Stress response induced by DNA damage leads to specific, delayed and untargeted mutations

Summary Cells of the mouse T-lymphoma line GRSL13 were treated with 8-methoxy-psoralen plus longwave ultraviolet light (PUVA) under conditions where the biological effects are mainly due to non-persistent DNA crosslinks (PUVA-CL treatment). Fluctuation analysis showed that PUVA-CL treatment resulted in an enhancement of the mutation rate in the progeny of treated cells, which persisted until the eleventh generation after treatment. Since only 5 cross-links are available to account for 52 mutational events observed in the coding region, about 90% of the induced mutational events must have been untargeted. This was confirmed by molecular analysis of these mutations, which showed that 53% of the point mutations arose at sites which are not a target for psoralens. This supports the hypothesis that stress responses may give rise to untargeted mutagenesis. Further support for this hypothesis is provided by the observation that 8-methoxy-psoralen (8-MOP) or UVA alone (both of which are known to induce many pleiotropic effects) each acted as indirect mutagen by enhancing the mutation rate 2–4 fold in the progeny of treated cells.     

The impact of the bystander effect on the low-dose hypersensitivity phenomenon

The aim of this study was to investigate the possible relationship between the bystander effect and the low-dose hypersensitivity/increased radio-resistance phenomenon in BJ fibroblast cells taking as response criteria different end points of radiation damage such as cell survival, chromosomal damage (as detected by using micronucleus assay) and double strand breaks (DSBs) of the DNA. Although γ-H2AX foci were observed in confluent bystander BJ cells, our data suggest that X-irradiation does not lead to a significant rate of DSBs in bystander cells. Thus, neither bystander effect induced unstable chromosomal aberrations nor bystander effect induced DSBs are sufficiently pronounced to explain the apparent relationship between bystander effect and low-dose hypersensitivity. The experiments described here suggest that the hyper-radiosensitivity phenomenon might be related to bystander factor induced cell inactivation in the low-dose region (lower than 1 Gy).     

Metakaryotic stem cell lineages in organogenesis of humans and other metazoans

A non-eukaryotic, metakaryotic cell with large, open mouthed, bell shaped nuclei represents an important stem cell lineage in fetal/juvenile organogenesis in humans and rodents. each human bell shaped nucleus contains the diploid human DNA genome as tested by quantitative Feulgen DNA cytometry and fluorescent in situ hybridization with human pan-telomeric, pan-centromeric and chromosome specific probes. From weeks ∼5–12 of human gestation the bell shaped nuclei are found in organ anlagen enclosed in sarcomeric tubular syncytia. Within syncytia bell shaped nuclear number increases binomially up to 16 or 32 nuclei; clusters of syncytia are regularly dispersed in organ anlagen. Syncytial bell shaped nuclei demonstrate two forms of symmetrical amitoses, facing or “kissing” bells and “stacking” bells resembling separation of two paper cups. Remarkably, DNA increase and nuclear fission occur coordinately. Importantly, syncytial bell shaped nuclei undergo asymmetrical amitoses creating organ specific ensembles of up to eight distinct closed nuclear forms, a characteristic required of a stem cell lineage. Closed nuclei emerging from bell shaped nuclei are eukaryotic as demonstrated by their subsequent increases by extra-syncytial mitoses populating the parenchyma of growing anlagen. From 9–14 weeks syncytia fragment forming single cells with bell shaped nuclei that continue to display both symmetrical and asymmetrical amitoses. These forms persist in the juvenile period and are specifically observed in bases of colonic crypts. Metakaryotic forms are found in organogenesis of humans, rats, mice and the plant Arabidopsis indicating an evolutionary origin prior to the divergence of plants and animals.     

Uroplakin Peptide-Specific Autoimmunity Initiates Interstitial Cystitis/Painful Bladder Syndrome in Mice

The pathophysiology of interstitial cystitis/painful bladder syndrome (IC/PBS) is enigmatic. Autoimmunity and impaired urothelium might lead the underlying pathology. A major shortcoming in IC/PBS research has been the lack of an appropriate animal model. In this study, we show that the bladder specific uroplakin 3A-derived immunogenic peptide UPK3A 65–84, which contains the binding motif for IA^d MHC class II molecules expressed in BALB/c mice, is capable of inducing experimental autoimmune cystitis in female mice of that strain. A highly antigen-specific recall proliferative response of lymph node cells to UPK3A 65–84 was observed, characterized by selectively activated CD4+ T cells with a proinflammatory Th1-like phenotype, including enhanced production of interferon γ and interleukin-2. T cell infiltration of the bladder and bladder-specific increased gene expression of inflammatory cytokines were observed. Either active immunization with UPK3A 65–84 or adoptive transfer of peptide-activated CD4+ T cells induced all of the predominant IC/PBS phenotypic characteristics, including increased micturition frequency, decreased urine output per micturition, and increased pelvic pain responses to stimulation with von Frey filaments. Our study demonstrates the creation of a more specific experimental autoimmune cystitis model that is the first inducible model for IC/PBS that manifests all of the major symptoms of this debilitating condition.     

