Spectrum of complex DNA damages depends on the incident radiation

Ionizing radiation induces bistranded clustered damages--two or more abasic sites, oxidized bases and strand breaks on opposite DNA strands within a few helical turns. Since clusters are refractory to repair and are potential sources of double-strand breaks (DSBs), they are potentially lethal and mutagenic. Although induction of single-strand breaks (SSBs) and isolated lesions has been studied extensively, little is known about the factors affecting induction of clusters other than DSBs. To determine whether the type of incident radiation could affect the yields or spectra of specific clusters, we irradiated genomic T7 DNA, a simple 40-kbp linear, blunt-ended molecule, with ion beams [iron (970 MeV/nucleon), carbon (293 MeV/nucleon), titanium (980 MeV/nucleon), silicon (586 MeV/nucleon), protons (1 GeV/nucleon)] or 100 kVp X rays and then quantified DSBs, Fpg-oxypurine clusters and Nfo-abasic clusters using gel electrophoresis, electronic imaging and number average length analysis. The yields (damages/Mbp Gy(-1)) of all damages decreased with increasing linear energy transfer (LET) of the radiation. The relative frequencies of DSBs compared to abasic and oxybase clusters were higher for the charged particles-including the high-energy, low-LET protons-than for the ionizing photons.     

c-myc as a mediator of accelerated apoptosis and involution in mammary glands lacking Socs3

Suppressor of cytokine signalling (SOCS) proteins are critical attenuators of cytokine-mediated signalling in diverse tissues. To determine the importance of Socs3 in mammary development, we generated mice in which Socs3 was deleted in mammary epithelial cells. No overt phenotype was evident during pregnancy and lactation, indicating that Socs3 is not a key physiological regulator of prolactin signalling. However, Socs3-deficient mammary glands exhibited a profound increase in epithelial apoptosis and tissue remodelling, resulting in precocious involution. This phenotype was accompanied by augmented Stat3 activation and a marked increase in the level of c-myc. Moreover, induction of c-myc before weaning using an inducible transgenic model recapitulated the Socs3 phenotype, and elevated expression of likely c-myc target genes, E2F-1, Bax and p53, was observed. Our data establish Socs3 as a critical attenuator of pro-apoptotic pathways that act in the developing mammary gland and provide evidence that c-myc regulates apoptosis during involution.     

Why long-lived species are more likely to be social: the role of local dominance

Recent studies have shown that individuals of species that livein groups tend to have high annual survival, but this link haslacked a theoretical explanation. We evaluate two hypothesesthat explain how longevity could have led to the evolution ofgroup living. The first is the territory inheritance hypothesis,and it proposes that longevity increases the probability ofnonbreeding subordinates surviving long enough to have the opportunityof inheriting their natal territory. Second, we propose a novelhypothesis, the reciprocal altruism hypothesis, which is thatlongevity increases local dominance by favoring nonaggressionpacts among neighboring residents because longevity increasesthe likelihood of reciprocal altruism. Birds thus accept subordinateresidency because the exclusion of nonlocal birds will meanthat, if they survive long enough, they will be likely to actuallyachieve territory inheritance. The reciprocal altruism hypothesisis supported by a wider array of evidence; becomes progressivelymore powerful as longevity increases, thus producing a positivefeedback; explains the evolution of local dominance (whereasthe territory inheritance hypothesis assumes its existence);and provides an explanation for why cooperative breeding shouldbe found more often in aseasonal environments.     

Discovery of novel inhibitors of the ZipA/FtsZ complex by NMR fragment screening coupled with structure-based design

ZipA is a membrane anchored protein in Escherichia coli that interacts with FtsZ, a homolog of eukaryotic tubulins, forming a septal ring structure that mediates bacterial cell division. Thus, the ZipA/FtsZ protein-protein interaction is a potential target for an antibacterial agent. We report here an NMR-based fragment screening approach which identified several hits that bind to the C-terminal region of ZipA. The screen was performed by 1H-15N HSQC experiments on a library of 825 fragments that are small, lead-like, and highly soluble. Seven hits were identified, and the binding mode of the best one was revealed in the X-ray crystal structure. Similar to the ZipA/FtsZ contacts, the driving force in the binding of the small molecule ligands to ZipA is achieved through hydrophobic interactions. Analogs of this hit were also evaluated by NMR and X-ray crystal structures of these analogs with ZipA were obtained, providing structural information to help guide the medicinal chemistry efforts.     

