Yeast as a model of human mitochondrial tRNA base substitutions: investigation of the molecular basis of respiratory defects

We investigate the relationships between acylation defects and structure alterations due to base substitutions in yeast mitochondrial (mt) tRNA(UUR)(Leu). The studied substitutions are equivalent to the A3243G and T3250C human pathogenetic tRNA mutations. Our data show that both mutations can produce tRNA(UUR)(Leu) acylation defects, although to a different extent. For mutant A14G (equivalent to MELAS A3243G base substitution), the presence of the tRNA and its defective aminoacylation could be observed only in the nuclear context of W303, a strain where the protein synthesis defects caused by tRNA base substitutions are far less severe than in previously studied strains. For mutant T20C (equivalent to the MM/CPEO human T3250C mutation), the acylation defect was less severe, and a thermosensitive acylation could be detected also in the MCC123 strain. The correlation between the severity of the in vivo phenotypes of yeast tRNA mutants and those obtained in in vitro studies of human tRNA mutants supports the view that yeast is a suitable model to study the cellular and molecular effects of tRNA mutations involved in human pathologies. Furthermore, the yeast model offers the possibility of modulating the severity of yeast respiratory phenotypes by studying the tRNA mutants in different nuclear contexts. The nucleotides at positions 14 and 20 are both highly conserved in yeast and human mt tRNAs; however, the different effect of their mutations can be explained by structure analyses and quantum mechanics calculations that can shed light on the molecular mechanisms responsible for the experimentally determined defects of the mutants.     

The Clinical Breast Care Project: an important resource in investigating environmental and genetic contributions to breast cancer in African American women

Age at diagnosis, pathological characteristics, and tumor behavior differ between African American (AAW) and Caucasian women (CW) with breast cancer, with AAW having more aggressive tumors and higher mortality rates. Although both societal and molecular contributions to these disparities have been suggested, the African American population has traditionally been under-represented in research and clinical protocols, limiting the power of epidemiologic and molecular studies to provide better understanding of disease pathogenesis in this minority population. The Clinical Breast Care Project (CBCP) has developed a large tissue and blood repository from patients undergoing treatment for breast cancer, with previous history of breast cancer, counseled in the Risk Reduction Clinic, screened by routine mammography, or undergoing elective reductive mammoplasty. Recruitment of AAW into the CBCP was successful; 25% of the 2,454 female patients were African American, including 35% disease-free, 3% high-risk, 40% benign, 8% preinvasive and 14% with invasive breast disease. More than 500 data fields regarding lifestyle choices, socioeconomic status, health history and geography were collected from all participants, and all consenting individuals provided blood specimens for genomic and proteomic studies. Tissues were collected from all patients undergoing surgical procedures using protocols that preserve the macromolecules for downstream research applications. In addition, patients were followed after diagnosis, with >85% of patients providing ongoing and updated demographic and clinical information. Thus, recruitment efforts in the CBCP have resulted in collection of well-annotated information and research-quality specimens from a large number of AAW. This unique resource will allow for the identification of biological and environmental factors associated with the identification of genetic and non-genetic factors associated with the occurrence, detection, prognosis, or survival of breast cancer in AAW. The opinion and assertions contained herein are the private views of the authors and are not to be construed as official or as representing the views of the Department of the Army or the Department of Defense.     

Survival of Infants Born to HIV-Positive Mothers,by Feeding Modality,in Rakai,Uganda

Background

Data comparing survival of formula-fed to breast-fed infants in programmatic settings are limited. We compared mortality and HIV-free of breast and formula-fed infants born to HIV-positive mothers in a program in rural, Rakai District Uganda.

Methodology/Principal Findings

One hundred eighty two infants born to HIV-positive mothers were followed at one, six and twelve months postpartum. Mothers were given infant-feeding counseling and allowed to make informed choices as to whether to formula-feed or breast-feed. Eligible mothers and infants received antiretroviral therapy (ART) if indicated. Mothers and their newborns received prophylaxis for prevention of mother-to-child HIV transmission (pMTCT) if they were not receiving ART. Infant HIV infection was detected by PCR (Roche Amplicor 1.5) during the follow-up visits. Kaplan Meier time-to-event methods were used to compare mortality and HIV-free survival. The adjusted hazard ratio (Adjusted HR) of infant HIV-free survival was estimated by Cox regression. Seventy-five infants (41%) were formula-fed while 107 (59%) were breast-fed. Exclusive breast-feeding was practiced by only 25% of breast-feeding women at one month postpartum. The cumulative 12-month probability of infant mortality was 18% (95% CI = 11%–29%) among the formula-fed compared to 3% (95% CI = 1%–9%) among the breast-fed infants (unadjusted hazard ratio (HR)  = 6.1(95% CI = 1.7–21.4, P-value<0.01). There were no statistically significant differentials in HIV-free survival by feeding choice (86% in the formula-fed compared to 96% in breast-fed group (Adjusted RH = 2.8[95%CI = 0.67–11.7, P-value = 0.16]

Conclusions/Significance

Formula-feeding was associated with a higher risk of infant mortality than breastfeeding in this rural population. Our findings suggest that formula-feeding should be discouraged in similar African settings.     

