Hydrolytic Reactions of 3′-Deoxy-3′-thioinosylyl-(3′→ >5′)-uridine; An RNA Dinucleotide Containing a 3′-S-Phosphorothiolate Linkage

Abstract

The course of hydrolysis of 3′-deoxy-3′-thioinosylyl-(3′ → 5′)-uridine (IspU) has been followed by HPLC over a wide pH-range. Two reactions of the internucleosidic thiophosphate linkage compete: (i) cleavage yielding thioinosine monophosphates and uridine, and (ii) isomerization to the 2′,5′-isomer of IspU. Under very acidic conditions, even acid-catalyzed depurination of the inosine moiety is observed. The stability of the thiophosphate linkage and the mechanisms of its rupture are discussed.     

Resolution of (±)– Cytallene — A Highly Active Anti-HIV Agent with Axial Dissymmetry

Abstract

Anti-HIV agent (±)-cytallene (1b + 2b) was resolved by enantioselective acylation of (±)-N⁴-benzoylcytallene (1d + 2d) with vinyl butyrate in tetrahydrofuran catalyzed by lipase AK from Pseudomonas sp. and subsequent deacylation of 4d and 1d with ammonia in methanol. Optically pure enantiomers 1b and 2b were obtained.     

The ortholog of the human proto‐oncogene ROS1 is required for epithelial development in C. elegans

The orphan receptor ROS1 is a human proto‐oncogene, mutations of which are found in an increasing number of cancers. Little is known about the role of ROS1, however in vertebrates it has been implicated in promoting differentiation programs in specialized epithelial tissues. In this study we show that the C. elegans ortholog of ROS1, the receptor tyrosine kinase ROL‐3, has an essential role in orchestrating the morphogenesis and development of specialized epidermal tissues, highlighting a potentially conserved function in coordinating crosstalk between developing epithelial cells. We also provide evidence of a direct relationship between ROL‐3, the mucin SRAP‐1, and BCC‐1, the homolog of mRNA regulating protein Bicaudal‐C. This study answers a longstanding question as to the developmental function of ROL‐3, identifies three new genes that are expressed and function in the developing epithelium of C. elegans, and introduces the nematode as a potentially powerful model system for investigating the increasingly important, yet poorly understood, human oncogene ROS1. genesis 51:545–561. © 2013 Wiley Periodicals, Inc.     

Parasexuality and Ploidy Change in Candida tropicalis

Candida species exhibit a variety of ploidy states and modes of sexual reproduction. Most species possess the requisite genes for sexual reproduction, recombination, and meiosis, yet only a few have been reported to undergo a complete sexual cycle including mating and sporulation. Candida albicans, the most studied Candida species and a prevalent human fungal pathogen, completes its sexual cycle via a parasexual process of concerted chromosome loss rather than a conventional meiosis. In this study, we examine ploidy changes in Candida tropicalis, a closely related species to C. albicans that was recently revealed to undergo sexual mating. C. tropicalis diploid cells mate to form tetraploid cells, and we show that these can be induced to undergo chromosome loss to regenerate diploid forms by growth on sorbose medium. The diploid products are themselves mating competent, thereby establishing a parasexual cycle in this species for the first time. Extended incubation (>120 generations) of C. tropicalis tetraploid cells under rich culture conditions also resulted in instability of the tetraploid form and a gradual reduction in ploidy back to the diploid state. The fitness levels of C. tropicalis diploid and tetraploid cells were compared, and diploid cells exhibited increased fitness relative to tetraploid cells in vitro, despite diploid and tetraploid cells having similar doubling times. Collectively, these experiments demonstrate distinct pathways by which a parasexual cycle can occur in C. tropicalis and indicate that nonmeiotic mechanisms drive ploidy changes in this prevalent human pathogen.     

Distinct binding interactions of HIV-1 Gag to Psi and non-Psi RNAs: Implications for viral genomic RNA packaging

Despite the vast excess of cellular RNAs, precisely two copies of viral genomic RNA (gRNA) are selectively packaged into new human immunodeficiency type 1 (HIV-1) particles via specific interactions between the HIV-1 Gag and the gRNA psi (ψ) packaging signal. Gag consists of the matrix (MA), capsid, nucleocapsid (NC), and p6 domains. Binding of the Gag NC domain to ψ is necessary for gRNA packaging, but the mechanism by which Gag selectively interacts with ψ is unclear. Here, we investigate the binding of NC and Gag variants to an RNA derived from ψ (Psi RNA), as well as to a non-ψ region (TARPolyA). Binding was measured as a function of salt to obtain the effective charge (Z_eff) and nonelectrostatic (i.e., specific) component of binding, K_d(1M). Gag binds to Psi RNA with a dramatically reduced K_d(1M) and lower Z_eff relative to TARPolyA. NC, GagΔMA, and a dimerization mutant of Gag bind TARPolyA with reduced Z_eff relative to WT Gag. Mutations involving the NC zinc finger motifs of Gag or changes to the G-rich NC-binding regions of Psi RNA significantly reduce the nonelectrostatic component of binding, leading to an increase in Z_eff. These results show that Gag interacts with gRNA using different binding modes; both the NC and MA domains are bound to RNA in the case of TARPolyA, whereas binding to Psi RNA involves only the NC domain. Taken together, these results suggest a novel mechanism for selective gRNA encapsidation.     

