Protease-Dead Separase Is Dominant Negative in the C. elegans Embryo

Separase is a protease that promotes chromosome segregation at anaphase by cleaving cohesin. Several non-proteolytic functions of separase have been identified in other organisms. We created a transgenic C. elegans line that expresses protease-dead separase in embryos to further characterize separase function. We find that expression of protease-dead separase is dominant-negative in C. elegans embryos, not previously reported in other systems. The C. elegans embryo is an ideal system to study developmental processes in a genetically tractable system. However, a major limitation is the lack of an inducible gene expression system for the embryo. We have developed two methods that allow for the propagation of lines carrying dominant-negative transgenes and have applied them to characterize expression of protease-dead separase in embryos. Using these methods, we show that protease-dead separase causes embryo lethality, and that protease-dead separase cannot rescue separase mutants. These data suggest that protease-dead separase interferes with endogenous separase function, possibly by binding substrates and protecting them from cleavage.     

Regulation of intracellular pH by a neuronal homolog of the erythrocyte anion exchanger

We have isolated AE3, a novel gene expressed primarily in brain neurons and in heart. The predicted AE3 polypeptide shares a high degree of identity with the anion exchange and cytoskeletal binding domains of the erythrocyte band 3 protein. Expression of AE3 cDNA in COS cells leads to chronic cytoplasmic acidification and to chloride- and bicarbonate-dependent changes in intracellular pH, confirming that this gene product is an anion exchanger. Characterization of an AE3 mutant lacking the NH2-terminal 645 amino acids demonstrates that the COOH-terminal half of the polypeptide is both necessary and sufficient for correct insertion into the plasma membrane and for anion exchange activity. The NH2-terminal domain may play a role in regulating the activity of the exchanger and may be involved in the structural organization of the cytoskeleton in neurons.     

Ankylosis of hock joints in group caged male B6C3F1 mice

Enlarged hock joints were observed during 1983 in B6C3F1 mice of chronic toxicity and carcinogenicity studies sponsored by the National Toxicology Program (NTP). Subsequently, approximately 9,500 B6C3F1 mice on 32 NTP chemical toxicity and carcinogenicity studies were evaluated for this condition by clinical examination. Group caged male B6C3F1 mice had thickening and reduced mobility of the hock joints at prevalences of 1.2% up to 6 months of age; 23% at 6 to 12 months of age; and 62% at 13 to 26 months of age. Group caged female B6C3F1 mice had a prevalence of 2% or less. Histologically, affected mice had periarticular exostoses on the bones of the hock joints, with formation of bony bridges around joints and deposition of new bone in joint spaces, resulting in partial or complete ankylosis. Individually caged male and female B6C3F1 mice were not affected. The cause of the ankylosis was not determined, but its occurrence in the NTP studies has been reduced by individual caging.     

A monoclonal antibody to glycoprotein gp85 inhibits fusion but not attachment of Epstein-Barr virus.

Epstein-Barr virus (EBV) codes for at least three glycoproteins, gp350, gp220, and gp85. The two largest glycoproteins are thought to be involved in the attachment of the virus to its receptor on B cells, but despite the fact that gp85 induces neutralizing antibody, no function has been attributed to it. As an indirect approach to understanding the role of gp85 in the initiation of infection, we determined the point at which a neutralizing, monoclonal antibody that reacted with the glycoprotein interfered with virus replication. The antibody had no effect on virus binding. To examine the effect of the antibody on later stages of infection, the fusion assay of Hoekstra and colleagues (D. Hoekstra, T. de Boer, K. Klappe, and J. Wilshaut, Biochemistry 23:5675-5681, 1984) was adapted for use with EBV. The virus was labeled with a fluorescent amphiphile that was self-quenched at the high concentration obtained in the virus membrane. When the virus and cell membrane fused, there was a measurable relief of self-quenching that could be monitored kinetically. Labeling had no effect on virus binding or infectivity. The assay could be used to monitor virus fusion with lymphoblastoid lines or normal B cells, and its validity was confirmed by the use of fixed cells and the Molt 4 cell line, which binds but does not internalize the virus. The monoclonal antibody to gp85 that neutralized virus infectivity, but not a second nonneutralizing antibody to the same molecule, inhibited the relief of self-quenching in a dose-dependent manner. This finding suggests that gp85 may play an active role in the fusion of EBV with B-cell membranes.     

