Elucidation of the topography of the thapsigargin binding site in the sarco-endoplasmic calcium ATPase

Removal of each of the acyl groups of thapsigargin at O-3, O-8 and O-10 significant reduces the affinity of the inhibitors to the SERCA1a pump. Replacement of the acyl groups at O-3 and O-10 with flexible residues could be performed with only a minor decrease of the affinity, whereas introduction of voluminous stiff residues caused dramatic reduction of the affinity. The results can be rationalized on the basis of the interactions of thapsigargin with the SERCA1a pump as revealed from 3D X-ray structural models of thapsigargin bound to the SERCA1a. In conclusion the results confirm and elaborate the previously suggested pharmocophore model of thapsigargin.     

Culture Conditions of Roseobacter Strain 27-4 Affect Its Attachment and Biofilm Formation as Quantified by Real-Time PCR

The fish probiotic bacterium Roseobacter strain 27-4 grows only as rosettes and produces its antibacterial compound under static growth conditions. It forms three-dimensional biofilms when precultured under static conditions. We quantified attachment of Roseobacter strain 27-4 using a direct real-time PCR method and demonstrated that the bacteria attached more efficiently to surfaces during static growth than under aerated conditions.     

Similar protein phosphatases control starch metabolism in plants and glycogen metabolism in mammals

We report that protein phosphorylation is involved in the control of starch metabolism in Arabidopsis leaves at night. sex4 (starch excess 4) mutants, which have strongly reduced rates of starch metabolism, lack a protein predicted to be a dual specificity protein phosphatase. We have shown that this protein is chloroplastic and can bind to glucans and have presented evidence that it acts to regulate the initial steps of starch degradation at the granule surface. Remarkably, the most closely related protein to SEX4 outside the plant kingdom is laforin, a glucan-binding protein phosphatase required for the metabolism of the mammalian storage carbohydrate glycogen and implicated in a severe form of epilepsy (Lafora disease) in humans.     

A new non-catalytic role for ubiquitin ligase RNF8 in unfolding higher-order chromatin structure

The ubiquitin ligases RNF8 and RNF168 orchestrate DNA damage signalling through the ubiquitylation of histone H2A and the recruitment of downstream repair factors. Here, we demonstrate that RNF8, but not RNF168 or the canonical H2A ubiquitin ligase RNF2, mediates extensive chromatin decondensation. Our data show that CHD4, the catalytic subunit of the NuRD complex, interacts with RNF8 and is essential for RNF8-mediated chromatin unfolding. The chromatin remodelling activity of CHD4 promotes efficient ubiquitin conjugation and assembly of RNF168 and BRCA1 at DNA double-strand breaks. Interestingly, RNF8-mediated recruitment of CHD4 and subsequent chromatin remodelling were independent of the ubiquitin-ligase activity of RNF8, but involved a non-canonical interaction with the forkhead-associated (FHA) domain. Our study reveals a new mechanism of chromatin remodelling-assisted ubiquitylation, which involves the cooperation between CHD4 and RNF8 to create a local chromatin environment that is permissive to the assembly of checkpoint and repair machineries at DNA lesions.     

Ecological and molecular consequences of prolonged drought and subsequent rehydration in Folsomia candida (Collembola)

Drought tolerance in water-permeable, soil-living Collembola (e.g. Folsomia candida) is achieved due to a unique water vapour absorption mechanism, where accumulation of sugars and polyols is essential. However, the molecular mechanisms underlying such adaptation as well as the maintenance of this survival strategy and the responses to rehydration after prolonged drought in these soil-living Collembola are unclear. In the present study, the functional relationships between ecological drought responses and expression of related target genes were investigated in F. candida exposed to mild and severe drought for up to 5 weeks by relating survival, moulting and reproduction rate with mRNA-level expression of 7 target genes during drought, dehydration and rehydration. Prolonged drought and subsequent rehydration induced significant changes in gene expression which could be related to the fitness traits studied. In F. candida the ecological and molecular responses to mild drought differed from those of severe drought. From the changes in gene expression, where significantly increased expression of Glucose-6-phosphate-isomerase (gpi) and Heat shock protein 70 (hsp70) was dominating, it is proposed that protection of cellular structure and function during prolonged mild drought (98.2% RH) is partly achieved from a continuous accumulation of compatible osmolytes in F. candida. To achieve protection during and after prolonged severe drought (96.1% RH), components related to cell division and development such as inositol monophosphatase and one of the small heat shock proteins (sHsps), Heat shock protein23 (hsp23), seem to play an important role in F. candida.     

