Kinetic analysis of interaction of BRCA1 tandem breast cancer c-terminal domains with phosphorylated peptides reveals two binding conformations

Tandem breast cancer C-terminal (BRCT) domains, present in many DNA repair and cell cycle checkpoint signaling proteins, are phosphoprotein binding modules. The best-characterized tandem BRCT domains to date are from the protein BRCA1 (BRCA1-BRCT), an E3 ubiquitin ligase that has been linked to breast and ovarian cancer. While X-ray crystallography and NMR spectroscopy studies have uncovered the structural determinants of specificity of BRCA1-BRCT for phosphorylated peptides, a detailed kinetic and thermodynamic characterization of the interaction is also required to understand how structure and dynamics are connected and therefore better probe the mechanism of phosphopeptide recognition by BRCT domains. Through a global analysis of binding kinetics data obtained from surface plasmon resonance (SPR) and stopped-flow fluorescence spectroscopy, we show that the recognition mechanism is complex and best modeled by two equilibrium conformations of BRCA1-BRCT in the free state that both interact with a phosphopeptide, with dissociation constants ( K d) in the micromolar range. We show that the apparent global dissociation constant derived from this kinetic analysis is similar to the K d values measured using steady-state SPR, isothermal titration calorimetry, and fluorescence anisotropy. The dynamic nature of BRCA1-BRCT may facilitate the binding of BRCA1 to different phosphorylated protein targets.     

Comparative activity and functional ecology of permafrost soils and lithic niches in a hyper‐arid polar desert

Permafrost in the high elevation McMurdo Dry Valleys of Antarctica ranks among the driest and coldest on Earth. Permafrost soils appear to be largely inhospitable to active microbial life, but sandstone lithic microhabitats contain a trophically simple but functional cryptoendolithic community. We used metagenomic sequencing and activity assays to examine the functional capacity of permafrost soils and cryptoendolithic communities in University Valley, one of the most extreme regions in the Dry Valleys. We found metagenomic evidence that cryptoendolithic microorganisms are adapted to the harsh environment and capable of metabolic activity at in situ temperatures, possessing a suite of stress response and nutrient cycling genes to fix carbon under the fluctuating conditions that the sandstone rock would experience during the summer months. We additionally identified genes involved in microbial competition and cooperation within the cryptoendolithic habitat. In contrast, permafrost soils have a lower richness of stress response genes, and instead the metagenome is enriched in genes involved with dormancy and sporulation. The permafrost soils also have a large presence of phage genes and genes involved in the recycling of cellular material. Our results underlie two different habitability conditions under extreme cold and dryness: the permafrost soil which is enriched in traits which emphasize survival and dormancy, rather than growth and activity; and the cryptoendolithic environment that selects for organisms capable of growth under extremely oligotrophic, arid and cold conditions. This study represents the first metagenomic interrogation of Antarctic permafrost and polar cryptoendolithic microbial communities.     

Mechanisms of subzero growth in the cryophile Planococcus halocryophilus determined through proteomic analysis

The eurypsychrophilic bacterium Planococcus halocryophilus is capable of growth down to ?15°C, making it ideal for studying adaptations to subzero growth. To increase our understanding of the mechanisms and pathways important for subzero growth, we performed proteomics on P. halocryophilus grown at 23°C, 23°C with 12% w/v NaCl and ?10°C with 12% w/v NaCl. Many proteins with increased abundances at ?10°C versus 23°C also increased at 23C‐salt versus 23°C, indicating a closely tied relationship between salt and cold stress adaptation. Processes which displayed the largest changes in protein abundance were peptidoglycan and fatty acid (FA) synthesis, translation processes, methylglyoxal metabolism, DNA repair and recombination, and protein and nucleotide turnover. We identified intriguing targets for further research at ?10°C, including PlsX and KASII (FA metabolism), DD‐transpeptidase and MurB (peptidoglycan synthesis), glyoxalase family proteins (reactive electrophile response) and ribosome modifying enzymes (translation turnover). PemK/MazF may have a crucial role in translational reprogramming under cold conditions. At ?10°C P. halocryophilus induces stress responses, uses resources efficiently, and carefully controls its growth and metabolism to maximize subzero survival. The present study identifies several mechanisms involved in subzero growth and enhances our understanding of cold adaptation.     