The effect of grasses on the quality of transmitted radiation and its influence on the growth of white clover Trifolium repens

Summary Plants of white clover Trifolium repens were grown under the canopies of three grass species, Lolium perenne, Agrostis tenuis and Holcus lanatus, and under simulated canopies of black polythene and controls were exposed to unfiltered natural radiation. The canopies were adjusted so that they transmitted equal intensities of Photosynthetically Active Radiation (P.A.R.). The ratio of red to far red radiation () was unchanged under the black polythene canopies but was reduced under canopies of Lolium and Agrostis and even more so under Holcus. The effect of canopy filtered radiation on the growth of clover was greatly to reduced internode length, mean number of nodes, the number of branched nodes and the number of rooted nodes and greatly to increase petiole length. The effect of canopies of Holcus was greater than that of the other grass species both in its effect on and on the responses of the clover plant to its shade.     

Syringe exchange schemes for drug users in England and Scotland

In 1987 experimental schemes for distributing injecting equipment to intravenous drug users to help prevent the spread of the human immunodeficiency virus were started by the government. After six months the schemes were found to have been reasonably successful in attracting clients but were less successful in keeping them. It has been shown that equipment can be distributed to drug users on an exchange basis. Many of the clients who were attracted to these schemes had had no treatment or other help for their drug problems.     

Medullary expiratory activity: influence of intercostal tendon organs and muscle spindle endings

Studies were conducted to determine the effects of intercostal muscle spindle endings (MSEs) and tendon organs (TOs) on medullary expiratory activity in decerebrate cats. Impeded intercostal muscle contractions, elicited by electrical stimulation of the peripheral cut end of the T6 ventral root, were used to stimulate intercostal TOs without MSEs. Impeded contractions of the intercostal muscles augmented expiratory laryngeal motoneuron activity, and either had no effect on or reduced the activity of bulbospinal expiratory neurons. Vibration was used to stimulate intercostal MSEs. Intercostal MSEs had no effect on medullary expiratory neuron activity. It is concluded that both external and internal intercostal TOs have an excitatory effect on expiratory laryngeal motoneuron activity and an inhibitory effect on a subpopulation of expiratory neurons driving intercostal and/or abdominal muscles, and intercostal MSEs have no direct influence on medullary expiratory activity.     

Characterization of envelope proteins of alcelaphine herpesvirus 1.

Alcelaphine herpesvirus 1 is a gammaherpesvirus which causes malignant catarrhal fever, an acute lymphoproliferative disorder of cattle and other susceptible Bovidae, which is almost invariably fatal. A preliminary analysis of proteins induced by the virus indicated that as many as six glycoproteins and one nonglycosylated molecule might be present in the virus envelope. Monoclonal antibodies selected for recognition of virion envelope proteins included two that recognized a complex of infected cell proteins, designated the gp115 complex, and neutralized virus infectivity in the absence of complement. The gp115 complex consisted of five glycoproteins of 115, 110, 105, 78, and 48 kilodaltons (kDa), and all except the 48-kDa species reacted with antibody in Western blots (immunoblots). Pulse-chase experiments analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions suggested that the 110-kDa protein was the precursor molecule which was processed by addition of sugars to 115 kDa. The 115-kDa protein was cleaved to form a disulfide-linked heterodimer of 78 and 48 kDa, which was the mature form of the molecule incorporated into the virion envelope. The glycoprotein contained N-linked sugars, but little or no O-linked sugar was present. The relative abundance of the mature protein and its ability to induce neutralizing antibodies suggest that it will prove useful to studies aimed at elucidating the biology and pathogenesis of alcelaphine herpesvirus 1.