DNA damage repair and transcription

DNA mutations and aberrations are a problem for all forms of life. Eukaryotes specifically have developed ways of identifying and repairing various DNA mutations in a complex and refractory chromatin environment. The chromatin structure is much more than a packaging unit for DNA; it is dynamic. Cells utilize and manipulate chromatin for gene regulation, genome organization and maintenance of genome integrity. Once a DNA aberration has occurred, the various DNA repair machineries interact with chromatin proteins, such as the histone variant H2A.X, and chromatin remodeling machines of the SWI/SNF family to gain access and repair the lesion in a timely manner. Recent studies have thus begun to address the roles of chromatin proteins in DNA repair as well as to dissect the functions of DNA repair machinery in vitro on more physiological, nucleosomal templates.     

Targeted gene alteration in Caenorhabditis elegans by gene conversion

Now that some genomes have been completely sequenced, the ability to direct specific mutations into genomes is particularly desirable. Here we present a method to create mutations in the Caenorhabditis elegans genome efficiently through transgene-directed, transposon-mediated gene conversion. Engineered deletions targeted into two genes show that the frequency of obtaining the desired mutation was higher using this approach than using standard transposon insertion-deletion approaches. We also targeted an engineered green fluorescent protein insertion-replacement cassette to one of these genes, thereby confirming that custom alleles of different types can be created in vitro to make the corresponding mutations in vivo. This approach should also be applicable to heterologous transposons in C. elegans and other organisms, including vertebrates.     

The role of increasing temperature variability in European summer heatwaves

Instrumental observations and reconstructions of global and hemispheric temperature evolution reveal a pronounced warming during the past approximately 150 years. One expression of this warming is the observed increase in the occurrence of heatwaves. Conceptually this increase is understood as a shift of the statistical distribution towards warmer temperatures, while changes in the width of the distribution are often considered small. Here we show that this framework fails to explain the record-breaking central European summer temperatures in 2003, although it is consistent with observations from previous years. We find that an event like that of summer 2003 is statistically extremely unlikely, even when the observed warming is taken into account. We propose that a regime with an increased variability of temperatures (in addition to increases in mean temperature) may be able to account for summer 2003. To test this proposal, we simulate possible future European climate with a regional climate model in a scenario with increased atmospheric greenhouse-gas concentrations, and find that temperature variability increases by up to 100%, with maximum changes in central and eastern Europe.     

Wet periods in northeastern Brazil over the past 210 kyr linked to distant climate anomalies

The tropics are the main source of the atmosphere's sensible and latent heat, and water vapour, and are therefore important for reconstructions of past climate. But long, accurately dated records of southern tropical palaeoclimate, which would allow the establishment of climatic connections to distant regions, have not been available. Here we present a 210,000-year (210-kyr) record of wet periods in tropical northeastern Brazil--a region that is currently semi-arid. The record is obtained from speleothems and travertine deposits that are accurately dated using the U/Th method. We find wet periods that are synchronous with periods of weak East Asian summer monsoons, cold periods in Greenland, Heinrich events in the North Atlantic and periods of decreased river runoff to the Cariaco basin. We infer that the wet periods may be explained with a southward displacement of the Intertropical Convergence Zone. This widespread synchroneity of climate anomalies suggests a relatively rapid global reorganization of the ocean-atmosphere system. We conclude that the wet periods probably affected rainforest distribution, as plant fossils show that forest expansion occurred during these intermittent wet intervals, and opened a forest corridor between the Amazonian and Atlantic rainforests.     

