Loss of homologous recombination or non-homologous end-joining leads to radial formation following DNA interstrand crosslink damage

High levels of interstrand cross-link damage in mammalian cells cause chromatid breaks and radial formations recognizable by cytogenetic examination. The mechanism of radial formation observed following DNA damage has yet to be determined. Due to recent findings linking homologous recombination and non-homologous end-joining to the action of the Fanconi anemia pathway, we speculated that radials might be the result of defects in either of the pathways of DNA repair. To test this hypothesis, we have investigated the role of homologous recombination proteins RAD51 and RAD52, non-homologous end-joining proteins Ku70 and LIG4, and protein MRE11 in radial formation and cell survival following interstrand crosslink damage with mitomycin C. For the studies we used small inhibitory RNA to deplete the proteins from cells, allowing for evaluation of radial formation and cell survival. In transformed normal human fibroblasts, depletion of these proteins increased interstrand crosslink sensitivity as manifested by decreased cell survival and increased radial formation. These results demonstrate that inactivation of proteins from either of the two separate DNA repair pathways increases cellular sensitivity to interstrand crosslinks, indicating each pathway plays a role in the normal response to interstrand crosslink damage. We can also conclude that homologous recombination or non-homologous end-joining are not required for radial formation, since radials occur with depletion of these pathways.     

Tip60 is required for DNA interstrand cross-link repair in the Fanconi anemia pathway

The disease Fanconi anemia is a genome instability syndrome characterized by cellular sensitivity to DNA interstrand cross-linking agents, manifest by decreased cellular survival and chromosomal aberrations after such treatment. There are at least 13 proteins acting in the pathway, with the FANCD2 protein apparently functioning as a late term effecter in the maintenance of genome stability. We find that the chromatin remodeling protein, Tip60, interacts directly with the FANCD2 protein in a yeast two-hybrid system. This interaction has been confirmed by co-immunoprecipitation and co-localization using both endogenous and epitope-tagged FANCD2 and Tip60 from human cells. The observation of decreased cellular survival after exposure to mitomycin C in normal fibroblasts depleted for Tip60 indicates a direct function in interstrand cross-link repair. The coincident function of Tip60 and FANCD2 in one pathway is supported by the finding that depletion of Tip60 in Fanconi anemia cells does not increase sensitivity to DNA cross-links. However, depletion of Tip60 did not reduce monoubiquitination of FANCD2 or its localization to nuclear foci following DNA damage. The observations indicate that Fanconi anemia proteins act in concert with chromatin remodeling functions to maintain genome stability after DNA cross-link damage.     

Characterization of the laminated layer of in vitro cultivated Echinococcus vogeli metacestodes

The metacestode (larval) stages of the cestode parasites Echinococcus vogeli and E. multilocularis were isolated from the peritoneal cavity of experimentally infected C57BL/6 mice and were cultured in vitro for a period of up to 4 mo under conditions normally applied for the in vitro cultivation of E. multilocularis metacestodes. In contrast to E. multilocularis, E. vogeli did not exhibit extensive exogenous budding and proliferation but increased in size with a final diameter of up to 10 mm. Most metacestodes contained protoscoleces, singly or in groups, either associated with brood capsules or growing directly out of the germinal layer. Each individual metacestode was covered by an acellular translucent laminated layer that was considerably thicker than the laminated layer of E. multilocularis metacestodes. The ultrastructural characteristics, protein content, and carbohydrate composition of the laminated layer of in vitro cultivated E. vogeli and E. multilocularis were assessed using transmission electron microscopy, lectin fluorescence labeling, and lectin blotting assays. The laminated layer of E. vogeli is, as previously described for E. multilocularis metacestodes, largely composed of N-acetyl-beta-D-galactosaminyl residues and alpha- and beta-D-galactosyl residues, as well as of the core structure of O-linked carbohydrate chains, N-acetylgalactosamine-beta-1,3-galactose. However, in contrast to E. multilocularis, N-linked glycopeptides and alpha-D-mannosyl and/or glucosyl residues were also associated with the laminated layer of E. vogeli. The laminated layer from both species was isolated from in vitro cultivated metacestodes, and the purified fractions were comparatively analyzed. The protein:carbohydrate ratio (1:1) was similar in both parasites; however, the protein banding pattern obtained by silver staining following sodium dodecyl sulfate polyacrylamide gel electrophoresis suggested intrinsic differences in protein composition. A polyclonal antiserum raised against the E. multilocularis laminated layer and a monoclonal antibody, G11, directed against the major E. multilocularis laminated layer antigen Em2 did not cross-react with E. vogeli, indicating distinct compositional and antigenic differences between these 2 parasites.     

