Vital role for Plasmodium berghei Kinesin8B in axoneme assembly during male gamete formation and mosquito transmission

Sexual development is an essential phase in the Plasmodium life cycle, where male gametogenesis is an unusual and extraordinarily rapid process. It produces 8 haploid motile microgametes, from a microgametocyte within 15 minutes. Its unique achievement lies in linking the assembly of 8 axonemes in the cytoplasm to the three rounds of intranuclear genome replication, forming motile microgametes, which are expelled in a process called exflagellation. Surprisingly little is known about the actors involved in these processes. We are interested in kinesins, molecular motors that could play potential roles in male gametogenesis. We have undertaken a functional characterization in Plasmodium berghei of kinesin‐8B (PbKIN8B) expressed specifically in male gametocytes and gametes. By generating Pbkin8B‐gfp parasites, we show that PbKIN8B is specifically expressed during male gametogenesis and is associated with the axoneme. We created a ΔPbkin8B knockout cell line and analysed the consequences of the absence of PbKIN8B on male gametogenesis. We show that the ability to produce sexually differentiated gametocytes is not affected in ΔPbkin8B parasites and that the 3 rounds of genome replication occur normally. Nevertheless, the development to free motile microgametes is halted and the life cycle is interrupted in vivo. Ultrastructural analysis revealed that intranuclear mitoses are unaffected whereas cytoplasmic microtubules, although assembled in doublets and elongated, fail to assemble in the normal axonemal ‘9+2' structure and become motile. Absence of a functional axoneme prevented microgamete assembly and release from the microgametocyte, severely reducing infection of the mosquito vector. This is the first functional study of a kinesin involved in male gametogenesis. These results reveal a previously unknown role for PbKIN8B in male gametogenesis, providing new insights into Plasmodium flagellar formation.     

Lack of correlation between plasminogen activator production and the continous expression of the transformed phenotype of chicken and mammalian cells as shown with S-adenosyl homocysteine analogues

The effect of S-adenosyl-homocysteine and some of its natural and synthetic analogues was studied on plasminogen activator production in chick embryo fibroblasts transformed by a wild type and by a temperature-conditional mutant of Rous Sarcoma Virus (RSV), in a RSV transformed hamster cell line and in human KB cells. When the analogues were added to the cells infected by RSV before the morphologic transformation took place, those molecules which were previously known as inhibitors of cell transformation, inhibited also plasminogen activator production. However when the same molecules were added to already transformed cells, plasminogen activator was still inhibited very strongly, but the cells conserved their transformed morphology. These results show a lack of coordination between plasminogen activator production and the maintenance of the transformed phenotype, and the possibility to inhibit plasminogen activator production by certain transmethylase inhibitors.     

Endogenous factors possessing lymphoid chalone properties, purified by chromatography

Lymphoid chalone extracts, obtained from bovine spleen were purified on chromatographic columns: in the first step of the procedure, exclusion chromatography on Sephadex G75, and ion exchange chromatography on DEAE Sephadex were applied. A purified, active material was thus obtained. Upon thin layer chromatography, this fraction was shown to contain 3-4 UV absorbing components and 2 ninhydrin positive components. Further purification on Biogel P2, on Sephadex G10 and preparative thin layer chromatography, showed that the biological activity was located in small molecular weight components which belong to the polypeptide series. The ultraviolet absorbing components were identified as known nucleosides. The purified material shows an inhibitory effect on the uptake of 3H-thymidine by lymphocytes, as well as on mitogen induced lymphocyte transformation and haemolysin plaque forming cells. Furthermore, this inhibitory effect is specific for lymphoid cells.     

Endothelial cell junctions

In the course of a freeze-cleave study on intercellular junctions in the regenerating rat liver, we observed an unusual array of intramembranous particles located in regions of contact between endothelial cells lining the hepatic sinusoids. These arrays were characterized by an accumulation of particles which resembled a zonula occludens in their linear deployment but differed in that the contact regions were composed of individual particles which remained separated from each other by regular particle-free intervals.