Radioprotection of human cell nuclear DNA by polyamines: radiosensitivity of chromatin is influenced by tightly bound spermine

The polyamines putrescine (PUT) and spermine (SPM) were examined for their ability to protect human cell DNA against the formation of radiation-induced double-strand breaks (DSBs). As observed previously, under conditions where polyamines were shown to be almost completely absent, association with nuclear matrix protein into a nucleoid, and organization into chromatin structure, protected DNA from induction of DSBs by factors of 4.5 and 95, respectively. At concentrations below 1 mM, PUT or SPM provided equivalent levels of protection to deproteinized nuclear DNA, consistent with their capacity to scavenge radiation-induced radicals. At constant ionic strength, 5 mM SPM protected deproteinized DNA and nucleoid DNA and DNA in nuclear chromatin by factors of 100 and 26, respectively. At 5 mM, SPM provided 15 times greater protection of deproteinized DNA than did PUT. Under physiologically relevant conditions, 5 mM SPM protected DNA in the intact nucleus from the induction of DSBs by a factor of 2 relative to DNA in the absence of SPM. Studies of SPM binding during cellular fractionation revealed that a significant fraction of the cellular SPM is tightly bound in the nucleus but can be removed by extended washing. Thus the association of SPM with nuclear chromatin appears to be a significant contributor to the resistance of the cell's DNA to the induction of DSBs.     

Muc5b and Muc5ac are the major oligomeric mucins in equine airway mucus

Horses frequently suffer from respiratory diseases, which, irrespective of etiology, are often associated with airway mucus accumulation. Studies on human airways have shown that the key structural components of the mucus layer are oligomeric mucins, which can undergo changes of expression and properties in disease. However, there is little information on these gel-forming glycoproteins in horse airways mucus. Therefore, the aims of this study were to isolate equine airways oligomeric mucins, characterize their macromolecular properties, and identify their gene products. To this end, pooled tracheal washes, collected from healthy horses and horses suffering from respiratory diseases, were solubilized with 6 M guanidinium chloride (GdmCl). The oligomeric mucins were purified by density gradient centrifugation followed by size exclusion chromatography. Biochemical and biophysical analyses showed the mucins were stiffened random coils in solution that were polydisperse in size (M(r) = 6-20 MDa, average M(r) = 14 MDa) and comprised of disulfide-linked subunits (average M(r) = 7 MDa). Agarose gel electrophoresis showed that the pooled mucus sample contained at least two populations of oligomeric mucins. Electrospray ionization tandem mass spectrometry of tryptic digests of the unfractionated mucin preparation showed that the oligomeric mucins Muc5b and Muc5ac were present. In summary, we have shown that equine airways mucus is a mixture of Muc5b and Muc5ac mucins that have a similar macromolecular organization to their human counterparts. This study will form the basis for future studies to analyze the contribution of these two mucins to equine airways pathology associated with mucus accumulation.     

Acidosis of severe falciparum malaria: heading for a shock?

Severe Plasmodium falciparum malaria encompasses a complex syndrome affecting many organs and causing physiological perturbations that have many features in common with children with sepsis. Among these, metabolic acidosis has emerged as a central feature of severe malaria and is the best independent predictor of a fatal outcome in both adults and children. There is now clear evidence that intravascular hypovolaemia (shock) is common in children with malarial acidosis. How it should be treated presents a therapeutic dilemma because acidosis often coexists with impaired consciousness (cerebral malaria). We summarize the results of recent clinical trials examining the safety and efficacy of volume expansion in children with 'cerebral malaria' complicated by acidosis.     

Molecular Mechanism of Resistance in a Clinically Significant Double-Mutant Variant of HCV NS3/4A Protease

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Carbon pools recover more quickly than plant biodiversity in tropical secondary forests

Although increasing efforts are being made to restore tropical forests, little information is available regarding the time scales required for carbon and plant biodiversity to recover to the values associated with undisturbed forests. To address this knowledge gap, we carried out a meta-analysis comparing data from more than 600 secondary tropical forest sites with nearby undisturbed reference forests. Above-ground biomass approached equivalence to reference values within 80 years since last disturbance, whereas below-ground biomass took longer to recover. Soil carbon content showed little relationship with time since disturbance. Tree species richness recovered after about 50 years. By contrast, epiphyte richness did not reach equivalence to undisturbed forests. The proportion of undisturbed forest trees and epiphyte species found in secondary forests was low and changed little over time. Our results indicate that carbon pools and biodiversity show different recovery rates under passive, secondary succession and that colonization by undisturbed forest plant species is slow. Initiatives such as the Convention on Biological Diversity and REDD+ should therefore encourage active management to help to achieve their aims of restoring both carbon and biodiversity in tropical forests.     

Polyadenylic acid synthesis activity of purified DNA-dependent RNA polymerase from Caulobacter

Characterization of purified DNA-dependent RNA polymerase (EC 2.7.7.6) of Caulobacter crescentus, strain CB15 has led to the conclusion that this enzyme catalyzes poly(A) synthesis in the absence of template. Poly(A) synthetase activity co-purifies with both holoenzyme and core polymerase on DNA-cellulose columns, and core polymerase purified to 98% homogeneity by glycerol gradient centrifugation is still capable of catalyzing poly(A) polymerization. Both RNA synthesis and poly(A) polymerization activities are sensitive to rifampicin. In addition, RNA polymerase purified from partially rifampicin-sensitive mutants exhibits the same partial sensitivity in vitro to the drug in the synthesis of RNA and poly(A). The enzyme used in these studies was prepared by a simple method which allows a high yield of pure RNA polymerase from large batches of exponential cells. The procedure includes high speed centrifugation of cell extracts, DEAE-cellulose column, DNA-affinity chromatography, and low salt glycerol gradient centrifugation. Holoenzyme can be resolved into core and sigma subunit by either DNA-cellulose chromatography or glycerol gradient centrifugation, and the latter step allows recovery of pure sigma factor.     

Molecular analysis of the inheritance and stability of the mitochondrial genome of an inbred line of maize

Summary We have investigated the inheritance of the mitochondrial DNA (mtDNA) restriction endonuclease digestion patterns of maize inbred line B37N in individual plants and pooled siblings in lineages derived from five separate plants in the third generation following successive self-pollinations. The restriction fragment patterns of the different mtDNA samples were compared after digestion with five endonucleases. No differences were visible in the mobilities of the 199 fragments scored per sample. Hybridization analysis with two different cloned mtDNA probes, one of which contains homologies to a portion of the S2 plasmid characteristic of cms-S maize, failed to reveal cryptic variation. The apparent rate of genomic change in maize mtDNA from inbred plants appears to be very slow, compared with the faster rates of change seen in maize tissue cultures and with the documented rapid rate of inter- and intraspecific variation for mammalian mtDNA.