Mitochondrial tRNA import in Toxoplasma gondii

Apicomplexan parasites have the smallest known mitochondrial genome. It consists of a repeated element of approximately 6-7 kb in length and encodes three mitochondrial proteins, a number of rRNA fragments, but no tRNAs. It has therefore been postulated that in apicomplexans all tRNAs required for mitochondrial translation are imported from the cytosol. To provide direct evidence for this process we have established a cell fractionation procedure allowing the isolation of defined organellar RNA fractions from the apicomplexan Toxoplasma gondii. Analysis of T. gondii total and organellar RNA by Northern hybridization showed that except for the cytosol-specific initiator tRNAMet all nucleus-encoded tRNAs tested were present in the cytosol and in the mitochondrion but not in the plastid. Thus, these results provide the first experimental evidence for mitochondrial tRNA import in apicomplexans. The only other taxon that imports the whole set of mitochondrial tRNAs are the trypanosomatids. Interestingly, the initiator tRNAMet is the only cytosol-specific tRNA in trypanosomatids, indicating that the import specificity is identical in both groups. In agreement with this, the T. gondii initiator tRNAMet remained in the cytosol when expressed in Trypanosoma brucei. However, in contrast to trypanosomatids, no thio-modifications were detected in the tRNAGln of T. gondii indicating that, unlike what is suggested in Leishmania, they are not involved in regulating import.     

Horse‐mounted invaders from the Russo‐Kazakh steppe or agricultural colonists from western Central Asia? A craniometric investigation of the Bronze Age settlement of Xinjiang

Numerous Bronze Age cemeteries in the oases surrounding the T?klamakan Desert of the Tarim Basin in the Xinjiang Uyghur Autonomous Region, western China, have yielded both mummified and skeletal human remains. A dearth of local antecedents, coupled with woolen textiles and the apparent Western physical appearance of the population, raised questions as to where these people came from. Two hypotheses have been offered by archaeologists to account for the origins of Bronze Age populations of the Tarim Basin. These are the "steppe hypothesis" and the "Bactrian oasis hypothesis." Eight craniometric variables from 25 Aeneolithic and Bronze Age samples, comprising 1,353 adults from the Tarim Basin, the Russo-Kazakh steppe, southern China, Central Asia, Iran, and the Indus Valley, are compared to test which, if either, of these hypotheses are supported by the pattern of phenetic affinities possessed by Bronze Age inhabitants of the Tarim Basin. Craniometric differences between samples are compared with Mahalanobis generalized distance (d2), and patterns of phenetic affinity are assessed with two types of cluster analysis (the weighted pair average linkage method and the neighbor-joining method), multidimensional scaling, and principal coordinates analysis. Results obtained by this analysis provide little support for either the steppe hypothesis or the Bactrian oasis hypothesis. Rather, the pattern of phenetic affinities manifested by Bronze Age inhabitants of the Tarim Basin suggests the presence of a population of unknown origin within the Tarim Basin during the early Bronze Age. After 1200 B.C., this population experienced significant gene flow from highland populations of the Pamirs and Ferghana Valley. These highland populations may include those who later became known as the Saka and who may have served as "middlemen" facilitating contacts between East (Tarim Basin, China) and West (Bactria, Uzbekistan) along what later became known as the Great Silk Road.     

Temporal dissection of Bax-induced events leading to fission of the single mitochondrion in Trypanosoma brucei

The protozoan Trypanosoma brucei has a single mitochondrion and lacks an apoptotic machinery. Here we show that expression of the proapoptotic protein Bax in T. brucei causes the release of cytochrome c, the depolarization of the mitochondrial membrane potential and mitochondrial fission. However, in contrast to mammalian cells, the three events are temporally well separated. The release of cytochrome c from the intermembrane space precedes mitochondrial fission, showing that it does not depend on mitochondrial fragmentation. Furthermore, halting Bax expression allows some cells to recover even after mitochondrial fission, the last recorded event, went to completion, indicating that all three Bax-induced events are, in principle, reversible.     

Vero cell surface proteoglycan interaction with the microneme protein NcMIC(3) mediates adhesion of Neospora caninum tachyzoites to host cells unlike that in Toxoplasma gondii

Neospora caninum and Toxoplasma gondii are characterised by a very low host cell specificity, thus they are able to infect a wide range of different cells in vivo and in vitro. Infection of the host cell by tachyzoites is a process which is preceded by adhesion onto the host cell surface. The receptors on the host cell surface which would allow N. caninum to establish a physical interaction have not been investigated so far. Here we report the role of host cell surface proteoglycans as receptors for the adhesion of N. caninum tachyzoites to Vero cell monolayers. We found that N. caninum tachyzoites, similar to T. gondii tachyzoites, can bind to sulphated proteoglycans which naturally occur on the surface of mammalian cells, including heparin/heparan sulphate, chondroitin sulphates, as well as to the artificially sulphated glycosaminoglycan dextran sulphate. Although removal of heparan sulphate from the host cell surface results in decreased adhesion of T. gondii tachyzoites, binding of N. caninum tachyzoites is not affected by this treatment. Conversely, enzymatic removal of chondroitin sulphate A, B and C decreases N. caninum adhesion but does not affect T. gondii binding to Vero cells. Thus, T. gondii and N. caninum tachyzoites exhibit differential adhesive properties with regard to host cell surface glycosaminoglycans. Additional experiments employing Triton X-100 solubilised NcSRS2 and NcMIC3 showed that NcSRS2 binds to the host cell surface, but not through those sulphated glycosaminoglycans investigated in this study. In contrast, NcMIC3 binding to the host cell surface is dramatically influenced by these modifications. Further experiments showed that the NcMIC3 adhesive motif comprised of four consecutive epidermal growth factor-like domains expressed as a recombinant protein exhibits a high binding activity for sulphated glycosaminoglycans. These results suggest that host cell surface proteoglycan interaction of N. caninum differs from that observed for T. gondii, and that the epidermal growth factor-like adhesive motif in NcMIC3 could be involved in this process.     

