A phenotype-sensitizing Apoe-deficient genetic background reveals novel atherosclerosis predisposition loci in the mouse

Therapeutic intervention for atherosclerosis has predominantly concentrated on regulating cholesterol levels; however, these therapeutics are not efficacious for all patients, suggesting that other factors are involved. This study was initiated to identify mechanisms that regulate atherosclerosis predisposition in mice other than cholesterol level regulation. To do so we performed quantitative trait locus analysis using two inbred strains that each carry the atherosclerosis phenotype-sensitizing Apoe deficiency and that have been shown to have widely disparate predilection to atherosclerotic lesion formation. One highly significant locus on chromosome 10 (LOD = 7.8) accounted for 19% of the variance in lesion area independent of cholesterol. Two additional suggestive loci were identified on chromosomes 14 (LOD = 3.2) and 19 (LOD = 3.2), each accounting for 7-8% of the lesion variance. In all, five statistically significant and suggestive loci affecting lesion size but not lipoprotein levels were identified. Many of these were recapitulated in an independent confirmatory cross. In summary, two independently performed crosses between C57BL/6 and FVB/N Apoe-deficient mice have revealed several previously unreported atherosclerosis susceptibility loci that are distinct from loci linked to lipoprotein levels.     

Morphology of cell injury: An approach to the EDIT programme by the use of tobacco pollen tubes. Evaluation-guided Development of New In Vitro Test Batteries

Tobacco pollen tubes were used as a standard in vitro system to investigate cell growth aberrations caused by some of the Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) programme chemicals and other toxic compounds. Changes in cytoskeletal pattern were observed in the tube cells by using tubulin immunofluorescence and rhodamin-phalloidin fluorescence for the localisation of microtubules and actin filaments, respectively. Four different types of cell malformation were found: screw-like growth, isodiametric tip swelling, hook formation, and pollen grain enlargement. We suggest that these malformations resulted from an interference by the chemicals with the cytosolic calcium gradient which controls tip growth and the orientation of the pollen tube. The results may contribute to a general understanding of toxicity-based cell malformations.     

Neural correlates of target choice for pursuit and saccades in the primate superior colliculus

We have examined the role of the superior colliculus (SC) in choosing targets for pursuit and saccades by comparing neuronal activity at sites representing the possible choices. After recording during a two-alternative forced-choice paradigm, we measured the difference in activity of the populations representing the two choices by computing receiver operating characteristic (ROC) curves on a millisecond timescale. A signal indicating the correct choice emerged from noise over time, forming a tradeoff between speed and accuracy. The observed performance corresponded to particular points along the predicted speed-accuracy curves-pursuit emphasizing speed and saccades emphasizing accuracy. These results show that activity from the same set of neurons in the superior colliculus can predict target choices for both pursuit and saccades.     

Structural basis for the beta lactam resistance of PBP2a from methicillin-resistant Staphylococcus aureus

The multiple antibiotic resistance of methicillin-resistant strains of Staphylococcus aureus (MRSA) has become a major clinical problem worldwide. The key determinant of the broad-spectrum beta-lactam resistance in MRSA strains is the penicillin-binding protein 2a (PBP2a). Because of its low affinity for beta-lactams, PBP2a provides transpeptidase activity to allow cell wall synthesis at beta-lactam concentrations that inhibit the beta-lactam-sensitive PBPs normally produced by S. aureus. The crystal structure of a soluble derivative of PBP2a has been determined to 1.8 A resolution and provides the highest resolution structure for a high molecular mass PBP. Additionally, structures of the acyl-PBP complexes of PBP2a with nitrocefin, penicillin G and methicillin allow, for the first time, a comparison of an apo and acylated resistant PBP. An analysis of the PBP2a active site in these forms reveals the structural basis of its resistance and identifies features in newly developed beta-lactams that are likely important for high affinity binding.     

Depleted uranium-catalyzed oxidative DNA damage: absence of significant alpha particle decay

Depleted uranium (DU) is a dense heavy metal used primarily in military applications. Published data from our laboratory have demonstrated that DU exposure in vitro to immortalized human osteoblast cells (HOS) is both neoplastically transforming and genotoxic. DU possesses both a radiological (alpha particle) and a chemical (metal) component. Since DU has a low-specific activity in comparison to natural uranium, it is not considered to be a significant radiological hazard. In the current study we demonstrate that DU can generate oxidative DNA damage and can also catalyze reactions that induce hydroxyl radicals in the absence of significant alpha particle decay. Experiments were conducted under conditions in which chemical generation of hydroxyl radicals was calculated to exceed the radiolytic generation by one million-fold. The data showed that markers of oxidative DNA base damage, thymine glycol and 8-deoxyguanosine could be induced from DU-catalyzed reactions of hydrogen peroxide and ascorbate similarly to those occurring in the presence of iron catalysts. DU was 6-fold more efficient than iron at catalyzing the oxidation of ascorbate at pH 7. These data not only demonstrate that DU at pH 7 can induced oxidative DNA damage in the absence of significant alpha particle decay, but also suggest that DU can induce carcinogenic lesions, e.g. oxidative DNA lesions, through interaction with a cellular oxygen species.     

