Characterization of herpesvirus sylvilagus glycoproteins released into the culture medium of infected cells: antisera to gp13 and gp32 neutralize viral infectivity in vitro and identify antigens on plasma membranes of infected cells.

Polypeptides released into the culture medium of herpesvirus sylvilagus-infected cells were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of extracellular fluid from [35S]methionine- and [3H]glucosamine-labeled cell cultures. Virus-induced glycoproteins 31, 32, and 33 (molecular weights of 62,000, 59,000, and 54,000, respectively) were the most abundant species and appeared predominantly in the culture medium. This observation, together with the known cell-associated nature of herpesvirus sylvilagus, suggested that virus-induced glycoproteins 31, 32, and 33 were specifically released. Immunization of rabbits with virus-induced glycoproteins 13 (molecular weight of 130,000) and 32 resulted in the production of antibodies that neutralized viral infectivity in vitro. Both antiserum to gp13 and antiserum to gp32 immunoprecipitated gp13, gp26, gp33a, gp45, and virus-induced polypeptide 39 (molecular weights of 130,000, 77,000, 49,000, 27,000, and 36,000, respectively) from [35S]methionine-labeled cell extracts as well as virus-induced glycoproteins 31, 32, and 33 from the culture medium. In addition, membrane immunofluorescence assays indicate that an antigen(s) reactive with anti-gp13/32 serum was located on the plasma membrane of infected cells.     

The role of oxygen availability in the embryonation of Heterakis gallinarum eggs.

The importance of oxygen availability in the embryonation of the infective egg stages of the gastrointestinal nematode parasite Heterakis gallinarum was studied in the laboratory. Unembryonated H. gallinarum eggs were kept under either aerobic conditions by gassing with oxygen, or anaerobic conditions by gassing with the inert gas nitrogen, under a range of constant temperatures. Oxygenated eggs embryonated at a rate influenced by temperature. Conversely, eggs treated with nitrogen showed no embryonation although when these eggs were transferred from nitrogen to oxygen gas after 60 days of treatment, embryonation occurred. This demonstrated that oxygen is an essential requirement for H. gallinarum egg development, although undeveloped eggs remain viable, even after 60 days in low oxygen conditions. The effects of climate on the biology of free-living stages studied under constant laboratory conditions cannot be applied directly to the field where climatic factors exhibit daily cycles. The effect of fluctuating temperature on development was investigated by including an additional temperature group in which H. gallinarum eggs were kept under daily temperature cycles between 12 and 22°C. Cycles caused eggs to develop significantly earlier than those in the constant mean cycle temperature, 17°C, but significantly slower than those in constant 22°C suggesting that daily temperature cycles had an accelerating effect on H. gallinarum egg embryonation but did not accelerate to the higher temperature. These results suggest that daily fluctuations in temperature influence development of the free-living stages and so development cannot be accurately predicted on the basis of constant temperature culture.     

Atomic force microscope imaging of phospholipid bilayer degradation by phospholipase A2.

We have investigated the time course of the degradation of a supported dipalmitoylphosphatidylcholine bilayer by phospholipase A2 in aqueous buffer with an atomic force microscope. Contact mode imaging allows visualization of enzyme activity on the substrate with a lateral resolution of less than 10 nm. Detailed analysis of the micrographs reveals a dependence of enzyme activity on the phospholipid organization and orientation in the bilayer. These experiments suggest that it is possible to observe single enzymes at work in small channels, which are created by the hydrolysis of membrane phospholipids. Indeed, the measured rate of hydrolysis of phospholipids corresponds very well with the enzyme activity found in kinetic studies. It was also possible to correlate the number of enzymes at the surface, as calculated from the binding constant to the number of starting points of the hydrolysis. In addition, the width of the channels was found to be comparable to the diameter of a single phospholipase A2 and thus further supports the single-enzyme hypothesis.