Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis.

The functions of many open reading frames (ORFs) identified in genome-sequencing projects are unknown. New, whole-genome approaches are required to systematically determine their function. A total of 6925 Saccharomyces cerevisiae strains were constructed, by a high-throughput strategy, each with a precise deletion of one of 2026 ORFs (more than one-third of the ORFs in the genome). Of the deleted ORFs, 17 percent were essential for viability in rich medium. The phenotypes of more than 500 deletion strains were assayed in parallel. Of the deletion strains, 40 percent showed quantitative growth defects in either rich or minimal medium.

Comparison of late results of balloon valvotomy in mitral stenosis with versus without mitral regurgitation.

Balloon mitral valvotomy (BMV) is safe and effective in patients with mitral stenosis (MS) and coexisting mild mitral regurgitation (MR). Influence of preexisting MR on late outcome of BMV is under evaluation. We included 77 patients without MR and 72 with MR in this study, and compared their immediate and late results in a mean follow-up of 33 +/- 24 months after BMV. Patients with coexisting MR were older and more frequently had significant valvular calcium and atrial fibrillation than patients without MR. After BMV, mitral valve gradient decreased, and cardiac output and mitral valve area by planimetry increased significantly (all p = 0.0001) in both groups. There was no difference in values of mitral valve gradient and cardiac output after BMV between the groups. Mitral valve area was significantly smaller in patients with preexisting MR. During follow-up, there were 11 patients (14%) in the group without MR and 24 (33%) in the group with MR developed cardiac events (p = 0.006). Cumulative event-free survival was 90% at the second year, 87% at the fourth year, and 69% at the sixth year, respectively, in the group without MR versus 78%, 62%, and 37%, respectively, in the group with MR (p = 0.0014). Cox regression showed that preexisting MR was a significant predictor for late cardiac events with a threefold increased hazard risk (p = 0.0025), but age, valvular calcium, echocardiographic score, and cardiac rhythm also played a culpable role. We conclude that preexisting MR is an important risk factor for poor, late outcome of BMV.

Cell-cycle-specific biosynthesis of the photosynthetic membrane of Rhodopseudomonas sphaeroides. Structural implications.

Structural changes association with the intracytoplasmic membrane during the cell cycle of the photosynthetic bacterium Rhodopseudomonas sphaeroides have been studied by freeze-fracture electron microscopy. The isolated intracytoplasmic membrane vesicles, chromatophores, were fused in order to obtain large fracture faces, allowing more precise measurements and statistical analysis of both intramembrane particle density and size determinations. The intramembrane particle density of the protoplasmic face (PF) of the intracytoplasmic membrane, (from 4970 to 8290/micrometers 2), was shown to be a linear function of the protein/phospholipid ratio (from 2.5 to 5.1, w/w) of the intracytoplasmic membrane. Under constant light intensity, both the average particle size and particle size distribution remained unchanged during the cell cycle. These results provide the structural basis for the earlier reported cell-cycle-specific variations in both protein/phospholipid ratio and alternation in phospholipid structure of the intracytoplasmic membrane of R. sphaeroides during photosynthetic growth. The average particle diameter in the PF face of the intracytoplasmic membrane was 8.25, 9.08 and 9.75 nm at incident light intensities of 4000, 500 and 30 ft X cd, respectively. When chromatophores were fused with small, unilamellar liposomes, the intramembrane particle density decreased as input liposome phospholipid increased, whereas the particle size remained constant and particle distribution became random.

Biosynthesis of the photosynthetic membranes of Rhodopseudomonas sphaeroides.

The steady-state biosynthesis of the photosynthetic membrane (ICM) of Rhodopseudomonas sphaeroides has been reviewed. At moderate light intensities, 500 ft-c, preexisting ICM serves as the insertion matrix for newly synthesized membrane components. Whereas the bulk of the membrane protein, protein-pigment complexes, and pigments are inserted into preexisting ICM throughout the cell cycle, phospholipid is transferred from outside the ICM to the ICM only at the time of cell division. Because the site of cellular phospholipid synthesis is the cytoplasmic membrane, these results infer that despite the physical continuity of cytoplasmic membrane and ICM, there must exist between these membranous domains a "barrier" to the free diffusion of cellular phospholipid. The cyclical alternation in protein to phospholipid ratio of the ICM infers major structural and functional alternations, such as changes in the protein to lipid ratio of the membrane, specific density of the membrane, lipid structure within the membrane, and the rate of cyclic electron flow. When biochemical studies are correlated with detailed electron microscopic investigations we can further conclude that the number of photosynthetic units within the plane of the membrane can vary by nearly a factor of two over the course of the cell cycle. The average physical size of the photosynthetic units is constant for a given light intensity but inversely proportional to light intensity. The distribution of photosynthetic unit size classes within the membrane can be interpreted as suggesting that the "core" of the photosynthetic unit (reaction center plus fixed antenna complex) is inserted into the membrane coordinately as a structural entity. The variable antenna complex is, on the other hand, inserted independent of the "core" and randomly associates with both old and new core complexes. Finally, we conclude that there is substantial substructure to te distribution of photosynthetic units within the ICM, ie, they are highly ordered and exist in a defined spatial orientation to one another.

Photosensitization of herpes simplex virus type 1 with neutral red.

Commercial neutral red (NR) originally containing at least 8 components was purified by thin layer chromatography. Herpes simplex virus type 1 (HSV-1) treated in vitro with 30 microgram/ml of purified NR became sensitive to light inactivation within 2 min but rapidly lost this sensitivity upon dilution. Similarly, virus grown in the presence of NR lost its photosensitivity upon dilution of the virus stock. In both cases the kinetics of inactivation appeared to be multi-hit. Photoinactivation of intracellular virus was most effective when NR was applied between 6 and 12 h post-infection. The most efficient inactivation occurred when virus at pH 8.8 was irradiated by light at a wavelength of 470 nm.