Mechanisms of action of green tea catechins, with a focus on ischemia-induced neurodegeneration

Catechins are dietary polyphenolic compounds associated with a wide variety of beneficial health effects in vitro, in vivo and clinically. These therapeutic properties have long been attributed to the catechins' antioxidant and free radical scavenging effects. Emerging evidence has shown that catechins and their metabolites have many additional mechanisms of action by affecting numerous sites, potentiating endogenous antioxidants and eliciting dual actions during oxidative stress, ischemia and inflammation. Catechins have proven to modulate apoptosis at various points in the sequence, including altering expression of anti- and proapoptotic genes. Their anti-inflammatory effects are activated through a variety of different mechanisms, including modulation of nitric oxide synthase isoforms. Catechins' actions of attenuating oxidative stress and the inflammatory response may, in part, account for their confirmed neuroprotective capabilities following cerebral ischemia. The versatility of the mechanisms of action of catechins increases their therapeutic potential as interventions for numerous clinical disorders. However, more epidemiological and clinical studies need to be undertaken for their efficacy to be fully elucidated.     

The saponin-mediated enhanced uptake of targeted saporin-based drugs is strongly dependent on the saponin structure

Saponins are a group of plant glycosides consisting of a steroid or triterpenoid aglycone to which one or more sugar chains are attached. They exhibit cell membrane-permeabilizing properties and, thus, have been investigated for their therapeutic potential. Recently, at a non-permeabilizing concentration saponinum album from Gypsophila paniculata L. has been described to enhance the cytotoxicity of a chimeric toxin in a cell culture model. To elucidate whether this enhancing effect is also mediated by other saponins, we analyzed the ability of seven different saponins to enhance the cytotoxicity of a targeted chimeric toxin. The chimeric toxin is composed of saporin, a plant ribosome-inactivating toxin, a cleavable adapter, and human epidermal growth factor (EGF). Cytotoxicity on EGF receptor (EGFR)-bearing cells was analyzed both alone and after combined application of saponin and chimeric toxin. Only two of the tested saponins, quillajasaponin and saponinum album, enhanced cytotoxicity by more than 1,000-fold, whereas the enhancement factors of the other saponins were only approximately 10-fold. In contrast to saponinum album, quillajasaponin enhanced the cytotoxicity both on control cells lacking EGFR and on target cells, indicating that, in this case, the enhancement is not target cell receptor specific. This is also the case for some of the saponins with low enhancement factors. Saponinum album resulted in a more than 13,600-fold receptor-specific enhancement, decreasing the 50% inhibitory concentration (IC(50)) from 2.4 nM to 0.18 pM, which renders it the best option to promote saporin-3-based drug uptake while retaining specificity for the EGFR.     

Immunoglobulin GM and KM allotypes and prevalence of anti-LKM1 autoantibodies in patients with hepatitis C virus infection

GM and KM allotypes-genetic markers of immunoglobulin (Ig) gamma and kappa chains, respectively-are associated with humoral immunity to several infection- and autoimmunity-related epitopes. We hypothesized that GM and KM allotypes contribute to the generation of autoantibodies to liver/kidney microsomal antigen 1 (LKM1) in hepatitis C virus (HCV)-infected persons. To test this hypothesis, we characterized 129 persons with persistent HCV infection for several GM and KM markers and for anti-LKM1 antibodies. The heterozygous GM 1,3,17 23 5,13,21 phenotype was significantly associated with the prevalence of anti-LKM1 antibodies (odds ratio, 5.13; P=0.002), suggesting its involvement in this autoimmune phenomenon in HCV infection.     

