Induction of replication protein A in bystander cells

The bystander effect is a biological phenomenon whereby cells not directly targeted by DNA-damaging agents elicit a response similar to that of targeted cells. Understanding the mechanisms underlying the bystander effect is important not only for radiation risk assessment but also for evaluation of protocols for radiotherapy of tumors. Identification of DNA repair and signal transduction proteins that are induced specifically in bystander cells may help in deducing the molecular mechanism(s) responsible for this complex phenomenon. With this objective, we have studied the expression of replication protein A (RPA), which is involved in various DNA metabolic activities such as replication, repair and recombination. We analyzed RPA expression by immunofluorescence and Western blot techniques in both gamma-irradiated primary human fibroblast cells and bystander cells that were recipients of conditioned growth medium harvested from gamma-irradiated cell cultures. A two- to threefold induction of RPA was observed in bystander MRC5 cells treated with conditioned medium collected from gamma-irradiated WI38 or MRC5 cells. Lack of induction of RPA in sham-manipulated MRC5 cells treated with irradiated medium alone (without cells) indicates that the signal elicited from the irradiated cells is responsible for induction of RPA in bystander cells. RPA was induced more effectively in bystander cells than in irradiated cells at the earliest time analyzed (30 min), and the RPA level declined to that of sham-treated control cells by 24 h after treatment. In addition to RPA, apurinic/apyrimidinic endonuclease (APE, a key enzyme of the base excision repair pathway) also showed enhanced expression in bystander cells. Our findings suggest that the induction of RPA and APE is due to a combination of DNA strand breaks and oxidized base lesions in the genomic DNA of bystander cells.     

A low-toxicity method for the separation of lanosterol and dihydrolanosterol from commercial mixtures

We describe an inexpensive, low-toxicity and high-yielding method for the production of pure lanosterol and dihydrolanosterol from the commercially available mixture. Optimum conditions are presented for the one-pot production of the intermediate 24,25 vicinal diol of lanosterol acetate (via either epoxidation or hydroxyhalogenation) which is readily separated from the unreacted dihydrolanosterol acetate. The lanosterol diol can then be converted to pure (>97%) lanosterol. Hypophosphorous acid was used for both the conversion of the epoxide to the diol, and as a catalyst for the hydroxyhalogenation by N-halosuccinimides of the olefinic bond.     

Stochasticity, invasions, and branching random walks

We link deterministic integrodifference equations to stochastic, individual-based simulations by means of branching random walks. Using standard methods, we determine speeds of invasion for both average densities and furthest-forward individuals. For density-independent branching random walks, demographic stochasticity can produce extinction. Demographic stochasticity does not, however, reduce the overall asymptotic speed of invasion or preclude continually accelerating invasions.     

Effects of heavy metals on immunocompetence of white-footed mice (Peromyscus leucopus)

Continued human population growth and industrialization result in increased contamination of wildlife habitats. Effects of such habitat deterioration on the well-being of natural populations are unclear. Exposure to contaminants may impair immunocompetence, thereby increasing disease susceptibility. The mammalian immune system is important in maintaining health and in its sensitivity to toxins. In our study conducted from May 1999 through May 2001, we examined assays of immnnoompetence in the white-footed mouse (Peromyscus leucopus) that inhabited reference sites and sites significantly contaminated with mixtures of heavy metals. We estimated potential exposure and uptake of heavy metals by measuring the level of each contaminant in representative soil and tissue samples. Intraindividual variation across mice, but not sex, explained a large portion of the overall variance in immune response, and spleen weight was significanltly afflicted by mouse age. We found no evidence that residence on contaminated sites had any effect on immunopathology and humoral immunity as measured in our study. We suggest that field and laboratory studies in ecotoxicology provide estimates of exposure to contaminants (i.e., tissue analyses) to establish a database suitable to clarify the dose-response relationship between contaminants and target systems.     

Polysaccharide processing and presentation by the MHCII pathway

The adaptive immune system functions through the combined action of antigen-presenting cells (APCs) and T cells. Specifically, class I major histocompatibility complex antigen presentation to CD8(+) T cells is limited to proteosome-generated peptides from intracellular pathogens while the class II (MHCII) endocytic pathway presents only proteolytic peptides from extracellular pathogens to CD4(+) T cells. Carbohydrates have been thought to stimulate immune responses independently of T cells; however, zwitterionic polysaccharides (ZPSs) from the capsules of some bacteria can activate CD4(+) T cells. Here we show that ZPSs are processed to low molecular weight carbohydrates by a nitric oxide-mediated mechanism and presented to T cells through the MHCII endocytic pathway. Furthermore, these carbohydrates bind to MHCII inside APCs for presentation to T cells. Our observations begin to elucidate the mechanisms by which some carbohydrates induce important immunologic responses through T cell activation, suggesting a fundamental shift in the MHCII presentation paradigm.     