Functional characterization of integrin alpha6beta4 adhesion interactions using soluble integrin constructs reveals the involvement of different functional domains in the beta4 subunit

Integrin alpha6beta4-mediated adhesion interactions play key roles in keratinocyte and epithelial tumor cell biology. In order to evaluate how alpha6beta4 adhesion interactions contribute to these important cellular processes, the authors generated soluble versions of the integrin by recombinant expression of the subunit ectodomains fused to a human immunoglobulin G (IgG) Fc constant domain. Coexpression of the appropriate subunits enabled dimerization, secretion and purification of stable Fc-containing alpha6beta4 heterodimers. The soluble proteins exhibited the same metal ion and ligand dependency in their binding characteristics as intact alpha6beta4. Using these reagents in combination with anti-beta4 antibodies, the authors identified two distinct functional epitopes on the beta4 subunit. They demonstrated the involvement of one epitope in adhesion interactions and the other in regulating adhesion-independent growth in alpha6beta4-expressing tumor cell lines. The availability of these soluble integrin reagents and the data provided herein help to further delineate the structure-function relationships regulating alpha6beta4 signaling biology.     

Mobiluncus curtisii bacteremia

Mobiluncus curtisii was isolated from the blood of a 35-year-old man with a medical history of ulcerative colitis who was admitted unconscious to the Intensive Care Unit (ICU). A CT scan revealed massive intracerebral hemorrhage in the left hemisphere. Temperature remained constant over 38.5 degrees C; therefore, two sets of blood cultures were collected. One anaerobic bottle BacT/ALERT SN (bioMerieux, France) was detected as positive after 5 days of incubation and a Gram stain confirmed a gram variable curved-shaped rod. The patient died 18 h after being admitted to the hospital.     

Wide confocal cytometry: a new approach to study proteomic and structural changes in the cell nucleus during the cell cycle

Wide-confocal-cytometry (WCC) is a new method developed in this paper that uses a standard confocal system to gather quantitative information on contents and fine structural details of cells. The system is operated under conditions of non-confocality, in order to capture the maximum amount of light emitted by the specimen (comparable to LSC). After analysis of macromolecule content (DNA, RNA, specific proteins, lipids, etc.), cells can be sampled using conventional confocal microscopy. We analyzed the illumination and acquiring capabilities of WCC. The quantitative power of WCC was validated by analysis of cell cycle stage in Hela cells, looking at DNA content and markers for S phase and mitosis. As an example of the potential of this methodology we have documented changes in cell nucleus during the cell cycle. After mitosis the cell nucleus changes its shape from elongated to ellipsoid and remains constant until G2. This change is associated with nuclear volume increase. As nuclear volume increases, chromatin becomes decondensed in an isometric manner, probably due to the increase in gene expression and factors necessary for RNA metabolism.     

Azospirillum amazonense inoculation: effects on growth, yield and N2 fixation of rice (Oryza sativa L.)

