Mediator structure and conformation change

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Amino-Terminal Charge Affects the Periplasmic Accumulation of Recombinant Heregulin/EGF Hybrids Exported Using theEscherichia coliAlkaline Phosphatase Signal Sequence

AnEscherichia coliexpression system that exploits the bacterial alkaline phosphatase (PhoA) signal sequence to translocate recombinant human epidermal growth factor (hEGF) to the periplasm was used to evaluate how changes in the composition and sequence of amino acids near the PhoA–hEGF junction influence the periplasmic accumulation of recombinant protein. A series of chimeric structural genes was generated byin vitroreplacement of hEGF sequence with analogous segments from the EGF-like domain of human heregulin (HRG), significantly altering the electrostatic character of the amino-terminal region of the mature protein. Quantitation of HRG/EGF protein inE. coliperiplasmic extracts, by RP-HPLC, showed a fourfold decrease after one of two acidic residues located in the amino-terminal region of the mature hEGF, near the PhoA junction, was replaced. An additional threefold decrease was observed when the second acidic residue was replaced with a positively charged lysine. Further extension of the amino-terminal HRG sequence, beyond the first six residues, resulted in net neutralization of a more distant EGF acidic residue with no additional effect on protein yield. The importance of having a negatively charged group in the amino-terminal region of the mature protein was confirmed when insertion of an aspartic acid near the amino-terminus of two poorly expressed hybrid protein sequences resulted in a five- to eightfold increase in their recovery from the periplasm. This study demonstrates the importance of having negatively charged residues near the fusion junction of recombinant proteins expressed inE. coliusing the PhoA signal sequence for protein export.     

Fluorescence anisotropy from diphenylhexatriene in rat liver plasma membranes

Rat livers were fractionated to obtain intracellular membrane preparations and a highly purified preparation of bile canaliculi. The fraction containing bile canaliculi was homogenized and subfractionated to give fractions representing fragments of contiguous membrane and of canalicular microvilli. The relative purity and extent of contamination of each preparation was determined. When the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene was incorporated into aliquots of each fraction at the same probe: lipid ratio and the steady-state anisotropy of its fluorescence measured, it was found that the plasma membrane preparations were much more ordered than the intracellular membrane preparations. Of the plasma membrane preparations, that containing the canalicular microvilli was the most ordered, even allowing for any contribution of contaminants. Thus the microvillus membrane of the bile canaliculus appears to be the most ordered domain of the plasma membrane of the hepatocyte. The high order in this domain may be a factor in reducing the susceptibility to bile salt damage during bile secretion, since it is this region which is exposed to high concentrations of bile salts in vivo.     

ROOT PRODUCTION IN FOUR COMMUNITIES IN THE GREAT DISMAL SWAMP

A sequential coring approach was used to measure root biomass and production over 1 year in four different communities within the Great Dismal Swamp. A second method, an implanted bag technique, was also used to measure root production, and values were generally lower using this technique. On all sites, fine roots were the most dynamic root component. Both biomass (1,887 g/m²) and production (354–989 g m ² yr^-1) were highest on the mixed hardwood site, the least flooded site, and second highest on the cedar site, the site with the longest duration of soil saturation (1,033 g/m² and 274–366 g m^-2 yr^-1). The maple-gum (696 g/m² and 59–91 g m^-2 yr^-1) and cypress (824 g/m² and 68–308 g m^-2 yr^-1) sites had similarly low amounts of biomass and rates of production. Environmental parameters that influenced production include frequency and duration of flooding, and soil type. Peaks in belowground production were observed on the most productive sites (mixed hardwood and cedar) in summer and late fall-winter; the other two sites exhibited little seasonal variability. The least flooded stand appears to allocate a greater percentage of net primary production belowground than the more extensively flooded stands. The ratio of above- and belowground allocation appears to change in response to a flooding gradient. This has major implications for ecosystem functions as carbon allocation patterns determine the array of litter types generated (leaves vs. roots) which affect decomposition rates and nutrient availability.