Apoptotic signaling in response to a single type of DNA lesion, O(6)-methylguanine

Until now, it has been difficult to establish exactly how a specific DNA lesion signals apoptosis because each DNA damaging agent produces a collection of distinct DNA lesions and produces damage in RNA, protein, and lipids. We have developed a system in human cells that focuses on the response to a single type of DNA lesion, namely O(6)-methylguanine (O(6)MeG). We dissect the signaling pathways involved in O(6)MeG-induced apoptosis, a response dependent on the MutSalpha heterodimer that is normally involved in DNA mismatch repair. O(6)MeG triggers robust activation of caspases associated with both death receptor- and mitochondrial-mediated apoptosis. Despite this, O(6)MeG/MutSalpha-triggered apoptosis is only partly dependent on caspase activation; moreover, it is mediated solely by mitochondrial signaling and not at all by death receptor signaling. Finally, while Bcl-2 and Bcl-x(L), negative regulators of mitochondrial-regulated apoptosis, could effectively block O(6)MeG/MutSalpha-dependent apoptosis, they were unable to prevent the cells from ultimately dying.     

Clonality and recombination in genetically differentiated subgroups of Cryptococcus gattii

Cryptococcus gattii is a pathogenic yeast that together with Cryptococcus neoformans causes cryptococcosis in humans and animals. High numbers of viable C. gattii propagules can be obtained from certain species of Australian Eucalyptus camaldulensis trees, and an epidemiological link between Eucalyptus colonization and human exposure has been proposed. However, the highest prevalence of C. gattii cryptococcosis occurs in Papua New Guinea and in regions of Australia where the eucalypt species implicated to date are not endemic. This study investigated the population structure of three geographically distinct clinical and veterinary populations of C. gattii from Australia and Papua New Guinea. All populations that consisted of a genotype found frequently in Australia (VGI) were strongly clonal and were highly differentiated from one another. Two populations of the less common VGII genotype from Sydney and the Northern Territory had population structures inferring recombination. In addition, there was some evidence of reduced genetic differentiation between these geographically remote regions. In a companion study presented in this issue, VGII isolates were overwhelmingly more fertile than those of the VGI genotype, giving biological support to the indirect assessment of sexual exchange. It appears that the VGI genotype propagates clonally on eucalypts in Australia and on an unknown substrate in Papua New Guinea, with infection initiated by an unidentified infectious propagule. VGII isolates are completing their life cycles and may be dispersed via sexually produced basidiospores, which are also likely to initiate respiratory infection.     

Molecular and structural characterization of fluorescent human parvovirus B19 virus-like particles

Although sharing a T=1 icosahedral symmetry with other members of the Parvoviridae family, it has been suggested that the fivefold channel of the human parvovirus B19 VP2 capsids is closed at its outside end. To investigate the possibility of placing a relatively large protein moiety at this site of B19, fluorescent virus-like particles (fVLPs) of B19 were developed. The enhanced green fluorescent protein (EGFP) was inserted at the N-terminus of the structural protein VP2 and assembly of fVLPs from this fusion protein was obtained. Electron microscopy revealed that these fluorescent protein complexes were very similar in size when compared to wild-type B19 virus. Further, fluorescence correlation spectroscopy showed that an average of nine EGFP domains were associated with these virus-like structures. Atomic force microscopy and immunoprecipitation studies showed that EGFP was displayed on the surface of these fVLPs. Confocal imaging indicated that these chimeric complexes were targeted to late endosomes when expressed in insect cells. The fVLPs were able to efficiently enter cancer cells and traffic to the nucleus via the microtubulus network. Finally, immunoglobulins present in human parvovirus B19 acute and past-immunity serum samples were able to detect antigenic epitopes present in these fVLPs. In summary, we have developed fluorescent virus-like nanoparticles displaying a large heterologous entity that should be of help to elucidate the mechanisms of infection and pathogenesis of human parvovirus B19. In addition, these B19 nanoparticles serve as a model in the development of targetable vehicles designed for delivery of biomolecules.     