The Sec34/35 Golgi transport complex is related to the exocyst, defining a family of complexes involved in multiple steps of membrane traffic.

The specificity of intracellular vesicle transport is mediated in part by tethering factors that attach the vesicle to the destination organelle prior to fusion. We have identified a protein, Dor1p, that is involved in vesicle targeting to the yeast Golgi apparatus and found it to be associated with seven further proteins. Identification of these revealed that they include Sec34p and Sec35p, the two known components of the Sec34/35 complex previously proposed to tether vesicles to the Golgi. Of the six previously uncharacterized components, four have homologs in higher eukaryotes, including a subunit of a mammalian Golgi transport complex. Furthermore, several of the proteins show distant homology to components of two other putative tethering complexes, the exocyst and the Vps52/53/54 complex, revealing that tethering factors involved in different membrane traffic steps are structurally related.     

Biodegradation of Variable-Chain-Length Alkanes at Low Temperatures by a Psychrotrophic Rhodococcus sp.

The psychrotroph Rhodococcus sp. strain Q15 was examined for its ability to degrade individual n-alkanes and diesel fuel at low temperatures, and its alkane catabolic pathway was investigated by biochemical and genetic techniques. At 0 and 5°C, Q15 mineralized the short-chain alkanes dodecane and hexadecane to a greater extent than that observed for the long-chain alkanes octacosane and dotriacontane. Q15 utilized a broad range of aliphatics (C₁₀ to C₂₁ alkanes, branched alkanes, and a substituted cyclohexane) present in diesel fuel at 5°C. Mineralization of hexadecane at 5°C was significantly greater in both hydrocarbon-contaminated and pristine soil microcosms seeded with Q15 cells than in uninoculated control soil microcosms. The detection of hexadecane and dodecane metabolic intermediates (1-hexadecanol and 2-hexadecanol and 1-dodecanol and 2-dodecanone, respectively) by solid-phase microextraction–gas chromatography-mass spectrometry and the utilization of potential metabolic intermediates indicated that Q15 oxidizes alkanes by both the terminal oxidation pathway and the subterminal oxidation pathway. Genetic characterization by PCR and nucleotide sequence analysis indicated that Q15 possesses an aliphatic aldehyde dehydrogenase gene highly homologous to the Rhodococcus erythropolis thcA gene. Rhodococcus sp. strain Q15 possessed two large plasmids of approximately 90 and 115 kb (shown to mediate Cd resistance) which were not required for alkane mineralization, although the 90-kb plasmid enhanced mineralization of some alkanes and growth on diesel oil at both 5 and 25°C.     

Detection and characterization of the fibroblast growth factor-related oncoprotein INT-2.

Products of the fibroblast growth factor-related proto-oncogene int-2 have been detected by using a monoclonal antibody and polyclonal antisera raised against synthetic peptides predicted from the DNA sequence. COS-1 monkey cells transfected with int-2 DNA linked to the simian virus 40 early promoter contained at least four int-2-specific proteins, presumably representing modified forms of the expected 27-kilodalton primary translation product. The level of expression was increased approximately six- to eightfold by mutation of sequences around the presumed initiation codon, negating their capacity to encode a short oligopeptide in the +1 reading frame. Both tunicamycin inhibition and in vitro translation experiments indicated that some of the modifications correspond to asparagine-linked glycosylation, for which the sequence predicts a single site. In line with the similarities between INT-2 and other fibroblast growth factors, the in vitro translation products functioned as weak mitogens for mammary epithelial cells.     

New markers for linkage analysis of X-linked hypophosphataemic rickets

Three polymorphic markers have been used to improve the genetic map of the region Xp22.1-p22.2, which contains the HYP (hypophosphataemic rickets) locus. DXS365 gave no recombinants with HYP, with a peak Lod score of 5.4 at = 0.0. A microsatellite marker mPA274 was derived for the DXS274 locus; it detects five alleles with a polymorphism information content of 0.55. Combining information from this microsatellite and the original DXS274 marker, probe CRI-L1391, the peak Lod score for DXS274 against HYP was 9.6 at = 0.02. A microsatellite associated with the DXS207 locus (mPA207) gave a peak lod score against HYP of 4.7 at = 0.14. A consideration of key recombinants and multilocus analysis suggests the gene order: Xpter-DXS207-DXS43-DXS197-(DXS365, HYP)-DXS274-DXS41-Xcen.     

