A potent and selective photoaffinity probe for the anti-estrogen binding site of rat liver

The anti-estrogen binding site (ABS) is an apparently ubiquitous component of cells that has been shown to be intimately linked with the antiproliferative effects of certain antiestrogenic compounds, like tamoxifen, which is currently used for the treatment of breast cancer. However, the identification and in vitro study of this novel protein has been hampered to date by a lack of convenient probes that will efficiently label the molecule in nonpurified preparations. Thus, using a selective ABS ligand (4-benzylphenoxy-N-ethylmorpholine, MBPE) as starting material, we synthesized a photosensitive azido derivative, [(2-azido-4-benzyl)phenoxy]N-ethylmorpholine (azido-MBPE) that can be prepared in a tritiated form. Azido-MBPE has a high affinity for ABS (Kd = 3 nM), identical to that of tamoxifen, and covalently labels 5 and 12% of membrane-bound and detergent-solubilized ABS, respectively. Its incorporation is selectively and competitively inhibited by other ABS ligands (tamoxifen greater than nitromifen greater than hydroxytamoxifen). [3H]Azido-MBPE potently photolabels either membrane-bound or detergent-solubilized ABS as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under denaturing conditions revealing specific photoincorporation in a protein band of Mr = 40,000. This molecular weight is approximately two times lower than what we observed previously for ABS preparations studied under nondenaturing conditions and postlabeled with [3H]tamoxifen (Mr = 80,000-110,000). In chromatofocusing experiments with photolabeled ABS, a single specifically labeled protein fraction migrating with a pI of 6.4 was found to exhibit a Mr of 40,000 when subsequently electrophoresed on sodium dodecyl sulfate-polyacrylamide gels. These results indicate that [3H]azido-MBPE is a specific high affinity probe of ABS that will prove useful in the ultimate identification of this protein.     

Actin acetylation in Drosophila tissue culture cells

In a permanent cell line derived from Drosophila embryos, cytoplasmic actin is produced as an unstable precursor, which is subsequently converted to a stable form. This conversion results in a reduction in isoelectric point, with no apparent change in molecular weight. The conversion involves an enzymatic acetylation, and results in an insensitivity to aminopeptidase digestion, suggesting N-terminal blockage. Both the acetylated and unacetylated actins can participate in the assembly of F-actin, but with different efficiencies.This work was supported by a grant from the NIH (GM 22866).     

Evaluation of Bottlenecks in the Late Stages of Protein Secretion in Bacillus subtilis

Despite a high capacity for secretion of homologous proteins, the secretion of heterologous proteins by Bacillus subtilis is frequently inefficient. In the present studies, we have investigated and compared bottlenecks in the secretion of four heterologous proteins: Bacillus lichenifomis α-amylase (AmyL), Escherichia coli TEM β-lactamase (Bla), human pancreatic α-amylase (HPA), and a lysozyme-specific single-chain antibody. The same expression and secretion signals were used for all four of these proteins. Notably, all identified bottlenecks relate to late stages in secretion, following translocation of the preproteins across the cytoplasmic membrane. These bottlenecks include processing by signal peptidase, passage through the cell wall, and degradation in the wall and growth medium. Strikingly, all translocated HPA was misfolded, its stability depending on the formation of disulfide bonds. This suggests that the disulfide bond oxidoreductases of B. subtilis cannot form the disulfide bonds in HPA correctly. As the secretion bottlenecks differed for each heterologous protein tested, it is anticipated that the efficient secretion of particular groups of heterologous proteins with the same secretion bottlenecks will require the engineering of specifically optimized host strains.     

Rab11b regulates the trafficking and recycling of the epithelial sodium channel (ENaC)

Expression of the epithelial sodium channel (ENaC) at the apical membrane of cortical collecting duct (CCD) principal cells is modulated by regulated trafficking mediated by vesicle insertion and retrieval. Small GTPases are known to facilitate vesicle trafficking, recycling, and membrane fusion events; however, little is known about the specific Rab family members that modify ENaC surface density. Using a mouse CCD cell line that endogenously expresses ENaC (mpkCCD), the channel was localized to both Rab11a- and Rab11b-positive endosomes by immunoisolation and confocal fluorescent microscopy. Expression of a dominant negative (DN) form of Rab11a or Rab11b significantly reduced the basal and cAMP-stimulated ENaC-dependent sodium (Na(+)) transport. The greatest reduction in Na(+) transport was observed with the expression of DN-Rab11b. Furthermore, small interfering RNA-mediated knockdown of each Rab11 isoform demonstrated the requirement for Rab11b in ENaC surface expression. These data indicate that Rab11b, and to a lesser extent Rab11a, is involved in establishing the constitutive and cAMP-stimulated Na(+) transport in mpkCCD cells.     