Fire, native species, and soil resource interactions influence the spatio-temporal invasion pattern of Bromus tectorum

Bromus tectorum (cheatgrass) is an invasive annual that occupies perennial grass and shrub communities throughout the western United States. Bronus tectorum exhibits an intriguing spatio‐temporal pattern of invasion in low elevation ponderosa pine Pinus ponderosa/bunchgrass communities in western Montana where it forms dense rings beneath solitary pines following fire. This pattern provides a unique opportunity to investigate several indirect effects of native vegetation that influence the invasion pattern of B. tectorum, and specifically how native species, disturbance, and soil resources interact to influence the spatio‐temporal pattern of invasion. We established four replicate field sites, each containing burned‐tree, burned‐grass, unburned‐tree, and unburned‐grass sampling locations, and initiated a series of field sampling and greenhouse experiments utilizing these locations. The objective of our first greenhouse experiment was to identify whether belowground factors contributed to the pattern of B. tectorum biomass observed in these field locations. This experiment generated a B. tectorum biomass response that was nearly identical to the invasion pattern observed in the field, suggesting further investigation of belowground factors was necessary. We measured resin‐sorbed NH₄⁺ and NO₃⁻ during one generation of B. tectorum, and measured a suite of P fractions through a sequential extraction procedure from these soils. These data revealed that a resource island of high N and P exists beneath pine trees. Through a second greenhouse experiment, we determined that N limited B. tectorum biomass in tree soil, whereas P limited biomass in bunchgrass soil. Finally, through a germination experiment we determined that pine litter strongly inhibited B. tectorum germination. These data suggest B. tectorum is regulated by P in bunchgrass soil, and by N and inhibition by pine litter beneath trees, effects that are likely alleviated by fire. These data demonstrate the combined role of direct and indirect interactions between native and invasive species in regulating biological invasions.     

Quaternary structure of the mitochondrial TIM23 complex reveals dynamic association between Tim23p and other subunits

Tim23p is an essential channel-forming component of the multisubunit TIM23 complex of the mitochondrial inner membrane that mediates protein import. Radiolabeled Tim23p monocysteine mutants were imported in vitro, incorporated into functional TIM23 complexes, and subjected to chemical cross-linking. Three regions of proximity between Tim23p and other subunits of the TIM23 complex were identified: Tim17p and the first transmembrane segment of Tim23p; Tim50p and the C-terminal end of the Tim23p hydrophilic region; and the entire hydrophilic domains of Tim23p molecules. These regions of proximity reversibly change in response to changes in membrane potential across the inner membrane and also when a translocating substrate is trapped in the TIM23 complex. These structural changes reveal that the macromolecular arrangement within the TIM23 complex is dynamic and varies with the physiological state of the mitochondrion.     

Primary Culture of Neural Cells Isolated from the Cerebellum of Newborn and Adult Mucopolysaccharidosis Type IIIA Mice

In order to evaluate the mechanisms leading to neuropathology in Mucopolysaccharidosis type IIIA (MPS-IIIA, Sanfilippo syndrome), we have harvested and cultured primary neural cells isolated from the cerebellum of newborn and adult MPS-IIIA and unaffected mice. Cell viability and plating efficiency were comparable for brain tissue obtained from either newborn or adult MPS-IIIA and unaffected mice. Cultures (newborn and adult) comprised a mixed brain cell population including astrocytes, oligodendrocytes, and neurons. Newborn MPS-IIIA cells contained inclusions and vacuoles consistent with the pathology present in affected brain tissue. Newborn and adult MPS-IIIA brain cells had approximately 5–7% of the sulfamidase activity present in primary neural cells cultured from unaffected newborn and adult mice. In addition, high levels of glucosamine-N-sulfate[α-1,4]hexuronic acid, a heparan sulfate-derived disaccharide, were detected in both newborn and adult MPS-IIIA brain cells. These results suggest that the primary MPS-IIIA brain cells exhibit characteristics of MPS-IIIA phenotype at the histopathological and biochemical level in culture.