Characterization of RNA primers synthesized by the human breast cancer cell DNA synthesome

We previously reported on the purification and characterization of a functional multi‐protein DNA replication complex (the DNA synthesome) from human cells and tissues. The synthesome is fully competent to carry‐out all phases of the DNA replication process in vitro. In this study, DNA primase, a component of the synthesome, is examined to determine its activity and processivity in the in vitro synthesis and extension of RNA primers. Our results show that primase activity in the P4 fraction of the synthesome is 30‐fold higher than that of crude cell extracts. The synthesome synthesizes RNA primers that are 7–10 ribonucleotides long and DNA primers that are 20–40 deoxyribonucleotides long using a poly(dT) template of exogenous single‐stranded DNA. The synthesome‐catalyzed RNA primers can be elongated by E. coli DNA polymerase I to form the complementary DNA strands on the poly(dT) template. In addition, the synthesome also supports the synthesis of native RNA primers in vitro using an endogenous supercoiled double‐stranded DNA template. Gel analysis demonstrates that native RNA primers are oligoribonucleotides of 10–20 nt in length and the primers are covalently link to DNA to form RNA‐primed nascent DNA of 100–200 nt. Our study reveals that the synthesome model is capable of priming and continuing DNA replication. The ability of the synthesome to synthesize and extend RNA primers in vitro elucidates the organizational and functional properties of the synthesome as a potentially useful replication apparatus to study the function of primase and the interaction of primase with other replication proteins. J. Cell. Biochem. 106: 798–811, 2009. © 2009 Wiley‐Liss, Inc.     

The alpha-amylase gene in Drosophila melanogaster: nucleotide sequence, gene structure and expression motifs.

We present the complete nucleotide sequence of a Drosophila alpha-amylase gene and its flanking regions, as determined by cDNA and genomic sequence analysis. This gene, unlike its mammalian counterparts, contains no introns. Nevertheless the insect and mammalian genes share extensive nucleotide similarity and the insect protein contains the four amino acid sequence blocks common to all alpha-amylases. In Drosophila melanogaster, there are two closely-linked copies of the alpha-amylase gene and they are divergently transcribed. In the 5'-regions of the two gene-copies we find high sequence divergence, yet the typical eukaryotic gene expression motifs have been maintained. The 5'-terminus of the alpha-amylase mRNA, as determined by primer extension analysis, maps to a characteristic Drosophila sequence motif. Additional conserved elements upstream of both genes may also be involved in amylase gene expression which is known to be under complex controls that include glucose repression.     

Study of coronary risk factors related to physical activity in 15 171 men.

Physical activity, both at work and during leisure, was assessed in 15 171 men aged 25-74 years. Heavy leisure activity was associated with lower mean serum cholesterol levels and blood pressure. In men under 60 years the same negative association was also noted between leisure activity and relative weight and cigarette smoking. Different degrees of physical activity at work were not associated with any differences in these risk factors, nor did the level of exercise at work seem to influence the negative association between leisure activity and risk factors. Heavy leisure activity in young and middle-aged men is associated with lower levels of certain coronary risk factors and, therefore, a lower risk of coronary heart disease. The often-reported reduction in coronary morbidity and mortality with physical exercise may not be the direct effect of the exercise itself.     

Neurolysin: From Initial Detection to Latest Advances

The mechanisms by which peptidergic signals are terminated have been the center of multiple studies leading to the discoveries of novel proteolytic activities. When studying the catabolic fate of neurotensin (NT) in brain and gastrointestinal tract, we detected a novel activity belonging to the metallopeptidases class and apparently distinct from previously known enzymes. Purification and cloning confirmed that this NT-degrading neutral metalloendopeptidase activity was indeed original. It was named endopeptidase 3.4.24.16 according to the IUBMB nomenclature and later, referred to as neurolysin. This review tells the history of neurolysin from its initial detection to its purification, cloning, design of specific inhibitors as well as in vitro and in vivo pharmacological studies aimed at delineating its role in the control of NT function. Finally, we discuss very recent advances suggesting a potential role of neurolysin in pathologies.     

Does stopping smoking delay onset of angina after infarction?

This study was designed to determine the relation between stopping smoking and angina after infarction in survivors of an acute coronary attack. The study population comprised 408 men aged under 60 who survived a first attack of unstable angina or myocardial infarction by 28 days and were smoking cigarettes at the time of their attack. These patients were followed up for an average of nine years. Three hundred and eighty four were alive at the one year follow up examination, when the presence or absence of angina together with habits of smoking were recorded. The prevalence of angina at one year was 19.5% in the 241 who had stopped smoking cigarettes compared with 32.2% in those who had continued (p less than 0.01). Six years later, however, the prevalence of angina after infarction was the same in the two groups. It is concluded that the onset of angina after infarction can be delayed by stopping smoking cigarettes but that this effect is not maintained in the long term.     

