Genome analysis of DNA repair genes in the alpha proteobacterium <Emphasis Type="Italic">Caulobacter crescentus</Emphasis>

Background  

The integrity of DNA molecules is fundamental for maintaining life. The DNA repair proteins protect organisms against genetic damage, by removal of DNA lesions or helping to tolerate them. DNA repair genes are best known from the gamma-proteobacterium Escherichia coli, which is the most understood bacterial model. However, genome sequencing raises questions regarding uniformity and ubiquity of these DNA repair genes and pathways, reinforcing the need for identifying genes and proteins, which may respond to DNA damage in other bacteria.     

Microsporidia, amitochondrial protists, possess a 70-kDa heat shock protein gene of mitochondrial evolutionary origin

An intronless gene encoding a protein of 592 amino acid residues with similarity to 70-kDa heat shock proteins (HSP70s) has been cloned and sequenced from the amitochondrial protist Encephalitozoon cuniculi (phylum Microsporidia). Southern blot analyses show the presence of a single gene copy located on chromosome XI. The encoded protein exhibits an N-terminal hydrophobic leader sequence and two motifs shared by proteobacterial and mitochondrially expressed HSP70 homologs. Phylogenetic analysis using maximum likelihood and evolutionary distances place the E. cuniculi sequence in the cluster of mitochondrially expressed HSP70s, with a higher evolutionary rate than those of homologous sequences. Similar results were obtained after cloning a fragment of the homologous gene in the closely related species E. hellem. The presence of a nuclear targeting signal-like sequence supports a role of the Encephalitozoon HSP70 as a molecular chaperone of nuclear proteins. No evidence for cytosolic or endoplasmic reticulum forms of HSP70 was obtained through PCR amplification. These data suggest that Encephalitozoon species have evolved from an ancestor bearing mitochondria, which is in disagreement with the postulated presymbiotic origin of Microsporidia. The specific role and intracellular localization of the mitochondrial HSP70-like protein remain to be elucidated.      

Molecular mechanisms that distinguish TFIID housekeeping from regulatable SAGA promoters

An important distinction is frequently made between constitutively expressed housekeeping genes versus regulated genes. Although generally characterized by different DNA elements, chromatin architecture and cofactors, it is not known to what degree promoter classes strictly follow regulatability rules and which molecular mechanisms dictate such differences. We show that SAGA‐dominated/TATA‐box promoters are more responsive to changes in the amount of activator, even compared to TFIID/TATA‐like promoters that depend on the same activator Hsf1. Regulatability is therefore an inherent property of promoter class. Further analyses show that SAGA/TATA‐box promoters are more dynamic because TATA‐binding protein recruitment through SAGA is susceptible to removal by Mot1. In addition, the nucleosome configuration upon activator depletion shifts on SAGA/TATA‐box promoters and seems less amenable to preinitiation complex formation. The results explain the fundamental difference between housekeeping and regulatable genes, revealing an additional facet of combinatorial control: an activator can elicit a different response dependent on core promoter class.     

Morphological changes in human melanoma cells following irradiation with thermal neutrons

Morphological changes in two human melanoma cell lines, MM96 and MM418, following irradiation with thermal neutrons, were studied using light and electron microscopy. The results show that the response of human malignant melanoma cells to neutron irradiation is both cell line dependent and dose dependent, and that in any given cell line, some cells are more resistant to irradiation than others, thus demonstrating heterogeneity in respect to radiosensitivity. Cells repopulating MM96 flasks after irradiation were morphologically similar to the cells of origin whereas in MM418 flasks cells differentiated into five morphologically distinct subgroups and showed increased melanization. The results also show that radiation causes distinctive morphological patterns of damage although ultrastructural changes unique to the high LET particles released from boron 10 neutron capture are yet to be identified.     

Indicators of biodiversity and ecosystem services: a synthesis across ecosystems and spatial scales

According to the Millennium Ecosystem Assessment, common indicators are needed to monitor the loss of biodiversity and the implications for the sustainable provision of ecosystem services. However, a variety of indicators are already being used resulting in many, mostly incompatible, monitoring systems. In order to synthesise the different indicator approaches and to detect gaps in the development of common indicator systems, we examined 531 indicators that have been reported in 617 peer‐reviewed journal articles between 1997 and 2007. Special emphasis was placed on comparing indicators of biodiversity and ecosystem services across ecosystems (forests, grass‐ and shrublands, wetlands, rivers, lakes, soils and agro‐ecosystems) and spatial scales (from patch to global scale). The application of biological indicators was found most often focused on regional and finer spatial scales with few indicators applied across ecosystem types. Abiotic indicators, such as physico‐chemical parameters and measures of area and fragmentation, are most frequently used at broader (regional to continental) scales. Despite its multiple dimensions, biodiversity is usually equated with species richness only. The functional, structural and genetic components of biodiversity are poorly addressed despite their potential value across habitats and scales. Ecosystem service indicators are mostly used to estimate regulating and supporting services but generally differ between ecosystem types as they reflect ecosystem‐specific services. Despite great effort to develop indicator systems over the past decade, there is still a considerable gap in the widespread use of indicators for many of the multiple components of biodiversity and ecosystem services, and a need to develop common monitoring schemes within and across habitats. Filling these gaps is a prerequisite for linking biodiversity dynamics with ecosystem service delivery and to achieving the goals of global and sub‐global initiatives to halt the loss of biodiversity.     