Engineered viruses to select genes encoding secreted and membrane-bound proteins in mammalian cells

We have developed a functional genomics tool to identify the subset of cDNAs encoding secreted and membrane-bound proteins within a library (the ‘secretome’). A Sindbis virus replicon was engineered such that the envelope protein precursor no longer enters the secretory pathway. cDNA fragments were fused to the mutant precursor and expression screened for their ability to restore membrane localization of envelope proteins. In this way, recombinant replicons were released within infectious viral particles only if the cDNA fragment they contain encodes a secretory signal. By using engineered viral replicons to selectively export cDNAs of interest in the culture medium, the methodology reported here efficiently filters genetic information in mammalian cells without the need to select individual clones. This adaptation of the ‘signal trap’ strategy is highly sensitive (1/200 000) and efficient. Indeed, of the 2546 inserts that were retrieved after screening various libraries, more than 97% contained a putative signal peptide. These 2473 clones encoded 419 unique cDNAs, of which 77% were previously annotated. Of the 94 cDNAs encoding proteins of unknown function, 24% either had no match in databases or contained a secretory signal that could not be predicted from electronic data.     

Expression of leptin and its receptor in the murine ovary: possible role in the regulation of oocyte maturation

Leptin is a product of the ob gene that is produced primarily by adipose tissue. Leptin and its receptors are found within the ovary, but it is unclear what function this hormone has in the ovary. Using immunohistochemistry, we determined that leptin is found in most cell types in the murine ovary, with the highest staining levels observed in the oocyte. Leptin receptor was also expressed in all of the main ovarian cell types, with the thecal cell layer exhibiting the highest staining levels. Leptin administration did not affect spontaneous or induced maturation of either isolated denuded oocytes or cumulus-oocyte complexes, but it did significantly increase the rate of meiotic resumption in preovulatory follicle-enclosed oocytes (P < 0.01). Measurements of cAMP within oocytes cultured with leptin showed that this enhanced ability to resume meiosis does not occur via activation of phosphodiesterase 3B and subsequent cAMP reduction. These results provide evidence that leptin affects oocyte maturation when the oocyte is cultured within its normal follicular environment. It is suggested that leptin may induce the production of another factor, possibly from thecal cells, that directly or indirectly acts on the oocyte to initiate germinal vesicle breakdown in this species.     

Cancer mortality risk among workers at the Mayak nuclear complex

At present, direct data on risk from protracted or fractionated radiation exposure at low dose rates have been limited largely to studies of populations exposed to low cumulative doses with resulting low statistical power. We evaluated the cancer risks associated with protracted exposure to external whole-body gamma radiation at high cumulative doses (the average dose is 0.8 Gy and the highest doses exceed 10 Gy) in Russian nuclear workers. Cancer deaths in a cohort of about 21,500 nuclear workers who began working at the Mayak complex between 1948 and 1972 were ascertained from death certificates and autopsy reports with follow-up through December 1997. Excess relative risk models were used to estimate solid cancer and leukemia risks associated with external gamma-radiation dose with adjustment for effects of plutonium exposures. Both solid cancer and leukemia death rates increased significantly with increasing gamma-ray dose (P < 0.001). Under a linear dose-response model, the excess relative risk for lung, liver and skeletal cancers as a group (668 deaths) adjusted for plutonium exposure is 0.30 per gray (P < 0.001) and 0.08 per gray (P < 0.001) for all other solid cancers (1062 deaths). The solid cancer dose-response functions appear to be nonlinear, with the excess risk estimates at doses of less than 3 Gy being about twice those predicted by the linear model. Plutonium exposure was associated with increased risks both for lung, liver and skeletal cancers (the sites of primary plutonium deposition) and for other solid cancers as a group. A significant dose response, with no indication of plutonium exposure effects, was found for leukemia. Excess risks for leukemia exhibited a significant dependence on the time since the dose was received. For doses received within 3 to 5 years of death the excess relative risk per gray was estimated to be about 7 (P < 0.001), but this risk was only 0.45 (P = 0.02) for doses received 5 to 45 years prior to death. External gamma-ray exposures significantly increased risks of both solid cancers and leukemia in this large cohort of men and women with occupational radiation exposures. Risks at doses of less than 1 Gy may be slightly lower than those seen for doses arising from acute exposures in the atomic bomb survivors. As dose estimates for the Mayak workers are improved, it should be possible to obtain more precise estimates of solid cancer and leukemia risks from protracted external radiation exposure in this cohort.     

Testing for Mycobacterium avium subsp. paratuberculosis infection in asymptomatic free-ranging tule elk from an infected herd

Forty-five adult tule elk (Cervus elaphus nannodes) in good physical condition were translocated from a population located at Point Reyes National Seashore, Marin County (California, USA), to a holding pen 6 mo prior to release in an unfenced region of the park. Because infection with Mycobacterium avium subsp. paratuberculosis (Mptb) had been reported in the source population, the translocated elk underwent extensive ante-mortem testing using three Johne's disease assays: enzyme linked immunosorbent assay (ELISA); agar gel immunodiffusion assay (AGID), and fecal culture. Isolation of Mptb was made from fecal samples in six of 45 elk (13%). All AGID results were negative while ELISA results for 18 elk (40%) were considered elevated. Elevated ELISA results or Mptb isolation from fecal samples were obtained for 22 of 45 elk (49%); these elk were euthanized and necropsied. Mycobacterium avium subsp. paratuberculosis was isolated from tissue in 10 of 22 euthanized elk (45%); of these 10 cases of confirmed infection, eight had elevated ELISA results (80%) and four were fecal culture positive (40%). One of 10 cases had histopathologic lesions consistent with Mptb infection. Mycobacterium avium subsp. paratuberculosis was also isolated from tissue from one of eight fetuses sampled. The number of tule elk found to be infected was unexpected, both because of the continued overall health of the source herd and the normal clinical status of all study animals.