Dimer–monomer equilibrium of human thymidylate synthase monitored by fluorescence resonance energy transfer

An ad hoc bioconjugation/fluorescence resonance energy transfer (FRET) assay has been designed to spectroscopically monitor the quaternary state of human thymidylate synthase dimeric protein. The approach enables the chemoselective engineering of allosteric residues while preserving the native protein functions through reversible masking of residues within the catalytic site, and is therefore suitable for activity/oligomerization dual assay screenings. It is applied to tag the two subunits of human thymidylate synthase at cysteines 43 and 43′ with an excitation energy donor/acceptor pair. The dimer–monomer equilibrium of the enzyme is then characterized through steady‐state fluorescence determination of the intersubunit resonance energy transfer efficiency.     

Altered Intracellular Localization of SOD1 in Leukocytes from Patients with Sporadic Amyotrophic Lateral Sclerosis

Several lines of evidence support the hypothesis of a toxic role played by wild type SOD1 (WT-SOD1) in the pathogenesis of sporadic amyotrophic lateral sclerosis (SALS). In this study we investigated both distribution and expression profile of WT-SOD1 in leukocytes from 19 SALS patients and 17 healthy individuals. Immunofluorescence experiments by confocal microscopy showed that SOD1 accumulates in the nuclear compartment in a group of SALS subjects. These results were also confirmed by western blot carried out on soluble nuclear and cytoplasmic fractions, with increased nuclear SOD1 level (p<0.05). In addition, we observed the presence of cytoplasmic SOD1 aggregates in agreement with an increased amount of the protein recovered by the insoluble fraction. A further confirmation of the overall increased level of SOD1 has been obtained from single cells analysis using flow cytometry as cells from SALS patients showed an higher SOD1 protein content (p<0.05). These findings add further evidence to the hypothesis of an altered WT-SOD1 expression profile in peripheral blood mononuclear cells (PBMCs) from patients with ALS suggesting that WT-SOD1 species with different degrees of solubility could be involved in the pathogenesis of the disease.     

Coupling between phosphoinositide breakdown and early mitogenic events in fibroblasts. Studies with fluoroaluminate, vanadate, and pertussis toxin

In the preceding paper (Paris, S., and Pouysségur J. (1987) J. Biol. Chem. 262, 1970-1976), AlF4- and vanadate have been shown to induce inositol phosphate formation in resting hamster fibroblasts (CCL39). In this study, we show that these two phosphate analogs are good tools to explore the causal relationship between phosphoinositide breakdown and early mitogenic events. AlF4- can activate, very similarly to the mitogen alpha-thrombin: the amiloride-sensitive Na+/H+ antiport, the bumetanide-sensitive Na+/K+/Cl- co-transport, and the expression of c-myc mRNA. The link between phospholipase C activation and these early events of the mitogenic response is demonstrated by the similarity of all dose-response curves for NaF and AlCl3 and by the common sensitivity of the four events to pertussis toxin. Vanadate likewise stimulates the Na+/H+ antiport through a pertussis toxin-sensitive pathway. On longer incubations, both fluoride and vanadate were found to be toxic and failed to induce DNA synthesis. Therefore, we have used pertussis toxin to investigate the link between phospholipase C activation and commitment to DNA synthesis. We show that pertussis toxin strikingly inhibits thrombin-induced reinitiation of DNA synthesis but does not affect the stimulation by the epidermal or fibroblast growth factors, two mitogens that do not stimulate phosphoinositide breakdown in CCL39 cells. In conclusion, these studies demonstrate that activation of phospholipase C, if not an obligatory step in the action of all growth factors, plays a crucial role in the mitogenic signaling pathway of alpha-thrombin.     

The three-dimensional structure of the zona pellucida in growing and atretic ovarian follicles of the mouse

Summary The present study provides further details on the fine-structural three-dimensional architecture of the zona pellucida (ZP) in growing and atretic follicles of mice by use of ruthenium red in combination with the detergents Triton X100 and saponin. These detergents were used for extraction of the soluble fraction of the zonal proteins in an attempt to expose the structural zonal glycoproteins, which in turn can be viewed as minute three-dimensional networks upon transmission- and scanning electron-microscopic examination. By use of these methods, the ZP of growing follicles appeared to be formed by interconnected filaments which also bind to globular structures building up a three-dimensional lattice. In contrast, the ZP of stage I as well as other (II and III) stages of atretic follicles showed a structure characterized by the presence of closely packed granules connected with short filaments to form a close-mesh reticulum. This structural change of the ZP, which in the present study is also associated with the disappearance of gap junctions within the granulosa and cumulus cell population, might represent one of the early events involved in the onset of atresia. These changes, most probably depending on an altered secretory activity of both oocytes and follicle cells, might lead to a degradation of the ZP network structure and to its subsequent increased density (condensation). All these morphodynamic events eventually contribute to a sequestration of the oocyte in the early stage of atresia.     

Nasal allergy to fungi in guinea pigs

Sensitized guinea pigs produced specific IgG and IgE antibodies toward Cladosporium and Alternaria. In presence of fungal extracts, nasal mast cells degranulate. Ultrastructural modifications of the cells during degranulation have been established. The ciliary epithelium and the ciliary beating are not affected by fungal allergens.     

Pericentric inversion of chromosome 19 in three families

Summary Pericentric inversion of chromosome 19 has been found in several members of three unrelated families from a restricted geographical region. In one of the families, an additional pericentric inversion of chromosome 9 was observed. Reproductive problems, multiple abortions in two families and a neonatal death in the third, were present. A review of previously described cases is included, and the genetic risk connected with this type of rearrangement is also discussed.