The Prp2 protein of Saccharomyces cerevisiae is an RNA-dependent ATPase required before the first transesterification reaction in pre-mRNA splicing. Prp2 binds to the spliceosome in the absence of ATP and is released following ATP hydrolysis. We determined what regions in Prp2 are essential for release from the spliceosome by analyzing dominant negative mutants in vivo and in vitro. We made mutations in conserved motif II (DExH) and motif VI (QRxGR) of the helicase (H) domain. Mutations that inactivated PRP2 had a dominant negative phenotype when overexpressed in vivo. To test whether mutations outside of the H domain could confer a dominant negative phenotype, we mutagenized a GAL1-PRP2 construct and screened for mutants unable to grow on galactose-containing media. Five dominant negative mutants were characterized; three mapped within the H domain and two mapped downstream of motif VI, indicating that an extended helicase domain is required for release of Prp2 from the spliceosome. Most mutants stalled in the spliceosome in vitro. However, not all mutants that were dominant negative in vivo were dominant negative in vitro, indicating that multiple mechanisms may cause a dominant negative phenotype. Structural modeling of the H domain of Prp2 suggests that mutants map to a cleft region found in helicases of known structure.

Wistar rat kidneys were perfused with some fixatives at different pressures of 100, 150, 200 or 250 cm H₂0 via the aorta and were studied by the quick-freezing and freeze-substitution (QF-FS) or deep-etching (QF-DE) methods, in addition to the conventional immersion or perfusion fixation method. Different parts of glomerular capillary walls were selected for morphometric analyses. It was demonstrated that the widths of glomerular slit diaphragms and the spaces between foot processes were more widely dilated at higher perfusion pressures (200 and 250 cm H₂0) than those seen at both normal perfusion pressure (150 cm H₂O) and lower perfusion pressure (100 cm H₂O). On the other hand, the glomerular basement membranes were thinner at higher perfusion pressures. By the QF-FS and QF-DE methods, the foot processes showed different shapes from those revealed by the conventional preparation methods, even at the same perfusion pressure. It is concluded that the widths of glomerular slit diaphragms and glomerular basement membranes and the spaces between foot processes may be significantly changed in vivo, depending on the hemodynamics in the glomerular capillary.

The 3-dimensional ultrastructure of mouse renal glomeruli under normal haemodynamic conditions was studied by scanning electron microscopy using an in vivo cryotechnique followed by freeze-substitution, and compared with glomeruli prepared by conventional fixation methods. Mouse kidneys were frozen with a cryoknife apparatus and a liquid isopentane-propane mixture (k193 mC). Surface areas of the frozen tissues were freeze-fractured with a scalpel in liquid nitrogen. The specimens were routinely freeze-substituted, freeze-dried, ion-sputtered, and then observed in a scanning electron microscope at an accelerating voltage of 5 kV. Renal glomeruli showed good ultrastructural preservation of the surface tissues. Podocytes with interdigitating foot processes covering capillary loops exhibited smooth surface contours and their cell surfaces were arranged more tightly than those seen by the conventional fixation method. Filtration slits between foot processes were found to be narrow. The internal structure of the glomerular tuft was seen in the freeze-fracture faces. The capillary lumen with variously shaped erythrocytes was kept open in frozen glomeruli under normal blood circulation conditions. The ultrastructure of renal glomeruli, as revealed by the in vivo cryotechnique with freeze-substitution, appears to be closer to that of the living state.

Mechanical stimulation is known to regulate the calcification of endplate chondrocytes. The Ank protein has a strong influence on anti-calcification by transports intracellular inorganic pyrophosphate (PPi) to the extracellular matrix. It is known that TGF-β1 is able to induce Ank gene expression and protect chondrocyte calcification. Intermittent cyclic mechanical tension (ICMT) could induce calcification of endplate chondrocytes by decrease the expression of Ank gene. In this study, we investigated the relation of intermittent cyclic mechanical unconfined compression (ICMC) and Ank gene expression. We found that ICMC decreased the Ank gene expression in the endplate chondrocytes, and there was an decreased in the TGF-β1 expression after ICMC stimulation. The Ank gene expression significantly increased when treated by transforming growth factor alpha 1 (TGF-β1) in a dose-dependent manner and decreased when treated by SB431542 (ALK inhibitor) in a dose-dependent manner. Our results implicate that ICMC-induced downregulation of Ank gene expression may be regulated by TGF-β1 in end-plate chondrocytes.

The ultrastructures of anionic sites in rat glomerular basement membranes (GBM) were studied at different perfusion pressures of 100, 150, 200 or 250 cm H₂O by a quick-freezing and deep-etching (QF-DE) method, in addition to conventional fixation methods, using polyethyleneimine (PEI) as a cationic tracer. By the QF-DE method, three-dimensional ultrastructures at each pressure were observed more clearly than those seen on conventional ultrathin sections. When the perfusion pressures were changed from low levels to higher ones, the total thickness of GBM with anionic sites became gradually thinner. Many PEI particles were observed around filaments, not only in the laminae lucidae, but also in the lamina densa. These findings indicated the existence of anionic sites in both laminae lucidae and lamina densa of GBM. The numbers of PEI particles in the lamina rara externa were counted on conventional ultrathin sections for morphometric analyses. The numbers per unit length of GBM were significantly decreased at higher perfusion pressures (200 and 250 cm H₂O) than those seen at both normal (150 cm H₂O) and lower (100 cm H₂O) pressures. It is concluded that the ultrastructures of anionic sites in the GBM may be changed in vivo, depending on the hemodynamics in the glomerular capillary.