Climate change impacts on potential recruitment in an ecosystem engineer

Climate variability and the rapid warming of seas undoubtedly have huge ramifications for biological processes such as reproduction. As such, gametogenesis and spawning were investigated at two sites over 200 km apart on the south coast of Ireland in an ecosystem engineer, the common cockle, Cerastoderma edule. Both sites are classed as Special Areas of Conservation (SACs), but are of different water quality. Cerastoderma edule plays a significant biological role by recycling nutrients and affecting sediment structure, with impacts upon assemblage biomass and functional diversity. It plays a key role in food webs, being a common foodstuff for a number of marine birds including the oystercatcher. Both before and during the study (early 2010–mid 2011), Ireland experienced its two coldest winters for 50 years. As the research demonstrated only slight variation in the spawning period between sites, despite site differences in water and environmental quality, temperature and variable climatic conditions were the dominant factor controlling gametogenesis. The most significant finding was that the spawning period in the cockle extended over a greater number of months compared with previous studies and that gametogenesis commenced over winter rather than in spring. Extremely cold winters may impact on the cockle by accelerating and extending the onset and development of gametogenesis. Whether this impact is positive or negative would depend on the associated events occurring on which the cockle depends, that is, presence of primary producers and spring blooms, which would facilitate conversion of this extended gametogenesis into successful recruitment.     

Initial Characterization of Micafungin Pulmonary Delivery via Two Different Nebulizers and Multivariate Data Analysis of Aerosol Mass Distribution Profiles

Pharmaceutical aerosols have been targeted to the lungs for the treatment of asthma and pulmonary infectious diseases successfully. Micafungin (Astellas Pharma US, Deerfield, IL, USA) has been shown to be an effective antifungal agent when administrated intravenously. Pulmonary delivery of micafungin has not previously been reported. In the present pilot study, we characterize the performance of two nebulizers and their potential for delivering micafungin to the lungs as well as the use of multivariate data analysis for mass distribution profile comparison. The concentration of micafungin sodium increased by 21% when delivered by the Acorn II nebulizer and by 20% when delivered by the LC Plus nebulizer, respectively, from the first to the second sampling period. The Acorn II nebulizer delivered a fine particle fraction FPF_5.8 (%<5.8 μm) of 92.5 ± 0.8 and FPF_3.3 (%<3.3 μm) of 82.3 ± 2.1 during the first sampling period. For the LC Plus nebulizer, FPF_5.8 was 92.3 ± 0.1 and FPF_3.3 was 67.0 ± 0.7 during the first sampling period. The mass median aerodynamic diameter (MMAD) increased from 1.67 ± 0.05 to 1.77 ± 0.04 μm (Acorn II nebulizer) and from 2.09 ± 0.01 to 2.20 ± 0.01 μm (Pari LC Plus nebulizer) from the first to the second sampling periods. These changes in MMAD were statistically significant by paired t test. Multivariate data analysis showed that this could be explained systematically by greater drug deposition on stages with larger cutoff sizes and reduced drug deposition on stages with smaller cutoff sizes rather than multimodal deposition or other anomalies in size distribution.     

Identifying habitat conservation priorities and gaps for migratory shorebirds and waterfowl in California

Conservation of migratory shorebirds and waterfowl presents unique challenges due to extensive historic loss of wetland habitats, and current reliance on managed landscapes for wintering and migratory passage. We developed a spatially-explicit approach to estimate potential shorebird and waterfowl densities in California by integrating mapped habitat layers and statewide bird survey data with expert-based habitat rankings. Using these density estimates as inputs, we used the Marxan site-selection program to identify priority shorebird and waterfowl areas at the ecoregional level. We identified 3.7 million ha of habitat for shorebirds and waterfowl, of which 1.4 million ha would be required to conserve 50% of wintering populations. To achieve a conservation goal of 75%, more than twice as much habitat (3.1 million ha) would be necessary. Agricultural habitats comprised a substantial portion of priority areas, especially at the 75% level, suggesting that under current management conditions, large areas of agricultural land, much of it formerly wetland, are needed to provide the habitat availability and landscape connectivity required by shorebird and waterfowl populations. These habitats were found to be largely lacking recognized conservation status in California (96% un-conserved), with only slightly higher levels of conservation for priority shorebird and waterfowl areas. Freshwater habitats, including wetlands and ponds, were also found to have low levels of conservation (67% un-conserved), although priority shorebird and waterfowl areas had somewhat higher levels of conservation than the state as a whole. Conserving migratory waterfowl and shorebirds will require a diversity of conservation strategies executed at a variety of scales. Our modeled results are complementary with other approaches and can help prioritize areas for protection, restoration and other actions. Traditional habitat protection strategies such as conservation easements and fee acquisitions may be of limited utility for protecting and managing significant areas of agricultural lands. Instead, conservation strategies focused on incentive-based programs to support wildlife friendly management practices in agricultural settings may have greater utility and conservation effectiveness.