Selectivity in biomineralization of barium and strontium

The desmid green alga Closterium moniliferum belongs to a small number of organisms that form barite (BaSO₄) or celestite (SrSO₄) biominerals. The ability to sequester Sr in the presence of an excess of Ca is of considerable interest for the remediation of ⁹⁰Sr from the environment and nuclear waste. While most cells dynamically regulate the concentration of the second messenger Ca²⁺ in the cytosol and various organelles, transport proteins rarely discriminate strongly between Ca, Sr, and Ba. Herein, we investigate how these ions are trafficked in C. moniliferum and how precipitation of (Ba,Sr)SO₄ crystals occurs in the terminal vacuoles. Towards this goal, we simultaneously visualize intracellular dynamics of multiple elements using X-ray fluorescence microscopy (XFM) of cryo-fixed/freeze-dried samples. We correlate the resulting elemental maps with ultrastructural information gleaned from freeze-fracture cryo-SEM of frozen-hydrated cells and use micro X-ray absorption near edge structure (micro-XANES) to determine sulfur speciation. We find that the kinetics of Sr uptake and efflux depend on external Ca concentrations, and Sr, Ba, and Ca show similar intracellular localization. A highly ion-selective cross-membrane transport step is not evident. Based on elevated levels of sulfate detected in the terminal vacuoles, we propose a “sulfate trap” model, where the presence of dissolved barium leads to preferential precipitation of (Ba,Sr)SO₄ due to its low solubility relative to SrSO₄ and CaSO₄. Engineering the sulfate concentration in the vacuole may thus be the most direct way to increase the Sr sequestered per cell, an important consideration in using desmids for phytoremediation of ⁹⁰Sr.     

Dissecting the mechanisms of Notch induced hyperplasia

The outcome of the Notch pathway on proliferation depends on cellular context, being growth promotion in some, including several cancers, and growth inhibition in others. Such disparate outcomes are evident in Drosophila wing discs, where Notch overactivation causes hyperplasia despite having localized inhibitory effects on proliferation. To understand the underlying mechanisms, we have used genomic strategies to identify the Notch‐CSL target genes directly activated during wing disc hyperplasia. Among them were genes involved in both autonomous and non‐autonomous regulation of proliferation, growth and cell death, providing molecular explanations for many characteristics of Notch induced wing disc hyperplasia previously reported. The Notch targets exhibit different response patterns, which are shaped by both positive and negative feed‐forward regulation between the Notch targets themselves. We propose, therefore, that both the characteristics of the direct Notch targets and their cross‐regulatory relationships are important in coordinating the pattern of hyperplasia.     

The PCNA Interaction Protein Box Sequence in Rad54 Is an Integral Part of Its ATPase Domain and Is Required for Efficient DNA Repair and Recombination

Rad54 is an ATP-driven translocase involved in the genome maintenance pathway of homologous recombination (HR). Although its activity has been implicated in several steps of HR, its exact role(s) at each step are still not fully understood. We have identified a new interaction between Rad54 and the replicative DNA clamp, proliferating cell nuclear antigen (PCNA). This interaction was only mildly weakened by the mutation of two key hydrophobic residues in the highly-conserved PCNA interaction motif (PIP-box) of Rad54 (Rad54-AA). Intriguingly, the rad54-AA mutant cells displayed sensitivity to DNA damage and showed HR defects similar to the null mutant, despite retaining its ability to interact with HR proteins and to be recruited to HR foci in vivo. We therefore surmised that the PCNA interaction might be impaired in vivo and was unable to promote repair synthesis during HR. Indeed, the Rad54-AA mutant was defective in primer extension at the MAT locus as well as in vitro, but additional biochemical analysis revealed that this mutant also had diminished ATPase activity and an inability to promote D-loop formation. Further mutational analysis of the putative PIP-box uncovered that other phenotypically relevant mutants in this domain also resulted in a loss of ATPase activity. Therefore, we have found that although Rad54 interacts with PCNA, the PIP-box motif likely plays only a minor role in stabilizing the PCNA interaction, and rather, this conserved domain is probably an extension of the ATPase domain III.     

