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.     

Risk of feather damage explains fault bar occurrence in a migrant hawk,the Swainson's hawk Buteo swainsoni

Fault bars are common stress‐induced feather abnormalities that could produce feather damage thus reducing flight performance. For that reason, it has been hypothesized that birds may have evolved adaptive strategies that reduce the costs of fault bars (the ‘fault bar allocation hypothesis’). An untested prediction of this hypothesis is that fault bars in important feathers for flight (wing and tail) should be less abundant where they produce more feather damage. We tested such a prediction using moulted wing and tail feathers of the long‐distance migrant Swainson's hawk Buteo swainsoni in its Argentinean wintering quarters. We recorded the occurrence of fault bars of different strengths (light, medium and strong) and the damage (lost of a portion of the vane) produced by them. The occurrence of fault bars was very variable, with strong ones being rare throughout and light and medium fault bars being more frequent in the tail than in the wing. Risk of feather damage was similarly high and low across feather groups for strong and light fault bars, respectively, and higher in the wing than in the tail for medium strength. The occurrence of fault bars of different strengths on different feather groups was negatively correlated with their propensity to produce feather damage. At low damage risk (<5%), the occurrence of fault bars was highly variable depending on the feather group, but above 5% of feather damage the occurrence of fault bars was highly reduced throughout. Our results supports the ‘fault bar allocation hypothesis’ of natural selection reducing fault bar occurrence where fault bars are more risky, but further suggest that selection pressure could be relaxed in other instances, leaving the way free for other mechanisms to shape fault bar occurrence.     

Long-term changes in the prevalence of two helminth parasites (Cestoda: Trypanorhyncha) infecting marine fish

Changes in prevalence of the plerocerci of Grillotia angeli Dollfus, 1969 in mackerel, Scomber scombrus L., and of Lacistorhynchus sp. in herring, Clupea harengus L., were recorded over periods of eight years (1978–1985) and 11 years (1974–1984), respectively. Data were collected from 21 year classes of mackerel in an area to the south-west of Britain and Ireland and from seven year classes of herring in the eastern North Sea. Both sets of data showed sharp decreases in parasite prevalence from periods at relatively high levels to others at much lower levels. The changes in prevalence occurred at the same time in both host-parasite systems and coincided with the end of the hydrographic phenomenon known as the mid-70s salinity anomaly. Possible explanations for the changes which are discussed include changes in abundance of first intermediate and definitive hosts, variations in host year class strength, changes in hydrographic conditions and changes in host diet.     

Factors Involved in Hydrolysis of Microcrystalline Cellulose by Acetivibrio cellulolyticus

Acetivibrio cellulolyticus cellulase obtained by the water elution of residual cellulose from the growth medium was compared with the cellulase activity present in culture supernatants. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that water elution released most of the protein bands which adhered to undigested cellulose from the culture medium. The enzyme in the culture supernatant and that eluted from residual cellulose had specific activities for Avicel hydrolysis that were 20- to 40-fold greater than that of Trichoderma reesei cellulase. However, Ca²⁺ and a reducing agent such as dithiothreitol were required for maximum Avicel hydrolysis rates by these A. cellulolyticus enzyme preparations. The effect of these agents on p-nitrophenyl lactopyranoside hydrolysis suggested that they were required by an exoglucanase component. Supernatant enzyme preparations contained large amounts of carbohydrate which was separated from most of the cellulase protein by phenyl-Sepharose chromatography. Removal of this carbohydrate, which interfered with protein fractionations, allowed for an activity stain analysis of the supernatant enzyme.     

On the mountain top with Mr. Osterhout

George E. Osterhout, an amateur botanist, collected mainly in Colorado between 1893 and 1936. A brief account of his career and character; a list of his proposed new species and new combinations; and his bibliography.