Dielectric Spectroscopy: a Rapid Method for the Determination of Solvent Biocompatibility During Biotransformations

Dielectric spectroscopy provides a convenient means of determining the degree of intactness of biological cells. 4-terminal dielectric measurements of suspensions of Saccharomyces cerevisiae at 0.4 MHz show that, as with all other biological cells, these organisms possess a substantial β-dispersion. The additional of octanol to such suspensions causes a rapid decrease in the electrical capacitance of the suspension, which parallels the cellular viability as determined by methylene blue staining. The kinetics of cell death are determined in part by the rate of dissolution of the organic solvent in the aqueous phase. The toxicity of several organic solvents to S. cerevisiae is studied using this technique, and is found to be dependent upon the polarity of the solvent. The present method provides a simple and rapid means for assessing the biocompatibility of solvents used in biotransformations.     

Patterns and controls of the variability of radiation use efficiency and primary productivity across terrestrial ecosystems

Aim The controls of gross radiation use efficiency (RUE), the ratio between gross primary productivity (GPP) and the radiation intercepted by terrestrial vegetation, and its spatial and temporal variation are not yet fully understood. Our objectives were to analyse and synthesize the spatial variability of GPP and the spatial and temporal variability of RUE and its climatic controls for a wide range of vegetation types. Location A global range of sites from tundra to rain forest. Methods We analysed a global dataset on photosynthetic uptake and climatic variables from 35 eddy covariance (EC) flux sites spanning between 100 and 2200 mm mean annual rainfall and between ?13 and 26°C mean annual temperature. RUE was calculated from the data provided by EC flux sites and remote sensing (MODIS). Results Rainfall and actual evapotranspiration (AET) positively influenced the spatial variation of annual GPP, whereas temperature only influenced the GPP of forests. Annual and maximum RUE were also positively controlled primarily by annual rainfall. The main control parameters of the growth season variation of gross RUE varied for each ecosystem type. Overall, the ratio between actual and potential evapotranspiration and a surrogate for the energy balance explained a greater proportion of the seasonal variation of RUE than the vapour pressure deficit (VPD), AET and precipitation. Temperature was important for determining the intra‐annual variability of the RUE at the coldest energy‐limited sites. Main conclusions Our analysis supports the idea that the annual functioning of vegetation that is adapted to its local environment is more constrained by water availability than by temperature. The spatial variability of annual and maximum RUE can be largely explained by annual precipitation, more than by vegetation type. The intra‐annual variation of RUE was mainly linked to the energy balance and water availability along the climatic gradient. Furthermore, we showed that intra‐annual variation of gross RUE is only weakly influenced by VPD and temperature, contrary to what is frequently assumed. Our results provide a better understanding of the spatial and temporal controls of the RUE and thus could lead to a better estimation of ecosystem carbon fixation and better modelling.     

Uridine phosphorylase activity among the class mollicutes

Uridine phosphorylase activity has been used to detect mycoplasmas in cell cultures by measuring formation of¹⁴C-uracil from¹⁴C-uridine. In this report we show that all species ofMycoplasma, Acholeplasma, andUreaplasma tested exhibited uridine phorphorylase activity. Among the genusSpiroplasma, serogroups I-1, I-3, I-5, I-7, I-8, IV, XIII, and XIV lacked uridine phosphorylase activity.Present address: Ciba-Geigy, Basel, Switzerland.     

Hypomorphic mutations in the gene encoding a key Fanconi anemia protein, FANCD2, sustain a significant group of FA-D2 patients with severe phenotype

FANCD2 is an evolutionarily conserved Fanconi anemia (FA) gene that plays a key role in DNA double-strand-type damage responses. Using complementation assays and immunoblotting, a consortium of American and European groups assigned 29 patients with FA from 23 families and 4 additional unrelated patients to complementation group FA-D2. This amounts to 3%-6% of FA-affected patients registered in various data sets. Malformations are frequent in FA-D2 patients, and hematological manifestations appear earlier and progress more rapidly when compared with all other patients combined (FA-non-D2) in the International Fanconi Anemia Registry. FANCD2 is flanked by two pseudogenes. Mutation analysis revealed the expected total of 66 mutated alleles, 34 of which result in aberrant splicing patterns. Many mutations are recurrent and have ethnic associations and shared allelic haplotypes. There were no biallelic null mutations; residual FANCD2 protein of both isotypes was observed in all available patient cell lines. These analyses suggest that, unlike the knockout mouse model, total absence of FANCD2 does not exist in FA-D2 patients, because of constraints on viable combinations of FANCD2 mutations. Although hypomorphic mutations arie involved, clinically, these patients have a relatively severe form of FA.