Satellite microwave detection of boreal forest recovery from the extreme 2004 wildfires in Alaska and Canada

The rate of vegetation recovery from boreal wildfire influences terrestrial carbon cycle processes and climate feedbacks by affecting the surface energy budget and land‐atmosphere carbon exchange. Previous forest recovery assessments using satellite optical‐infrared normalized difference vegetation index (NDVI) and tower CO₂ eddy covariance techniques indicate rapid vegetation recovery within 5–10 years, but these techniques are not directly sensitive to changes in vegetation biomass. Alternatively, the vegetation optical depth (VOD) parameter from satellite passive microwave remote sensing can detect changes in canopy biomass structure and may provide a useful metric of post‐fire vegetation response to inform regional recovery assessments. We analyzed a multi‐year (2003–2010) satellite VOD record from the NASA AMSR‐E (Advanced Microwave Scanning Radiometer for EOS) sensor to estimate forest recovery trajectories for 14 large boreal fires from 2004 in Alaska and Canada. The VOD record indicated initial post‐fire canopy biomass recovery within 3–7 years, lagging NDVI recovery by 1–5 years. The VOD lag was attributed to slower non‐photosynthetic (woody) and photosynthetic (foliar) canopy biomass recovery, relative to the faster canopy greenness response indicated from the NDVI. The duration of VOD recovery to pre‐burn conditions was also directly proportional (P < 0.01) to satellite (moderate resolution imaging spectroradiometer) estimated tree cover loss used as a metric of fire severity. Our results indicate that vegetation biomass recovery from boreal fire disturbance is generally slower than reported from previous assessments based solely on satellite optical‐infrared remote sensing, while the VOD parameter enables more comprehensive assessments of boreal forest recovery.     

Investigating the long‐term legacy of drought and warming on the soil microbial community across five European shrubland ecosystems

We investigated how the legacy of warming and summer drought affected microbial communities in five different replicated long‐term (>10 years) field experiments across Europe (EU‐FP7 INCREASE infrastructure). To focus explicitly on legacy effects (i.e., indirect rather than direct effects of the environmental factors), we measured microbial variables under the same moisture and temperature in a brief screening, and following a pre‐incubation at stable conditions. Specifically, we investigated the size and composition of the soil microbial community (PLFA) alongside measurements of bacterial (leucine incorporation) and fungal (acetate in ergosterol incorporation) growth rates, previously shown to be highly responsive to changes in environmental factors, and microbial respiration. We found no legacy effects on the microbial community size, composition, growth rates, or basal respiration rates at the effect sizes used in our experimental setup (0.6 °C, about 30% precipitation reduction). Our findings support previous reports from single short‐term ecosystem studies thereby providing a clear evidence base to allow long‐term, broad‐scale generalizations to be made. The implication of our study is that warming and summer drought will not result in legacy effects on the microbial community and their processes within the effect sizes here studied. While legacy effects on microbial processes during perturbation cycles, such as drying–rewetting, and on tolerance to drought and warming remain to be studied, our results suggest that any effects on overall ecosystem processes will be rather limited. Thus, the legacies of warming and drought should not be prioritized factors to consider when modeling contemporary rates of biogeochemical processes in soil.     

Development of a competitive immunoassay for the determination of N-(2-hydroxypropyl)valine adducts in human haemoglobin and its application in biological monitoring

Abstract

Propylene oxide (PO) is an important industrial compound and a directly acting mutagen. Human exposure to PO can be monitored by the determination of haemoglobin adducts. An immunoassay that quantifies the N-terminal adduct N-(2-hydroxypropyl)valine in whole haemoglobin was developed and its potential usefulness as a tool for biologically monitoring occupational exposure was demonstrated. Analytical reliability was confirmed in a comparative study with GC-MS (range 3.7–992 nmol g^?1 haemoglobin (Hb), correlation coefficient 0.99, n=10). The assay has been configured as a competitive enzyme-linked immunosorbent assay to facilitate the rapid throughput of samples. The assay employs a whole blood matrix and has a working range of 2–250 pmol g^?1 Hb. It does not appear to be affected by structurally similar metabolites and has been used to determine adducts in human blood samples. The first results in potentially exposed workers indicate the assay's high potential usefulness in routine occupational biomonitoring of exposure to PO.