Neospora caninum protein disulfide isomerase is involved in tachyzoite-host cell interaction

We have previously shown that treatment of Neospora caninum tachyzoites with the aspartyl protease inhibitor pepstatin A reduces host cell invasion [Naguleswaran, A., Muller, N., Hemphill, A., 2003. Neospora caninum and Toxoplasma gondii: a novel adhesion/invasion assay reveals distinct differences in tachyzoite-host cell interactions. Exp. Parasitol. 104, 149-158]. Pepstatin A-affinity-chromatography led to the isolation of a major band of approximately 52 kDa which was identified as a homologue of a previously described Toxoplasma gondii putative protein disulfide isomerase (TgPDI) through tandem mass spectrometry. A BLAST search against N. caninum expressed sequence tags (ESTs) on the ApiDots server using TgPDI cDNA as query sequence revealed a 2251 bp PDI-like consensus (NcPDI), which shows 94% identity to the T. gondii homologue. In N. caninum tachyzoites, NcPDI was found mainly in the soluble hydrophilic fraction. Immunofluorescence showed that expression of NcPDI was dramatically down-regulated in the bradyzoite stage, and immunogold-EM on tachyzoites localised the protein to the cytoplasm, mostly in close vicinity to the nuclear membrane, to the micronemes, and to the parasite cell surface. However, NcPDI was absent in rhoptries and dense granules. Preincubation of tachyzoites with the sulfhydryl blocker 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), p-chloromercuribenzoic acid (pCMBA), and with the PDI inhibitor bacitracin reduced adhesion of parasites to host cells. In addition, incubation of N. caninum tachyzoites with affinity-purified anti-NcPDI antibodies reduced host cell adhesion. PDIs catalyse the formation, reduction or isomerisation of disulfide bonds. Many major components of the adhesion and invasion machinery of apicomplexan parasites are cysteine-rich and dependent on correct folding via disulfide bond formation. Thus, our data points towards an important role for surface-associated NcPDI in Neospora-host cell interaction.     

Differential effects of interferon-gamma and tumor necrosis factor-alpha on Toxoplasma gondii proliferation in organotypic rat brain slice cultures

Organotypic slice culture explants of rat cortical tissue infected with Toxoplasma gondii tachyzoites were applied as an in vitro model to investigate host-pathogen interactions in cerebral toxoplasmosis. The kinetics of parasite proliferation and the effects of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) in infected organotypic cultures were monitored by light microscopy, transmission electron microscopy (TEM), and quantitative polymerase chain reaction (PCR) assay. As assessed by the loss of the structural integrity of the glial fibrillary acidic protein-intermediate filament network, tachyzoites infected and proliferated mainly within astrocytes, whereas neurons and microglia remained largely unaffected. Toxoplasma gondii proliferation was severely inhibited by IFN-y. However, this inhibition was not linked to tachyzoite-to-bradyzoite stage conversion. In contrast, TNF-alpha treatment resulted in a dramatically enhanced proliferation rate of the parasite. The cellular integrity in IFN-gamma-treated organotypic slice cultures was severely impaired compared with untreated and TNF-alpha-treated cultures. Thus, on infection of organotypic neuronal cultures, IFN-gamma and TNF-alpha exhibit largely detrimental effects, which could contribute to either inhibition or acceleration of parasite proliferation during cerebral toxoplasmosis.     

Foreign elites from the Oxus civilization? A craniometric study of anomalous burials from Bronze Age Tepe Hissar

Discovery of a small number of individuals in the Period III necropolis at the large northern Iranian Bronze Age site of Tepe Hissar directly associated with a complex of imported artifacts raises the question of whether these individuals represent elites who had access to these exotic commodities, or an imposed foreign elite that may have brought these unusual artifacts from their homelands. The source of the atypical objects is believed to be the Oxus civilization of central Asia. This study investigates the identity of these individuals by employing canonical discriminant function analysis of 20 craniometric variables among 174 adult males from Tepe Hissar Period III and Oxus civilization males from Sapalli tepe and Djarkutan. Dicriminant function analysis provides a strong separation between Tepe Hissar Period III males and Oxus civilization males and a high level of correct assignation by sample (95.8%). Imposition of the five males associated with imported central Asian artifacts from the Period III necropolis indicates that the majority (4/5) are phenetically indistinguishable from other Period III Tepe Hissar males. The results indicate that these individuals most likely represent local elite inhabitants of Tepe Hissar, rather than the presence of an imposed foreign elite. However, given the scarcity of crucial specimens, especially females, and comparative skeletal series, this conclusion must remain tentative.