Synthesis and characterization of the biological activity of the cisplatin analogs, cis-PtCl(2)(dexrazoxane) and cis-PtCl(2)(levrazoxane), of the topoisomerase II inhibitors dexrazoxane (ICRF-187) and levrazoxane (ICRF-186)

The individual stereoisomers cis-PtCl(2)(dexrazoxane) and cis-PtCl(2)(levrazoxane) were synthesized and their structures were determined by X-ray crystallography. Dexrazoxane and levrazoxane inhibit cell growth because they are strong catalytic inhibitors of DNA topoisomerase II, whereas cisplatin acts through the formation of DNA cross-links. It was hypothesized that platinum(II) complexes of dexrazoxane and levrazoxane would retain both activities and yield drugs with a dual mode of action. Both cis-PtCl(2)(dexrazoxane) and cis-PtCl(2)(levrazoxane) inhibited Chinese hamster ovary cell growth, but more weakly than dexrazoxane and levrazoxane did. Based on their weak topoisomerase II inhibitory activity, it was concluded that these compounds did not inhibit cell growth by targeting topoisomerase II. A comparison of the conformation of cis-PtCl(2)(dexrazoxane) to that of dexrazoxane bound to the dimer interface of topoisomerase II showed that the highly constrained cis-PtCl(2)(dexrazoxane) was in a highly unfavorable conformation for binding. Neither of the platinum complexes were able to cross-link DNA. Thus the cell growth inhibitory activity of these complexes was also not likely due to any cisplatin-type cross-linking activity.     

Tissue distribution of cholinesterases and anticholinesterases in native and transgenic tomato plants

Acetylcholinesterase, a major component of the central and peripheral nervous systems, is ubiquitous among multicellular animals, where its main function is to terminate synaptic transmission by hydrolyzing the neurotransmitter, acetylcholine. However, previous reports describe cholinesterase activities in several plant species and we present data for its presence in tomato plants. Ectopic expression of a recombinant form of the human enzyme and the expression pattern of the transgene and the accumulation of its product in transgenic tomato plants are described. Levels of acetylcholinesterase activity in different tissues are closely effected by and can be separated from -tomatine, an anticholinesterase steroidal glycoalkaloid. The recombinant enzyme can also be separated from the endogenous cholinesterase activity by its subcellular localization and distinct biochemical properties. Our results provide evidence for the co-existence in tomato plants of both acetylcholinesterase activity and a steroidal glycoalkaloid with anticholinesterase activity and suggest spatial mutual exclusivity of these antagonistic activities. Potential functions, including roles in plant-pathogen interactions are discussed.     

Genomic analysis of mutants affecting xanthophyll biosynthesis and regulation of photosynthetic light harvesting in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

When the absorption of light energy exceeds the capacity for its utilization in photosynthesis, regulation of light harvesting is critical in order for photosynthetic organisms to minimize photo-oxidative damage. Thermal dissipation of excess absorbed light energy, measured as non-photochemical quenching (NPQ) of chlorophyll fluorescence, is induced rapidly in response to excess light conditions, and it is known that xanthophylls such as zeaxanthin and lutein, the transthylakoid pH gradient, and the PsbS protein are involved in this mechanism. Although mutants affecting NPQ and the biosynthesis of zeaxanthin and lutein were originally isolated and characterized at the physiological level in the unicellular green alga Chlamydomonas reinhardtii, the molecular basis of several of these mutants, such as npq1 and lor1, has not been determined previously. The recent sequencing of the C. reinhardtii nuclear genome has facilitated the search for C. reinhardtii homologs of plant genes involved in xanthophyll biosynthesis and regulation of light harvesting. Here we report the identification of C. reinhardtii genes encoding PsbS and lycopene ɛ-cyclase, and we show that the lor1 mutation, which affects lutein synthesis, is located within the lycopene ɛ-cyclase gene. In contrast, no homolog of the plant violaxanthin de-epoxidase (VDE) gene was found. Molecular markers were used to map the npq1 mutation, which affects VDE activity, as a first step toward the map-based cloning of the NPQ1 gene.     

Conserving tropical nature: current challenges for ecologists

Tropical biodiversity continues to erode unabated, which calls for ecologists to address the problem directly, placing less reliance on indirect interventions, such as community-based development schemes. Ecologists must become more assertive in providing scientifically formulated and adaptively managed interventions, involving biodiversity payments, to serve local, regional and global interests in tropical nature. Priorities for tropical ecologists thus include the identification of key thresholds to ecological resilience, and the formulation of clear monitoring protocols and management strategies for implementation by local resource managers. A particular challenge is to demonstrate how nature reserves contribute to the adaptive capacity of regional land-use matrices and, hence, to the provision of sustainable benefits at multiple spatial and temporal scales.     

A second human antiretroviral factor, APOBEC3F, is suppressed by the HIV-1 and HIV-2 Vif proteins

The HIV-1 Vif protein suppresses the inhibition of viral replication caused by the human antiretroviral factor APOBEC3G. As a result, HIV-1 mutants that do not express the Vif protein are replication incompetent in 'nonpermissive' cells, such as primary T cells and the T-cell line CEM, that express APOBEC3G. In contrast, Vif-defective HIV-1 replicates effectively in 'permissive' cell lines, such as a derivative of CEM termed CEM-SS, that do not express APOBEC3G. Here, we show that a second human protein, APOBEC3F, is also specifically packaged into HIV-1 virions and inhibits their infectivity. APOBEC3F binds the HIV-1 Vif protein specifically and Vif suppresses both the inhibition of virus infectivity caused by APOBEC3F and virion incorporation of APOBEC3F. Surprisingly, APOBEC3F and APOBEC3G are extensively coexpressed in nonpermissive human cells, including primary lymphocytes and the cell line CEM, where they form heterodimers. In contrast, both genes are quiescent in the permissive CEM derivative CEM-SS. Together, these data argue that HIV-1 Vif has evolved to suppress at least two distinct but related human antiretroviral DNA-editing enzymes.