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.     

Genetic and biological analyses of a herpes simplex virus intertypic recombinant reduced specifically for neurovirulence.

RS6 is a herpes simplex virus intertypic recombinant derived from type 1 strain 17 syn+ and type 2 strain HG52. With a 50% lethal dose of about 10(5) PFU after intracerebral inoculation of mice, RS6 was approximately 100,000 times less neurovirulent than either of its wild-type parental viruses were. When compared with strains 17 syn+ and HG52, RS6 replicated intermediately in primary mouse embryo fibroblasts in vitro at 38.5 degrees C (mouse temperature) and to wild-type peak titers in mouse feet in vivo. In contrast, following intracranial inoculation of mice, RS6 replicated significantly less well than did either of its parental viruses in brains. The genetic defect(s) responsible for the reduced neurovirulence of RS6 was stable after in vitro and in vivo serial passage, was not manifested as temperature-sensitive plaquing in vitro, and did not affect thymidine kinase expression. These data indicate that RS6 has a genetic defect(s) specifically affecting its ability to replicate in the mouse brain. Using marker rescue technologies, we increased the neurovirulence of RS6 and localized one genetic determinant(s) involved with the reduced neurovirulence of this agent to 0.72 to 0.87 map units (and, tentatively, to 0.79 to 0.83 map units) of the herpes simplex virus genome. When coupled with the work suggesting that thymidine kinase expression is essential for efficient replication in nerve tissues and earlier reports from this laboratory and others, the results presented in this study indicate that more than one herpes simplex virus gene is involved with neurovirulence.     

A 5'----3' exoribonuclease of Saccharomyces cerevisiae: size and novel substrate specificity

The purification scheme for a 5'----3' exoribonuclease of Saccharomyces cerevisiae has been modified to facilitate purification of larger amounts of enzyme and further extended to yield highly purified enzyme by use of poly(A)-agarose chromatography. As determined by either sodium dodecyl sulfate-polyacrylamide gel electrophoresis or physical characterization, the enzyme has a molecular weight of about 160,000. Further studies of its substrate specificity show that poly(C) and poly(U) preparations require 5' phosphorylation for activity and that poly(A) with a 5'-triphosphate end group is hydrolyzed at only 12% of the rate of poly(A) with a 5'-monophosphate end group. DNA is not hydrolyzed, but synthetic polydeoxyribonucleotides are strong competitive inhibitors of the hydrolysis of noncomplementary ribopolymers. Poly(A).poly(U) and poly(A).poly(dT) are hydrolyzed at 60 and 50%, respectively, of the rate of poly(A) at 37 degrees C. The RNase H activity of the enzyme can also be demonstrated using an RNA X M13 DNA hybrid as a substrate. When poly(dT).poly(dA) with a 5'-terminal poly(A) segment on the poly(dA) is used as a substrate, the enzyme hydrolyzes the poly(A) "tail," removing the last ribonucleotide, but does not hydrolyze the poly(dA).     

The metabolism of deoxyguanosine in mitochondria. Characterization of the uptake process

Summary The uptake of deoxyguanosine by rat liver mitochondria was characterized. The process required an intact mitochondrial membrane and exhibited a dependence on added phosphate. Deoxyguanosine uptake was minimally influenced by Mg²⁺ or Mn²⁺, but Ca²⁺ at concentrations above 0.5 mM were detrimental. Of the deoxynucleosides tested, only deoxyinosine inhibited the uptake of deoxyguanosine. The ribonucleoside guanosine was not observed to compete with its deoxynucleoside analog. Known inhibitors of nucleoside transport, cytochalasin B and NBMPR, did not block deoxyguanosine uptake, but the sulfhydryl reagents NEM and pCMB were both inhibitory. The uptake of deoxyguanosine was shown to be a saturable process and an apparent Km of 0.64 M was calculated from a Hanes plot.     

The relative changes in isometric force and work during fatigue and recovery in isolated toad sartorius muscle

Toad sartorius muscle was subjected to sinusoidal varying length changes at 2 Hz to measure work. Both isometric tetanic force and work per cycle were measured before, during, and after a 3-min fatigue. Both isometric tetanic force and positive work, the work done by the muscle during the shortening part of the cycle, rapidly decreased in parallel in the first 40 s of fatigue. Thereafter, force continued to decrease, but at a slower rate, to about 10% of prefatigue values, whereas positive work levelled off at about 30% of prefatigue values. Negative work, the work done on the muscle during the lengthening part of the cycle, increased during fatigue to the extent that net work became negative. This was due to a prolonged relaxation, which resulted in active force still being generated while the muscle was being stretched. Work and force recovered at about the same rate. Isometric force measurements alone do not give any clear indication that net work will be negative under a particular set of experimental conditions.