Effects of the combination of a proteasome inhibitor (PSI) and an inhibitor of ubiquitin-ligases (Leu-Ala) on the ultrastructure of human leukemic U937 cells.

We have used the dipeptide Leu-Ala in an attempt to prevent the formation of ubiquitin-protein conjugates in U937 cells by inhibition of cellular E3 enzymes (ubiquitin ligases). Proteasome inhibitors induce the formation of perinuclear aggregates of ubiquitinated proteins and proteasomes (aggresomes) in the area of the proteolytic center of the cell. Leu-Ala did not prevent the forrmation of those aggregates under the action of PSI (peptidyl aldehyde, selective inhibitor of the chymotrypsin-like activity of the proteasome), however it induced an accumulation of lipid droplets in treated cells, suggesting a previously unknown involvement of Leu-Ala in lipid metabolism. We conclude, that either Leu-Ala is not able to completely inhibit the cellular E3 enzymes or some of those enzymes are insensitive to this dipeptide, allowing therefore the build-up of ubiquitin-conjugates in the proteolytic centre of the cell.

Proteasome localization and ultrastructure of spermatozoa from patients with varicocele--immunoelectron microscopic study.

Localization of proteasomes in spermatozoa from patients with varicocele-associated sterility was studied by means of immunolabeling with the MPC21 monoclonal antibody detecting the C3 subunit of the 20S proteasome. The reaction was visualized for electron microscopy using the secondary Nano-Gold-coupled antibody with Gold-Enhancement in pre-embedding technique. We found that semen samples from varicocele patients contained a large amount of abnormal spermatozoa characterized by the presence of dispersed chromatin and large residual bodies (cytoplasmic droplets) as well as spermatids at various stages of spermiogenesis. In normal spermatozoa, the immunolabeling was found in the acrosome, postacrosomal regions, nuclear vacuoles, in the neck and in the middle-piece as well as in the residual bodies, while chromatin remained unlabeled. In varicocele spermatozoa, the immunolabeling was also associated with chromatin and large residual bodies (cytoplasmic droplets). In contrast to normal, mature spermatozoa, the chromatin of the cells at earlier stages of spermiogenesis was strongly immunolabeled. The association of proteasomes with sperm chromatin and large residual bodies can be the sign of abnormality and disturbances in spermatogenesis associated with varicocele.

Localization of a proteasomal antigen in human spermatozoa: immunohistochemical electron microscopic study.

The ultrastructural localization of a proteasomal antigen in human spermatozoa was studied by means of immunolabeling with the MPC21 monoclonal antibody and secondary gold labeled antibody with 1.4 nm gold particles in combination with silver enhancement reaction using pre-embedding technique. The labeling was found in the acrosomal and postacrosomal regions, in the connecting-piece (neck) and, in some cases, in the middle-piece and also in the residual bodies. There was no significant reaction in condensed chromatin. In some abnormal forms of spermatozoa, in which the chromatin was not well condensed, the labeling in nuclei was present. The nuclear vacuoles with looser chromatin were usually strongly labeled. The nuclei of cells representing different stages of spermatogenesis, that were present in semen samples, were also labeled.

Quantitative ultrastructural evaluation of satellite cells in soleus muscle from rats kept in hypokinesia.

The aim of the present study is a morphometric, ultrastructural evaluation of satellite cells (SC) derived from the soleus muscle (SOL) of rats exposed to conditions of hypokinesia for a period of 7 or 21 days. Qualitative and quantitative analysis of 320 electron micrographs of SC from each group was carried out. After 7 and 21 days of immobilization, profiles of the SC, in contrast to the control group, had a lower mean surface area, had a cylindrical shape, and exhibited more folded membrane. Analysis of electron micrographs of SC showed that after immobilization, a lower number of SC contained profiles of mitochondria, rough endoplasmic reticulum (RER), and Golgi. Volume fractions of RER were twofold lower after 7 days of hypokinesia and fivefold lower after 21 days compared with the control group. The SC of SOL of rats subjected to hypokinesia differed from the control group by a markedly decreased number of ribosomes and RER profiles. After 21 days of immobilization the ultrastructural characteristics of SC were typical for cells in an inactive state showing various degrees of degeneration. The results of our study presented here permit the conclusion that 21 days of hypokinesia induce a depression of SC activity, whereas subtle changes in SC appear only after 7 days of immobilization.

Morphometric analysis of satellite cells in rat skeletal muscles: soleus and extensor digitorum longus.

Satellite cells (SC) are present in all types of skeletal muscles. They can exhibit DNA replication and mitotic divisions throughout their life. Depending on the kind of muscle, a different number of SC is present. The aim of this study was to establish whether the differences in muscle metabolism are reflected in the quantitative ultrastructural evaluation of their SC. Two skeletal muscles of rats, soleus (SOL) with predominant slow fibers and extensor digitorum longus (EDL) with predominant fast ones, were studied. Morphometric analysis was performed according to the modified method of Weibel [Stereological Methods, vol 1: Practical Methods for Biological Morphometry. London, Academic Press, 1979]. In the SC of EDL mitochondria, rough endoplasmic reticulum and Golgi apparatus were less frequent as compared with the SOL. The volume fractions of these organelles were much higher in SC of SOL than in EDL. The ribosomes and polyribosomes were more densely packed in SC of SOL than in EDL. Ultrastructural morphometric evaluation demonstrated significant differences between the SC components of two different muscles: a slow (SOL) and a fast (EDL) one.

Morphometric ultrastructural evaluation of satellite cells in musculus soleus and musculus extensor digitorum longus of rats subjected to hypokinesia.

NASA: Soleus and extensor digitorum longus muscles were studied in rats confined to cages which restricted their movement. Ultrastructural study by electron microscopy of satellite cells did not reveal major differences between the cytoplasmic organelles of confined animals and those of controls. Some changes in mitochondria were noted. Results of the ultrastructural analysis are discussed.^ieng

Morphometric ultrastructural evaluation of secondary oocytes of mice receiving vincristine.

Secondary oocytes from mice receiving vincristine in amounts corresponding to therapeutic doses in human, in neoplasm treatment, were evaluated morphometrically and ultrastructurally. In the group of animals treated with vincristine an increase of the volume fraction of vesicular complexes and a decrease of the volume fraction of fibrous material were observed. It has also been demonstrated that, in secondary oocytes from mice receiving vincristine, the number of both multivesicular bodies and vesicles falling to one vesicular complex profile was higher than in the control group. The present results suggest that vincristine applied to females, even in therapeutic doses, may affect egg cells.