Growth activation of the microalga Isochrysis galbana by the aqueous extract of the seaweed Monostroma nitidum

Cell growth of the marine microalga Isochrysis galbana was regulated by the addition of seaweed extracts in the culture medium. Methanol-soluble extracts from 27 species of seaweed showed growth activation only from Enteromorpha linza, and growth inhibition from Ishige foliacea and Sargassum sagamianum. Water-soluble extracts from Grateloupia turuturu and Monostroma nitidum showed growth activation, while none of the seaweed showed growth inhibition. From results of growth activation of extracts on I. galbana, the water extract of M. nitidum was the most effective up to two-fold increase in cell density with the addition of 1 mg mL^-1 of extract to the medium. The cell growth rate was increased from 0.52 to 0.65 d^-1. Cell size, gross biochemical compositions, fatty acid compositions, and digestion efficiency by shellfish differed marginally between cultures of I. galbana grown with and without the M. nitidum aqueous extract. This extract has also enhanced the growth of other feed microalgae tested, including Dunaliella salina. This revised version was published online in August 2006 with corrections to the Cover Date.     

Osteopenia in 37 members of seven families: analysis based on a model of dominant inheritance.

BACKGROUND: The genetic factors involved in determining bone mineral density (BMD) have not been fully elucidated. We have begun genetic linkage analysis of seven families in which many members are osteopenic, in order to identify chromosomal loci that are potentially involved in determining BMD. MATERIALS AND METHODS: Spine BMD was measured in 143 members of seven kindred with familial osteopenia. The absolute BMD values for the spine (L2-L4) were converted to the age-, gender-, and weight-adjusted Z scores, and this corrected value was used as the quantitative trait on which to base subsequent genetic analyses. Simulations of linkage were performed in order to determine the information content of the pedigree set, and actual linkage analysis was conducted using polymorphic markers either within or near three candidate loci: COL1A1, COL1A2, and vitamin D receptor (VDR). RESULTS: The distribution of the corrected Z scores was bimodal (p = 0.001) suggesting a monogenic mode of inheritance of the low BMD trait. Simulation of linkage analysis suggested that the family data set was sufficient to detect linkage under a single major gene model. Actual linkage analysis did not support linkage to the three candidate loci. In addition, the VDR genotype was not statistically associated with low bone density at the spine. CONCLUSIONS: Loci other than COL1A1, COL1A2 and VDR are very likely responsible for the low BMD trait observed in these families. These families are suitable for a genome-wide screen using microsatellite repeats in order to identify the loci that are involved in osteopenia.     

An attempt to estimate the predatory pressure exerted by the lesser weever, Trachinus vipera Cuvier, in the southern North Sea

In order to evaluate the impact of the lesser weever on the ecosystem of the southern North Sea, geographical distribution, density, growth, production and food requirements have been estimated. High densities were found on and around the Brown Ridge, an area with high tidal current velocities, medium grain-size of the sediment and a poor benthic fauna. Growth is restricted to the months of June October. During the winter cessation of growth a considerable loss of weight (about 20%) takes place. Mortality has been estimated by using the average size frequency distribution of all catches made from 1972 to 1984. The resulting convex type of survival curve indicates a high survival rate of the II to IV-group fishes. The production (estimated with Allen's graphical method) of a population of 100 individuals including all age groups (0-VI) amounts to 123.7 g AFDW-year'. In areas with highest densities, consequently, production amounts to 0.018–0.078 g AFDW-m² -year^-1. With an assumed transfer efficiency of 10% through the year, food requirements amounts to 0.18–0.78 g AFDW-m ² -year ^-1. Since the lesser weever feeds mainly on fish (85.6%), almost exclusively on gobies (Pomatoschistus sp.), and with an assumed transfer efficiency of approximately 10%, the indirect predatory pressure exerted by it may amount to 1.6 6.7g AFDW-m ².year ^-1. A possible feeding by gobies on pelagic organisms (calanoids, mysids) is discussed.