Changes in the size and shape of the synaptic vesicles in the sensorimotor cortex of the rat brain in the initial phases of kindling

The sensorimotor area of rat cerebral cortex was subjected to repeated electrical stimulation at 10-min intervals, with resultant formation and progressive lengthening of self-sustained after-discharges (SSAD). One and 60 min after the third SSAD ended, we carried out an electron microscopy morphometric analysis of the agranular synaptic vesicles in type I synapses (after Gray) in the second cortical layer of the homotopic area of the unstimulated hemisphere. One minute after the seizure ended, 5.8% enlargement of the synaptic vesicles compared with the control was demonstrated in zone II of the synapse (0.1-0.2 micron from the active zone of the synapse). Neither the size nor the shape of the synaptic vesicles in the other parts of the synaptic apparatus altered. Sixty min after the seizure ended, a 5.5% enlargement of the synaptic vesicles in zone I (0.0-0.1 micron) and a 5.4% enlargement of those in zone II was found. The synaptic vesicles in zone I in the experimental animals were more oval than in the controls. Our findings support the vesicular theory and testify that hyperfunction, up to temporary exhaustion of the synaptic apparatuses, produces a change in the transmitter content of the synaptic vesicles. A raised amount of transmitter in the synaptic vesicles near the active zone could be one of the factors responsible for continued hyperexcitability of the tissue one hour after the seizure had ended. The results likewise support the concept of two mechanisms of synaptic vesicle formation, and hence of the existence of two different vesicle populations.     

Amiloride fluxes across erythrocyte membranes

Amiloride is known to inhibit both the influx of Na+ and the activation of mitogenesis in many cultured cell lines. This paper describes experiments in which the permeability coefficient of amiloride was determined from measurements of tracer fluxes across human erythrocytes and resealed ghosts. From an analysis of these fluxes, a permeability coefficient of 10(-7) cm/s for the uncharged form of amiloride was deduced. Based upon this measured permeability value, we present calculations of intracellular accumulation times of amiloride in cells of differing surface-to-volume ratio.     

Inhibition of apoptosis by calcium ionophores in IL-3-dependent bone marrow cells is dependent upon production of IL-4+.

Calcium ionophores inhibit apoptosis in the IL-3-dependent cell line BAF3 and maintain the cells in a viable noncycling state. In this report, an identical effect of ionophore was also demonstrated on the multipotent IL-3-dependent progenitor cell line FDCP-MIX and on the primary IL-3-dependent cell population that could be cultured from murine bone marrow. Inhibition of apoptosis required extracellular calcium and could be blocked by cyclosporin A. Nuclei from IL-3-dependent cells were found to lack a calcium-activatable nuclease that degrades chromatin in the linker region between nucleosomes, unlike the nuclei of lymphoid cells. The mechanism of action of calcium ionophore could be divided into two distinct steps. First, ionophore induced the production of a survival factor that stimulated DNA synthesis and was identified as IL-4. Second, ionophore inhibited the cell cycle of the various IL-3-dependent cells. IL-4 production could be inhibited by cyclosporin A and required extracellular calcium, whereas cell cycle arrest did not. This implied that factor production was the step that was necessary for inhibition of apoptosis and maintenance of cell viability. This was confirmed by the use of an anti-IL-4R antibody, which blocked the inhibition of apoptosis induced by calcium ionophores.     

Murine Ia-associated invariant chain's processing to complex oligosaccharide forms and its dissociation from the I-Ak complex

The processing of murine invariant chain (Ii) to a cell surface form bearing complex N-linked oligosaccharides has been demonstrated in the B cell lymphoma, AKTB-1b. In addition, the rate of processing of pulse-labeled Ii has been determined relative to its rate of dissociation from the alpha/beta complex of I-Ak. Ii, alpha-, and beta-chains were immunoprecipitated with anti-I-Ak or anti-Ii monoclonal antibodies. The heretofore uncharacterized complex oligosaccharide form of Ii (Ii-c) was identified in gel-purified immunoprecipitates by peptide mapping with reverse-phase HPLC. Ii-c is resistant to deglycosylation by Endo H, which is specific for high-mannose N-linkages, but can be digested with Endo F, a glycosidase capable of cleaving both complex and high-mannose N-linked oligosaccharides. Immunoprecipitation of surface iodinated cells indicates that Ii-c is expressed on the plasma membrane. Pulse-chase metabolic labeling data show that the processing of Ii to Ii-c occurs with a t1/2 of about 120 min. In contrast, the processing of both alpha- and beta-chains of I-Ak to complex forms occurs with a t1/2 of 15 to 20 min. Our data show that Ii-hm begins to dissociate rapidly from the I-Ak complex after 100 to 120 min of chase. Only a small amount (less than 5% on a per mole basis) of Ii-c was found associated with the I-Ak complexes after 300 min of continuous metabolic labeling. These results are consistent with Ii serving as a carrier for Ia antigens as they are transported to the cell surface. In addition, they suggest that the processing of Ii to Ii-c, or a late processing event of the alpha- and beta-chains, such as their sialylation, may be a possible mechanism for inducing the dissociation of Ii from the I-Ak complex.     

Interaction between the Msh2 and Msh6 Nucleotide-binding Sites in the Saccharomyces cerevisiae Msh2-Msh6 Complex

Indirect evidence has suggested that the Msh2-Msh6 mispair-binding complex undergoes conformational changes upon binding of ATP and mispairs, resulting in the formation of Msh2-Msh6 sliding clamps and licensing the formation of Msh2-Msh6-Mlh1-Pms1 ternary complexes. Here, we have studied eight mutant Msh2-Msh6 complexes with defective responses to nucleotide binding and/or mispair binding and used them to study the conformational changes required for sliding clamp formation and ternary complex assembly. ATP binding to the Msh6 nucleotide-binding site results in a conformational change that allows binding of ATP to the Msh2 nucleotide-binding site, although ATP binding to the two nucleotide-binding sites appears to be uncoupled in some mutant complexes. The formation of Msh2-Msh6-Mlh1-Pms1 ternary complexes requires ATP binding to only the Msh6 nucleotide-binding site, whereas the formation of Msh2-Msh6 sliding clamps requires ATP binding to both the Msh2 and Msh6 nucleotide-binding sites. In addition, the properties of the different mutant complexes suggest that distinct conformational states mediated by communication between the Msh2 and Msh6 nucleotide-binding sites are required for the formation of ternary complexes and sliding clamps.