. This oxidative status was partially prevented by the antioxidants, vitamin E, reduced glutathione, and by melatonin. However, NT2 rho0 cells (that lack mitochondrial DNA) in the absence of A beta showed an increase in ROS production, lipid and protein oxidation, as compared with parental rho+ cells. Upon A beta 25-35 treatment, in rho+ cells, a decrease in glutathione reductase activity and in GSH levels was observed, whereas glutathione peroxidase activity was shown to be increased. In NT2 rho0 cells, in the absence of A beta, GSH levels were maintained, whereas glutathione reductase and peroxidase activities were increased. The exposure of A beta to rho0 cells did not induce any change in these parameters. We observed that melatonin prevented caspase activation and DNA fragmentation in rho+ cells treated with A beta. Considering the evidence presented, we argue that the glutathione cycle impairment is a key event in A beta-induced cell toxicity.     

Utility of genetic screening in hypertrophic cardiomyopathy: prevalence and significance of novel and double (homozygous and heterozygous) beta-myosin mutations

Genetic screening of the beta-myosin heavy chain gene (MYH7) was evaluated in 100 consecutive unrelated patients with hypertrophic cardiomyopathy (HCM) and 200 normal unrelated subjects. Seventeen beta-myosin mutations were identified in 19 patients. Notably, 13, or 76%, were novel. Mutations were detected in both alleles in two patients: homozygous for Lys207Gln in one, and heterozygous for Pro211 Leu and Arg663His in another. No mutation was detected in the controls. MYH7-associated HCM was associated with more marked left atrial enlargement and syncope than non-MYH7-related HCM. Our findings indicate that: (1) screening methods should allow identification of novel mutations; and (2) more than one sarcomeric mutation may be present in a patient more commonly than is appreciated. Further studies are necessary to ascertain the clinical consequences of the novel and compound gene abnormalities, and to determine whether correlating functional domain to phenotype provides more useful information about the clinical significance of the molecular defects.     

A caudorostral wave of RALDH2 conveys anteroposterior information to the cardiac field

Establishment of anteroposterior (AP) polarity is one of the earliest decisions in cardiogenesis and plays an important role in the coupling between heart and blood vessels. Recent research implicated retinoic acid (RA) in the communication of AP polarity to the heart. We utilized embryo culture, in situ hybridization, morphometry, fate mapping and treatment with the RA pan-antagonist BMS493 to investigate the relationship between cardiac precursors and RA signalling. We describe two phases of AP signalling by RA, reflected in RALDH2 expression. The first phase (HH4-7) is characterized by increasing proximity between sino-atrial precursors and the lateral mesoderm expressing RALDH2. In this phase, RA signalling is consistent with diffusion of the morphogen from a large field rather than a single hot spot. The second phase (HH7-8) is characterized by progressive encircling of cardiac precursors by a field of RALDH2 originating from a dynamic and evolutionary-conserved caudorostral wave pattern in the lateral mesoderm. At this phase, cardiac AP patterning by RA is consistent with localized action of RA by regulated activation of the Raldh2 gene within an embryonic domain. Systemic treatment with BMS493 altered the cardiac fate map such that ventricular precursors were found in areas normally devoid of them. Topical application of BMS493 inhibited atrial differentiation in left anterior lateral mesoderm. Identification of the caudorostral wave of RALDH2 as the endogenous source of RA establishing cardiac AP fates provides a useful model to approach the mechanisms whereby the vertebrate embryo confers axial information on its organs.     

Decolourisation of Remazol Brilliant Blue R via a novel Bjerkandera sp. strain

A novel strain of Bjerkandera sp. (B33/3), with particularly high decolourisation activities upon Poly R-478 and Remazol Brilliant Blue R (RBBR) dyes, was isolated. The role of the ligninolytic extracellular enzymes produced by this strain on decolourisation of RBBR was studied in some depth. The basis of decolourisation is an enzyme-mediated process, in which the main enzyme responsible is a recently described peroxidase with capacity for oxidation of manganese, as well as veratryl alcohol and 2,6-dimethoxyphenol in a manganese-independent reaction.