Dall-Miles plating for periprosthetic B1 fractures of the femur.

Eight patients (9 fractures) who have been treated with Dall-Miles plating in this unit between April 1996 and October 1997 for ipsilateral periprosthetic fractures around total hip replacement (7 cases) and total knee replacement (2 cases) have been reviewed. Four were men, and 4 were women. The average age at the time of operation was 77 years (range, 65-89 years). The average follow-up period was 14.6 months (range, 6-24 months). Three fractures healed satisfactorily with no evidence of malunion (3 of 9). The final result was unsatisfactory in the other 6 fractures. The femoral component had been inserted in a varus position in all the failures but was in a neutral position in the 3 successes. Procedures other than Dall-Miles plating might be more appropriate in the management of periprosthetic fractures in which the femoral component is in a varus position.

Regulated secretion of a serine protease that activates an extracellular matrix-degrading metalloprotease during fertilization in Chlamydomonas.

During fertilization in the biflagellated alga, Chlamydomonas reinhardtii, gametes of opposite mating types adhere to each other via agglutinin molecules located on their flagellar surfaces, generating a sexual signal that induces several cellular responses including cell wall release. This cell contact-generated signal is mediated by cAMP and release of the wall, which is devoid of cellulose and contains several hydroxyproline-rich glycoproteins, is due to the activation of a metalloprotease, lysin. Although we originally assumed that lysin would be stored intracellularly in a compartment structurally separate from its substrate, recently we showed that lysin is stored in the periplasm as an inactive, higher relative molecular mass precursor, prolysin (Buchanan, M.J., S. H. Imam, W. A. Eskue, and W. J. Snell. 1989. J. Cell Biol. 108:199-207). Here we show that conversion of prolysin to lysin is due to a cellular, nonperiplasmic enzyme that has the properties of a serine protease. Release of this serine protease into the periplasm is induced by incubation of gametes in dibutyryl cAMP. This may be one of the few examples of regulated secretion of a protease in a eucaryotic microorganism and a novel example of regulated secretion in a plant system.

Activation of the cell wall degrading protease, lysin, during sexual signalling in Chlamydomonas: the enzyme is stored as an inactive, higher relative molecular mass precursor in the periplasm.

During the mating reaction in Chlamydomonas reinhardtii mating type plus and mating type minus gametes adhere to each other via adhesion molecules on their flagellar surfaces. This adhesive interaction induces a sexual signal leading to release of a cell wall degrading enzyme, lysin, that causes wall release and degradation. In this article, we describe the preparation of a polyclonal antibody against the 60,000-Mr lysin polypeptide excised from SDS-PAGE gels. After absorption of the IgG with cell walls to remove antibodies against a carbohydrate epitope common to several Chlamydomonas glycoproteins, the immune IgG reacted with the 60,000-Mr polypeptide, and a 47,000-Mr species that we show here was immunologically cross-reactive with the 60,000-Mr molecule. By use of several fractionation methods including ion exchange and molecular sieve chromatography, sucrose gradient centrifugation, and affinity chromatography, we showed that the 60,000-Mr antigen copurified with lysin activity, thereby demonstrating that the antibody was indeed directed against the enzyme. Immunoblot experiments on suspensions of nonmating and mating gametes showed that the 60,000-Mr antigen was missing in the nonmating gametes. Instead, they contained a 62,000-Mr antigen that was not present in suspensions of mating gametes that had undergone sexual signalling. Furthermore, nonmating gametes whose walls were removed with exogenously added lysin did not contain either form of the antigen. We also found that the 62,000-Mr form of the antigen, which could be released from gametes by freeze-thawing, did not have wall degrading activity. These results indicate that lysin in gametes is stored in the periplasm as a higher relative molecular mass, inactive precursor and also that sexual signalling induces conversion of this molecule to a lower relative molecular mass, active enzyme. This may be a novel example of processing of an extracellular protease induced by cell contact.

Biochemical studies on lysin, a cell wall degrading enzyme released during fertilization in Chlamydomonas.

New methods have been developed for the purification and characterization of the cell wall degrading enzyme, lysin, which is released into the medium during the mating reaction of the biflagellated alga Chlamydomonas reinhardtii. A quantitative spectrophotometric assay that detects the number of cells losing walls was used to devise a procedure for the 60-fold purification of lysin from the medium of mating gametes with a 30% yield of activity. Molecular sieve and ion exchange chromatography in combination with SDS-PAGE showed that lysin was a single polypeptide with an Mr of 60,000. High-performance liquid chromatography and sucrose density gradient centrifugation of lysin activity were used to obtain an estimate of 66,000 D for the nondenatured molecular weight of lysin, indicating that lysin behaves as a monomer.

The Chlamydomonas cell wall: characterization of the wall framework.

The cell wall of the biflagellate alga Chlamydomonas reinhardtii is a multilayered, extracellular matrix composed of carbohydrates and 20-25 polypeptides. To learn more about the forces responsible for the integrity of this cellulose-deficient cell wall, we have begun studies to identify and characterize the framework of the wall and to determine the effects of the cell wall-degrading enzyme, lysin, on framework structure and protein composition. In these studies we used walls released into the medium by mating gametes. When isolated shed walls are degraded by exogenously added lysin, no changes are detected in the charge or molecular weight of the 20-25 wall proteins and glycoproteins when analyzed on one- and two-dimensional polyacrylamide gels, which suggests that degradation of these shed walls is due either to cleavage of peptide bonds very near the ends of polypeptides or that degradation occurs via a mechanism other than proteolysis. Incubation of walls with Sarkosyl-urea solutions removes most of the proteins and yields thin structures that appear to be the frameworks of the walls. Analysis by polyacrylamide gel electrophoresis shows that the frameworks are highly enriched in a polypeptide of Mr 100,000. Treatment of frameworks with lysin leads to their degradation, which indicates that this part of the wall is a substrate for the enzyme. Although lysin converts the Mr 100,000 polypeptide from an insoluble to a soluble form, there is no detectable change in Mr of the framework protein. Solubilization in the absence of lysin requires treatment with SDS and dithiothreitol at 100 degrees C. These results suggest that the Chlamydomonas cell wall is composed of two separate domains: one containing approximately 20 proteins held together by noncovalent interactions and a second domain, containing only a few proteins, which constitutes the framework of the wall. The result that shed walls can be solubilized by boiling in SDS-dithiothreitol indicates that disulfide linkages are critical for wall integrity. Using an alternative method for isolating walls from mechanically disrupted gametes, we have also shown that a wall-shaped portion of these unshed walls is insoluble under the same conditions in which shed walls are soluble. One interpretation of these results is that wall release during mating and the wall degradation that follows may involve distinct biochemical events.