Quantification of dipalmitoyl lecithin in lung wash and extracts

A quantitative method for the measurement of dipalmitoyl lecithin in lung wash and extracts has been described. A known amount of tritiated dipalmitoyl lecithin is added to lung wash or tissues prior to homogenization and extraction. The lipid extract is subjected to a sequence of chromatographic separations, and after each separation the amount of radioactivity and phosphorus is determined. Specific activity is calculated in terms of disintegrations per minute per micromole of phosphorus. Constant specific activity is achieved in the last two thin-layer separations. The losses incurred in the procedure are determined by the recovery of radioactivity in the final purification step; the total amount of dipalmitoyl lecithin present in the pulmonary extract is calculated after correcting for losses. The accuracy, sensitivity, and precision of the method have been evaluated by using adult and fetal lung extracts as well as fetal lung wash. The sensitivity was found to be 0.24 mg and the coefficient of variance ranged between 3.0 and 6.9% with an average of 5%. Changes in dipalmitoyl lecithin levels as well as wet lung weight, dry lung weight, protein, and DNA have been reported.     

Biophysical characterization of an integrin-targeted lipopolyplex gene delivery vector

Nonviral gene delivery vectors now show good therapeutic potential: however, detailed characterization of the composition and macromolecular organization of such particles remains a challenge. This paper describes experiments to elucidate the structure of a ternary, targeted, lipopolyplex synthetic vector, the LID complex. This consists of a lipid component, Lipofectin (L) (1:1 DOTMA:DOPE), plasmid DNA (D), and a dual-function, cationic peptide component (I) containing DNA condensation and integrin-targeting sequences. Fluorophore-labeled lipid, peptide, and DNA components were used to formulate the vector, and the stoichiometry of the particles was established by fluorescence correlation spectroscopy (FCS). The size of the complex was measured by FCS, and the sizes of LID, L, LD, and ID complexes were measured by dynamic light scattering (DLS). Fluorescence quenching experiments and freeze-fracture electron microscopy were then used to demonstrate the arrangement of the lipid, peptide, and DNA components within the complex. These experiments showed that the cationic portion of the peptide, I, interacts with the plasmid DNA, resulting in a tightly condensed DNA-peptide inner core; this is surrounded by a disordered lipid layer, from which the integrin-targeting sequence of the peptide partially protrudes.     

Plant community responses to environmentally friendly piste management in northeast Iran

It is well‐known that pistes have adverse effects on alpine ecosystems. Previous studies urged that pistes should be installed and managed in the ways to minimize negative impacts on natural habitats. However, the impacts of this type of management on the plant communities are not widely studied. The aim of this study was to examine species composition and biodiversity changes in an environmentally friendly managed piste in northeast Iran. This piste has been established in a previously degraded alpine landscape. For the vegetation survey, we sampled 44 within and 28 off‐piste plots. Except for the piste management, other environmental factors were similar between the piste and off‐piste plots. Dominant species were determined, and variation in community composition of the two areas was visualized. Also, native species, phylogenetic, and functional Hill diversity of the two areas were compared. The results showed that there was a moderate differentiation in the species composition of the piste and off‐piste. Two palatable species (i.e., Bupleurum falcatum and Melica persica) were dominant in the piste and were not recorded in the off‐piste. The diversity calculations results showed that the species diversity of the piste was higher than that of the off‐piste. Phylogenetic diversity at the level of frequent and dominant plants showed a similar result. The piste had a higher functional diversity in terms of functional richness, and functional diversity of frequent and dominant plants. Our findings imply, after 10 years, species, phylogenetic, and functional diversity of the piste is significantly improved. Environmentally friendly piste management (EFPM) induced species composition change that led to emerging species that were absent in the off‐piste. We can conclude that EFPM led to restoration of a degraded landscape. Long‐term impacts of EFPM are still unknown, therefore, caution should be undertaken regarding the installation of new environmentally friendly pistes in other areas.