Improved production of spiramycin by mutant Streptomyces ambofaciens

Strain improvement and medium optimization to increase the productivity of spiramycin were carried out. Of oil tolerant mutant strains screened, one mutant, Streptomyces ambofaciens XC 2-37, produced 9% more spiramycin than the parent strain S. ambofaciens XC 1-29. The effects of soybean oil and propyl alcohol on spiramycin production with S.ambofaciens XC 2-37 were studied. The potency orS. ambofaciens XC 2-37 was improved by 61.8% with addition of 2% soybean oil in the fermentation medium and 0.4% propyl alcohol at 24 hours after incubation. The suitable time for feeding propyl alcohol is at 24 hours after incubation in flask fermentation and at 20 hours after incubation in fermentor fermentation The new process with S. ambofaciens XC 2-37 was scaled up for industrial scale production of spiramycin in a 60 m^3 fermentor in Xinchang Pharmaceutical Factory, Zhejiang Medicine Company, Ltd., China, and the potency and productivity of fermentation were improved by 42.9%.     

Coulomb blockade and the Kondo effect in single-atom transistors

Using molecules as electronic components is a powerful new direction in the science and technology of nanometre-scale systems. Experiments to date have examined a multitude of molecules conducting in parallel, or, in some cases, transport through single molecules. The latter includes molecules probed in a two-terminal geometry using mechanically controlled break junctions or scanning probes as well as three-terminal single-molecule transistors made from carbon nanotubes, C(60) molecules, and conjugated molecules diluted in a less-conducting molecular layer. The ultimate limit would be a device where electrons hop on to, and off from, a single atom between two contacts. Here we describe transistors incorporating a transition-metal complex designed so that electron transport occurs through well-defined charge states of a single atom. We examine two related molecules containing a Co ion bonded to polypyridyl ligands, attached to insulating tethers of different lengths. Changing the length of the insulating tether alters the coupling of the ion to the electrodes, enabling the fabrication of devices that exhibit either single-electron phenomena, such as Coulomb blockade, or the Kondo effect.     

Nitric oxide regulates the heart by spatial confinement of nitric oxide synthase isoforms

Subcellular localization of nitric oxide (NO) synthases with effector molecules is an important regulatory mechanism for NO signalling. In the heart, NO inhibits L-type Ca2+ channels but stimulates sarcoplasmic reticulum (SR) Ca2+ release, leading to variable effects on myocardial contractility. Here we show that spatial confinement of specific NO synthase isoforms regulates this process. Endothelial NO synthase (NOS3) localizes to caveolae, where compartmentalization with beta-adrenergic receptors and L-type Ca2+ channels allows NO to inhibit beta-adrenergic-induced inotropy. Neuronal NO synthase (NOS1), however, is targeted to cardiac SR. NO stimulation of SR Ca2+ release via the ryanodine receptor (RyR) in vitro, suggests that NOS1 has an opposite, facilitative effect on contractility. We demonstrate that NOS1-deficient mice have suppressed inotropic response, whereas NOS3-deficient mice have enhanced contractility, owing to corresponding changes in SR Ca2+ release. Both NOS1-/- and NOS3-/- mice develop age-related hypertrophy, although only NOS3-/- mice are hypertensive. NOS1/3-/- double knockout mice have suppressed beta-adrenergic responses and an additive phenotype of marked ventricular remodelling. Thus, NOS1 and NOS3 mediate independent, and in some cases opposite, effects on cardiac structure and function.     

Phagocytosis and clearance of apoptotic cells is mediated by MER

Apoptosis is fundamental to the development and maintenance of animal tissues and the immune system. Rapid clearance of apoptotic cells by macrophages is important to inhibit inflammation and autoimmune responses against intracellular antigens. Here we report a new function for Mer, a member of the Axl/Mer/Tyro3 receptor tyrosine kinase family. mer(kd) mice with a cytoplasmic truncation of Mer had macrophages deficient in the clearance of apoptotic thymocytes. This was corrected in chimaeric mice reconstituted with bone marrow from wild-type animals. Primary macrophages isolated from mer(kd) mice showed that the phagocytic deficiency was restricted to apoptotic cells and was independent of Fc receptor-mediated phagocytosis or ingestion of other particles. The inability to clear apoptotic cells adequately may be linked to an increased number of nuclear autoantibodies in mer(kd) mice. Thus, the Mer receptor tyrosine kinase seems to be critical for the engulfment and efficient clearance of apoptotic cells. This has implications for inflammation and autoimmune diseases such as systemic lupus erythematosus.