Effect of estrogen on tendon collagen synthesis, tendon structural characteristics, and biomechanical properties in postmenopausal women

The knowledge about the effect of estradiol on tendon connective tissue is limited. Therefore, we studied the influence of estradiol on tendon synthesis, structure, and biomechanical properties in postmenopausal women. Nonusers (control, n = 10) or habitual users of oral estradiol replacement therapy (ERT, n = 10) were studied at rest and in response to one-legged resistance exercise. Synthesis of tendon collagen was determined by stable isotope incorporation [fractional synthesis rate (FSR)] and microdialysis technique (NH(2)-terminal propeptide of type I collagen synthesis). Tendon area and fibril characteristics were determined by MRI and transmission electron microscopy, whereas tendon biomechanical properties were measured during isometric maximal voluntary contraction by ultrasound recording. Tendon FSR was markedly higher in ERT users (P < 0.001), whereas no group difference was seen in tendon NH(2)-terminal propeptide of type I collagen synthesis (P = 0.32). In ERT users, positive correlations between serum estradiol (s-estradiol) and tendon synthesis were observed, whereas change in tendon synthesis from rest to exercise was negatively correlated to s-estradiol. Tendon area, fibril density, fibril volume fraction, and fibril mean area did not differ between groups. However, the percentage of medium-sized fibrils was higher in ERT users (P < 0.05), whereas the percentage of large fibrils tended to be greater in control (P = 0.10). A lower Young's modulus (GPa/%) was found in ERT users (P < 0.05). In conclusion, estradiol administration was associated with higher tendon FSR and a higher relative number of smaller fibrils. Whereas this indicates stimulated collagen turnover in the resting state, collagen responses to exercise were negatively associated with s-estradiol. These results indicate a pivotal role for estradiol in maintaining homeostasis of female connective tissue.     

Variants of beta-microglobulin cleaved at lysine-58 retain the main conformational features of the native protein but are more conformationally heterogeneous and unstable at physiological temperature

Cleavage of the small amyloidogenic protein beta2-microglobulin after lysine-58 renders it more prone to unfolding and aggregation. This is important for dialysis-related beta2-microglobulin amyloidosis, since elevated levels of cleaved beta2-microglobulin may be found in the circulation of dialysis patients. However, the solution structures of these cleaved beta2-microglobulin variants have not yet been assessed using single-residue techniques. We here use such methods to examine beta2-microglobulin cleaved after lysine-58 and the further processed variant (found in vivo) from which lysine-58 is removed. We find that the solution stability of both variants, especially of beta2-microglobulin from which lysine-58 is removed, is much reduced compared to wild-type beta2-microglobulin and is strongly dependent on temperature and protein concentration. 1H-NMR spectroscopy and amide hydrogen (1H/2H) exchange monitored by MS show that the overall three-dimensional structure of the variants is similar to that of wild-type beta2-microglobulin at subphysiological temperatures. However, deviations do occur, especially in the arrangement of the B, D and E beta-strands close to the D-E loop cleavage site at lysine-58, and the experiments suggest conformational heterogeneity of the two variants. Two-dimensional NMR spectroscopy indicates that this heterogeneity involves an equilibrium between the native-like fold and at least one conformational intermediate resembling intermediates found in other structurally altered beta2-microglobulin molecules. This is the first single-residue resolution study of a specific beta2-microglobulin variant that has been found circulating in dialysis patients. The instability and conformational heterogeneity of this variant suggest its involvement in beta2-microglobulin amyloidogenicity in vivo.     

Zinc reduces the electrophysiological responses in vitro to basolateral receptor mediated secretagogues in piglet small intestinal epithelium

Dietary zinc treatment has a preventive impact on diarrhoea in newly weaned piglets and in undernourished children. The mechanisms behind this effect of zinc are however, still not fully understood. The aim of the present study was to assess if zinc has a direct effect on porcine intestinal secretory responses to different secretagogues in vitro. The study included two Ussing chamber experiments, where the short circuit current responses to different secretagogues were measured in piglet small intestinal epithelium. Exp. 1 aimed to study the effect of increased zinc concentrations in the bathing media on the secretory responses to 5-HT and theophylline. The objective of exp. 2 was to study the effect of zinc in the bathing media on the secretory responses induced by vasoactive intestinal polypeptide (VIP), Substance P (SP), Carbachol, and theophylline. The results showed that there were significant decreasing effects of zinc on the secretion, stimulated by 5-HT, VIP and carbachol, from piglet intestinal epithelium in vitro, whereas the secretion caused by SP and theophylline was not significantly affected. The data indicate that the inhibitory mechanism of zinc ions may take place at the receptors situated at the basolateral membrane of the epithelial cells.