SPATIAL AND TEMPORAL VARIATION OF EPIPSAMMIC DIATOMS IN A SPRING-FED BROOK1

Epipsammic diatom communities were sampled and quantified bimonthly from June 1983 to April 1985 at four sites in a small brook in northwest Ohio. The primary objective was to determine the extent of temporal and spatial variability in the epipsammon. The four sites, approximately 250 m apart, differed in current velocity, illumination and the amount of sand-associated detritus. Diatoms were significantly more abundant in the portion of the brook flowing through an unshaded marsh than at three heavily shaded sites located upstream. Fragilaria leptostauron (Ehr.) Hust. and Achnanthes lanceolata var. dubia Grun. dominated the epipsammic assemblage at all sites throughout the entire study period. Only Meridion circulare (Grev.) Ehr. displayed a marked seasonal distribution. Although the same species were generally found at all sites, there were sufficient differences in relative abundance that communities could be discriminated according to site. Using two canonical variate axes, all 48 samples were correctly assigned to their proper sampling site based on community composition. Data from this epipsammic assemblage support the idea developed from the river continuum concept that species comprising riverine benthic assemblages continually persist and rarely become completely absent.     

RPA not that different from SSB

Replication protein A, the heterotrimeric single-stranded DNA binding factor in eukaryotic cells, interacts with DNA in both an unstable and stable mode. Biochemical, mutational, and crystallographic analyses have provided insights into the switch between these two states.     

Optimization of the examination posture in spinal curvature assessment

ABSTRACT: To decrease the influence of postural sway during spinal measurements, an instrumented fixation posture (called G) was proposed and tested in comparison with the free standing posture (A) using the DTP-3 system in a group of 70 healthy volunteers. The measurement was performed 5 times on each subject and each position was tested by a newly developed device for non-invasive spinal measurements called DTP-3 system. Changes in postural stability of the spinous processes for each subject/the whole group were evaluated by employing standard statistical tools. Posture G, when compared to posture A, reduced postural sway significantly in all spinous processes from C3 to L5 in both the mediolateral and anterioposterior directions. Posture G also significantly reduced postural sway in the vertical direction in 18 out of 22 spinous processes. Importantly, posture G did not significantly influence the spinal curvature.     

Pharmacokinetics and pharmacodynamics of terguride following intramuscular administration in cows and goats

The pharmacokinetics of intramuscular terguride (transdihydrolisuride) was evaluated in a single-dose study in cows (doses 100, 62 and 31 micrograms/kg b.w.) and goats (dose 100 micrograms/kg b.w.). A radioreceptor assay was used to quantitative plasma terguride concentrations. The peak plasma concentrations of terguride were attained within 0.6 h of the drug administration and then decreased monoexponentially with half-life of 1.3 h (cows) and 2 h (goats). The pharmacokinetics of terguride in cows is nearly linear. Pharmacodynamics of terguride was expressed as reduction in plasma prolactin levels. Maximal decline in prolactin was observed 3-4 h following terguride administration and the effect lasted for about 24 h.     

Molecular architecture of acetylcholinesterase collagen-tailed forms; construction of a glycolipid-tailed tetramer.

Asymmetric forms of Torpedo acetylcholinesterase (AChE) are produced in COS cells by the simultaneous expression of collagenic subunits (Q) and catalytic T subunits (AChET). Truncated AChET delta subunits, from which most of the C-terminal peptide (TC) had been deleted by mutagenesis, did not associate with Q subunits. The TC peptide is therefore necessary for the association of the AChET and Q subunits. In order to determine the orientation of the Q subunit in the collagen-tailed forms, we have developed an antiserum against its non-collagenic C-terminal domain, expressed as a fusion protein in Escherichia coli. This antiserum, which recognized the Q subunit in Western blots, was found to react with intact asymmetric forms, but not with collagenase-treated forms, from which the distal part of the tail had been cleaved, suggesting that the N-terminal non-collogenic domain (QN) is responsible for the interaction with the AChET subunits. This was confirmed by creating a chimeric subunit (QN/HC), in which QN was linked to the C-terminal peptide of the H subunit of Torpedo AChE, which contains the glycophosphatidylinositol (GPI) cleavage/attachment signal: co-expression of AChET and QN/NC produced GPI-anchored tetramers, which were sensitive to PI-PLC and largely exposed to the external surface of the cells. We thus demonstrate that: (i) the HC peptide is sufficient to determine the addition of a glycolipid anchor and (ii) the QN domain is sufficient to bind a catalytic AChET tetramer by interacting with the TC peptide.