Neutron irradiation of human melanoma cells

The biological characteristics and in vitro radiosensitivity of melanoma cells to thermal neutrons were investigated as a guide to the effectiveness of boron neutron capture therapy. Plateau phase cultures of three human malignant melanoma-established cell lines were examined for cell density at confluence, doubling time, cell cycle parameters, chromosome constitution, and melanin content. Cell survival dose-response curves, for cells preincubated in the presence or absence of p-boronophenylalanine. HCl (10B1-BPA), were measured over the dose range 0.6-8.0 Gy (N + gamma). The neutron fluence rate was 2.6 x 10(9) n/cm2/s and the total dose rate 3.7 Gy/h (31% gamma). Considerable differences were observed in the morphology and cellular properties of the cell lines. Two cell lines (96E and 96L) were amelanotic, and one was melanotic (418). An enhanced killing for neutron irradiation was found only for the melanotic cells after 20 h preincubation with 10 micrograms/ml 10B1-BPA. In view of the doubling times of the cell lines of about 23 h (96E and 96L) or of 36 h (418), it seems likely that an increased boron uptake, and hence increased radiosensitivity, might result if the preincubation period with 10B1-BPA is extended to several hours longer than the respective cell cycle times.     

Long-term changes in lowland calcareous grassland plots using <Emphasis Type="Italic">Tephroseris integrifolia</Emphasis> subsp. <Emphasis Type="Italic">integrifolia</Emphasis> as an indicator species

We investigated the changes to calcareous grassland plots within protected sites, and whether Tephroseris integrifolia subsp. integrifolia can act as a useful indicator species for re-visitation studies within vegetation predicted to remain relatively stable. Twenty-two plots located across lowland England and all formerly containing T. integrifolia were re-surveyed following the methodology used in the original survey undertaken in the 1960s. Pseudo-turnover and between-observer bias were minimised by sampling replicate quadrats at each fixed plot using a single surveyor and at a similar time of year as the original survey. Qualitative details concerning grazing management were obtained for all sites. In contrast to other long-term re-visitation studies, all our study plots were intact and retained diverse, herb-rich vegetation, demonstrating the value of site protection. However, there were clear shifts in vegetation composition, most notably where T. integrifolia was absent, as shown by an increase in Ellenberg fertility and moisture signifying nutrient enrichment, and a decrease in the cover of low-growing, light-demanding specialists, with a change likely to be associated predominantly with grazing management. Whereas in the mid-20th century the greatest threat to calcareous grassland was habitat loss, undergrazing or temporary neglect now appears to pose the principal threat. Distinctive species such as T. integrifolia with marked sensitivity to habitat change provide a potentially useful tool for rapid assessment and monitoring of site quality. Focusing monitoring on such species allows non-expert observers to recognise the early stages of habitat degradation, providing, in effect, a “health check” on individual sites and groups of sites.     

Rapid identification of Candida species by using nuclear magnetic resonance spectroscopy and a statistical classification strategy

Nuclear magnetic resonance (NMR) spectra were acquired from suspensions of clinically important yeast species of the genus Candida to characterize the relationship between metabolite profiles and species identification. Major metabolites were identified by using two-dimensional correlation NMR spectroscopy. One-dimensional proton NMR spectra were analyzed by using a staged statistical classification strategy. Analysis of NMR spectra from 442 isolates of Candida albicans, C. glabrata, C. krusei, C. parapsilosis, and C. tropicalis resulted in rapid, accurate identification when compared with conventional and DNA-based identification. Spectral regions used for the classification of the five yeast species revealed species-specific differences in relative amounts of lipids, trehalose, polyols, and other metabolites. Isolates of C. parapsilosis and C. glabrata with unusual PCR fingerprinting patterns also generated atypical NMR spectra, suggesting the possibility of intraspecies discontinuity. We conclude that NMR spectroscopy combined with a statistical classification strategy is a rapid, nondestructive, and potentially valuable method for identification and chemotaxonomic characterization that may be broadly applicable to fungi and other microorganisms.     

Studies on the binding site of the galactose-specific agglutinin PA-IL from Pseudomonas aeruginosa

The binding properties of Pseudomonas aeruginosa agglutinin-I (PA-IL) with glycoproteins (gps) and polysaccharides were studied by both the biotin/avidin-mediated microtiter plate lectin-binding assay and the inhibition of agglutinin-glycan interaction with sugar ligands. Among 36 glycans tested for binding, PA-IL reacted best with two glycoproteins containing Galalpha1-->4Gal determinants and a human blood group ABO precursor equivalent gp, but this lectin reacted weakly or not at all with A and H active gps or sialylated gps. Among the mammalian disaccharides tested by the inhibition assay, the human blood group Pkactive Galalpha1-->4Gal, was the best. It was 7.4-fold less active than melibiose (Galalpha1-->6Glc). PA-IL has a preference for the alpha-anomer in decreasing order as follows: Galalpha1-->6 >Galalpha1-->4 >Galalpha1-->3. Of the monosaccharides studied, the phenylbeta derivatives of Gal were much better inhibitors than the methylbeta derivative, while only an insignificant difference was found between the Galalpha anomer of methyl- and p -NO2-phenyl derivatives. From these results, it can be concluded that the combining size of the agglutinin is as large as a disaccharide of the alpha-anomer of Gal at nonreducing end and most complementary to Galalpha1-->6Glc. As for the combining site of PA-IL toward the beta-anomer, the size is assumed to be less than that of Gal; carbon-6 in the pyranose form is essential, and hydrophobic interaction is important for binding.