The efficacy of BCG-induced tumor immunity in guinea pigs with regional and systemic malignancy

Summary It has been previously demonstrated that transplanted syngeneic line-10 hepatocarcinoma established in the skin of inbred guinea pigs (strain 2) regressed and regional lymph node metastases were eliminated after intratumoral injection of viable Mycobacterium bovis strain BCG. During the course of this reaction there is the development of systemic tumor immunity. The purpose of this study was to determine the relative efficacy of the induced tumor immunity to eliminate regional as well as systemic tumor burden. The approach to evaluate the efficacy of BCG-induced systemic tumor immunity in vivo, for regional as well as systemic tumor, was to develop a competition assay using increasing doses of intravascular disseminated line-10 tumor cells in animals with established regional tumors. The results clearly show that the efficacy of intratumoral BCG injection in producing regression of regional tumor is abrogated by initial intravascular doses of 10³–10⁶ line-10 cells. That the vascular systemic tumor burden diminished the effective systemic tumor immunity was demonstrated by the inability of animals with systemic tumor burdens to reject contralateral challenge of line-10 tumor cells. The capability of BCG-treated animals to reject contralateral line-10 challenge was inversely proportional to the initial intravascular tumor dose. Survival studies clearly demonstrate that a significant therapeutic effect could be achieved in guinea pigs with regional skin tumors and limited vascular metastases when the modality of therapy included BCG-intratumoral injection, followed 6 weeks later by surgery of the established skin tumor and regional lymph node. These results suggest that the development of tumor immunity after BCG-intratumoral injection is not impaired by the systemic tumor burden, but rather that it is preempted at distant sites. Abbreviations used in this paper: BCG, Bacillus Calmette-Guérin; i.a., intraarterially; i.d., intradermally; i.v., intravenously; SDA, superficial distal axillary.     

The suicidal DNA repalr methyltransferases of microbes

Virtually every organism so far tested has been found to possess an extremely efficient DNA repair mechanism to ensure that certain alkylated oxygens do not accumulate in the genome. The repair is executed by DNA methyltransferases (MTases) which repair DNA O6-methylguanine (O6MeG), O4-methylthymine (O4MeT) and methylphosphotriesters (MePT). The mechanism is rather extravagant because an entire protein molecule is expended for the repair of just one, or sometimes two, O-alkyl DNA adduct(s). Cells profit from such an expensive transaction by earning protection against death and mutation by alkylating agents. This review considers the structure, function and biological roles of a number of well-characterized microbial DNA repair MTases.     

Analysis of sperm chromosome complements from a man heterozygous for a pericentric inversion of chromosome 1

Human sperm chromosomes were studied in a man heterozygous for a pericentric inversion of chromosome (1)(p31q12). Q-banded pronuclear chromosomes were analyzed after in vitro penetration of golden hamster oocytes. A total of 159 sperm were examined: 54% bearing the inverted chromosome 1 and 46% the normal chromosome 1. These frequencies are not significantly different from the theoretical 11 ratio. There were no recombinant sperm with duplications or deficiencies, suggesting that a pairing loop failed to form or that crossing-over was suppressed. The frequency of abnormalities unrelated to the inversion was 5% for numerical, 8.8% for structural, 2.5% for numerical and structural, values not significantly different from control donors studied in our lab. The frequencies of X- and Y-bearing sperm were 46% and 54%, respectively, not significantly different from the expected value of 50%. This is the fifth pericentric inversion studied by human sperm chromosome analysis; recombinant chromosomes have been observed in two of the five cases. Some of the factors associated with an increased risk of recombinant sperm appear to be inversion size greater than 30% of the chromosome and chromosome breakpoints in G-light bands.     

Microvascular network remodeling in dura mater of ovariectomized pigs: role for angiopoietin-1 in estrogen-dependent control of vascular stability

Estrogen is a key regulator of vascular responses and angioadaptation in multiple organs and tissues, including brain. However, the consequences of a loss of ovarian steroid hormone secretion on the status of microvascular networks in brain and meninges are largely unknown. Here, using the perfused dura mater model coupled with high-resolution digital epifluorescence and laser scanning confocal microscopy and computer-assisted morphometric analysis, we demonstrate that cessation of ovarian hormone production causes dramatic vascular remodeling in meningeal microvascular networks characterized by a threefold decrease in microvessel density and capillary rarefaction and an almost fourfold increase in vascular permeability. These changes were accompanied by a significant decrease in angiopoietin-1 (Ang-1) expression and Ang-1/Tie-2 ratio (1.4-fold, P < 0.01, and 1.5-fold, P < 0.05, respectively) in ovariectomized animals compared with intact females, but no changes were detected in the expression of estrogen receptors (ER)-alpha and -beta. We conclude that estrogen-dependent control of Ang-1 expression plays an important role in stabilizing meningeal microvessel and maintaining healthy microvascular networks.