Bacteria of the genus Azospirillum are considered to be plant-growth promoting bacteria (PGPR) and stimulate plant growth directly either by synthesising phyto-hormones or by promoting nutrition by the process of biological nitrogen fixation (BNF). Although this genus extensively studied, the effects of inoculation and the possible BNF contribution of the Azospirillum amazonense specie are very scarce. The aim of this study was to isolate, characterise and evaluate auxin production and nitrogenase activity of this species and to select, by inoculation of rice plants, promising isolates based on their ability to improve plant growth, yield and the BNF contribution. One hundred and ten isolates obtained from rice were characterised and grouped according to colony features. Forty-two isolates, confirmed as A. amazonense by the fluorescent in situ hybridization (FISH) technique, were tested for auxin production and nitrogenase activity in vitro. Subsequently plant growth promotion related to plant nutrition effect was evaluated, in vitro and in greenhouse experiments. The BNF contribution to plant growth was evaluated using the ¹⁵N isotope dilution technique. All A. amazonense strains tested produced indoles, but only 10% of them showed high production, above 1.33 μM mg protein⁻¹. The nitrogenase activity also was variable and only 9% of isolates showed high nitrogenase activity and the majority (54%) exhibited a low potential. The inoculation of selected strains in rice under gnotobiotic conditions reduced the growth of root and aerial part when compared to the control, showing the negative effects of excess of phytohormone accumulation in the medium. However, in the greenhouse experiment, inoculation of strains of A. amazonense increased grain dry matter accumulation (7 to 11.6%), the number of panicles (3 to 18.6%) and nitrogen accumulation at grain maturation (3.5 to 18.5%). BNF contributions up to 27% were observed for A. amazonense Y2 (wild type strain). The data presented here is the first report that the PGPR effect of A. amazonense for rice plants grown under greenhouse conditions is mainly due the BNF contribution as measured by ¹⁵N isotope dilution technique, in contrast to the hormonal effect observed with other Azospirillum species studied.     

Physical and biological organ dosimetry analysis for international space station astronauts

In this study, we analyzed the biological and physical organ dose equivalents for International Space Station (ISS) astronauts. Individual physical dosimetry is difficult in space due to the complexity of the space radiation environment, which consists of protons, heavy ions and secondary neutrons, and the modification of these radiation types in tissue as well as limitations in dosimeter devices that can be worn for several months in outer space. Astronauts returning from missions to the ISS undergo biodosimetry assessment of chromosomal damage in lymphocyte cells using the multicolor fluorescence in situ hybridization (FISH) technique. Individual-based pre-flight dose responses for lymphocyte exposure in vitro to gamma rays were compared to those exposed to space radiation in vivo to determine an equivalent biological dose. We compared the ISS biodosimetry results, NASA's space radiation transport models of organ dose equivalents, and results from ISS and space shuttle phantom torso experiments. Physical and biological doses for 19 ISS astronauts yielded average effective doses and individual or population-based biological doses for the approximately 6-month missions of 72 mSv and 85 or 81 mGy-Eq, respectively. Analyses showed that 80% or more of organ dose equivalents on the ISS are from galactic cosmic rays and only a small contribution is from trapped protons and that GCR doses were decreased by the high level of solar activity in recent years. Comparisons of models to data showed that space radiation effective doses can be predicted to within about a +/-10% accuracy by space radiation transport models. Finally, effective dose estimates for all previous NASA missions are summarized.     

Global response of terrestrial ecosystem structure and function to CO2 and climate change: results from six dynamic global vegetation models

The possible responses of ecosystem processes to rising atmospheric CO₂ concentration and climate change are illustrated using six dynamic global vegetation models that explicitly represent the interactions of ecosystem carbon and water exchanges with vegetation dynamics. The models are driven by the IPCC IS92a scenario of rising CO₂ ( Wigley et al. 1991 ), and by climate changes resulting from effective CO₂ concentrations corresponding to IS92a, simulated by the coupled ocean atmosphere model HadCM2‐SUL. Simulations with changing CO₂ alone show a widely distributed terrestrial carbon sink of 1.4–3.8 Pg C y^?1 during the 1990s, rising to 3.7–8.6 Pg C y^?1 a century later. Simulations including climate change show a reduced sink both today (0.6–3.0 Pg C y^?1) and a century later (0.3–6.6 Pg C y^?1) as a result of the impacts of climate change on NEP of tropical and southern hemisphere ecosystems. In all models, the rate of increase of NEP begins to level off around 2030 as a consequence of the ‘diminishing return’ of physiological CO₂ effects at high CO₂ concentrations. Four out of the six models show a further, climate‐induced decline in NEP resulting from increased heterotrophic respiration and declining tropical NPP after 2050. Changes in vegetation structure influence the magnitude and spatial pattern of the carbon sink and, in combination with changing climate, also freshwater availability (runoff). It is shown that these changes, once set in motion, would continue to evolve for at least a century even if atmospheric CO₂ concentration and climate could be instantaneously stabilized. The results should be considered illustrative in the sense that the choice of CO₂ concentration scenario was arbitrary and only one climate model scenario was used. However, the results serve to indicate a range of possible biospheric responses to CO₂ and climate change. They reveal major uncertainties about the response of NEP to climate change resulting, primarily, from differences in the way that modelled global NPP responds to a changing climate. The simulations illustrate, however, that the magnitude of possible biospheric influences on the carbon balance requires that this factor is taken into account for future scenarios of atmospheric CO₂ and climate change.