Identification of Cell Cycle Dependent Interaction Partners of the Septins by Quantitative Mass Spectrometry

The septins are a conserved family of GTP-binding proteins that, in the baker''s yeast, assemble into a highly ordered array of filaments at the mother bud neck. These filaments undergo significant structural rearrangements during the cell cycle. We aimed at identifying key components that are involved in or regulate the transitions of the septins. By combining cell synchronization and quantitative affinity-purification mass-spectrometry, we performed a screen for specific interaction partners of the septins at three distinct stages of the cell cycle. A total of 83 interaction partners of the septins were assigned. Surprisingly, we detected DNA-interacting/nuclear proteins and proteins involved in ribosome biogenesis and protein synthesis predominantly present in alpha-factor arrested that do not display an assembled septin structure. Furthermore, two distinct sets of regulatory proteins that are specific for cells at S-phase with a stable septin collar or at mitosis with split septin rings were identified.Complementary methods like SPLIFF and immunoprecipitation allowed us to more exactly define the spatial and temporal characteristics of selected hits of the AP-MS screen.     

Oat mesophyll protoplasts: their response to various feeder cultures

Oat (Avena sativa L.) mesophyll protoplasts were recently demonstrated to be capable of dedifferentiation, repeated divisions, and colony formation. Since the development of oat mesophyll protoplasts is decisively influenced by the nature of the used feeder culture (species, variety and concentration), we conducted a systematic study of this parameter. Generally, graminaceous feeders promoted protoplast proliferation, while dicot species repressed protoplast divisions. The beneficial effect of those feeders that promote divisions was counterbalanced by a factor that causes necrosis. The correct balance between promotion of divisions or necrosis depended on the nature of the feeder and its plating density.     

Expression of the parasite protein Pc90 in plasma membranes of erythrocytes infected with Plasmodium chabaudi

Erythrocytes infected with the malaria parasite Plasmodium chabaudi contain the neo-protein Pc90 in their plasma membrane. We investigate origin, membrane disposition, and intraerythrocytic traffic of this Pc90. Metabolic labeling of P.-infected erythrocytes, combined with cell fractionation as well as Western blot analysis and immunoprecipitation using a Pc90-recognizing monoclonal antibody, show that Pc90 is synthesized by early to mid trophozoites and is transported without any apparent processing steps to the erythrocyte membrane. Based upon the inaccessibility of Pc90 from the outside in intact erythrocytes and the water solubility of membrane-associated Pc90, it is concluded that Pc90 is localized on the cytoplasmic face of the host erythrocyte membrane. Immunoelectron microscopy using a Pc90-specific monoclonal antibody and the occurrence of soluble Pc90 in host cell cytosol indicate that the Pc90 is transported in both a 'vesicle-bound' and a 'free' form through the erythrocyte cytoplasm.     

Midbrain auditory sensitivity in the spring peeper (Pseudacris crucifer): correlations with behavioral studies

1. We derived audiograms from recordings of multiunit activity in the torus semicircularis of 10 males and 6 females of the spring peeper from central Missouri, USA. We used free-field stimulation with tone bursts that had temporal properties similar to typical advertisement calls and that ranged in frequency from 500-6000 Hz. 2. Audiograms from different electrode positions in the same animal had the same general shape. There was no evidence of tonotopy. 3. Audiograms showed two regions of maximal sensitivity: a low-frequency region (500-700 Hz); and a high-frequency region (2000-4000 Hz). Absolute thresholds and frequencies of maximum sensitivity varied considerably from individual to individual. 4. Audiograms derived from all individuals of each sex indicated that in the high-frequency region, corresponding to the frequency range of advertisement calls, males were more broadly tuned than females. However, tuning in both sexes was relatively weak, and the data predict relatively little selectivity in behavioral responses over the entire range of variation in frequency of the advertisement call in local populations. 5. The results are discussed in terms of behavioral experiments with both males and females from the same populations in central Missouri. We show that merely summarizing the audiograms based on estimates of minimum thresholds of a population or species may mask significant individual differences in tuning. Moreover, most behavioral studies are conducted at playback levels considerably above threshold. For these reasons, behavioral selectivity is not always accurately predicted by inspection of "average" audiograms.     

Specific cross-protective antigonococcal immunity in the murine genital tract

Specific acquired immunity to gonococci was studied in systemically immunized mice, challenged with 10(7) gonococci by intrauterine inoculation. Protection after intraperitoneal immunization was monitored by vaginal cultures taken 24 h post-challenge, since events during the first 24 h postexposure to gonococci are crucial in determining the outcome of infection. Mice were protected against gonococcal challenge by two inoculations with either live or boiled gonococci given 4 weeks apart, whereas immunization with one inoculation did not protect against challenge 1 week later. Protection was correlated with high titers of IgG antibody in serum after two immunizations, but not with the high titers of serum IgM antibody found after the one immunization. IgG antibodies, but not IgM antibodies, were shown to pass into genital secretions. Protection could be passively transferred by serum with high titers of antibody. Of most practical importance was the finding that not only were heat-stable antigens protective, but also heterologous protection resulted after immunization with three strains differing in source (disseminated gonococcal infection versus gonorrhea), opacity-transparency characteristics, and serum sensitivity. The data indicate that IgG antibodies resulting from systemic immunization with heat-stable antigens may be able to provide cross-protection immunity against gonorrhea.     

Sulfated oligosaccharides block antibodies to many Dictyostelium discoideum acid hydrolases

The lysosomal hydrolases of the cellular slime mold, Dictyostelium discoideum, possess a common posttranslational modification which is extremely antigenic in rabbits and mice. Rabbit antisera and mouse monoclonal antibodies that recognize this determinant cross-react with a group of at least 40-50 highly negatively charged proteins which include most or all of the lysosomal enzymes. (Knecht, D. A., Dimond, R. L., Wheeler, S., and Loomis, W. F. (1984) J. Biol. Chem. 259, 10633-10640). The present study demonstrates that the determinant is found on certain N-linked oligosaccharides derived from one of these proteins. An esterified sulfate is absolutely required for antigenicity.     

Natural immunity to murine gonococcal bacteremia: roles of complement, leucocytes, and sex

The roles of the serum bactericidal system, inflammatory cells, and sex in resisting gonococcal infection were studied in a murine model of gonococcal bacteremia. The role of serum killing in defense was investigated with complement component 5 deficient (C5-deficient) (B1O.D2/OSN) and normal (B1O.D2/NSN) mice. No significant differences were found between LD50's with either murine serum-sensitive or serum-resistant gonococci in those two mouse strains. However, in vitro experiments revealed a heat-stable factor in mouse serum which killed gonococci. Thus it appeared that the C5-deficient mouse is not a good model for the study of the role of C-mediated killing in resistance to gonococcal infection. Mice with Chediak-Higashi disease were used to study the role of phagocytes and natural killer cells. The difference in LD50's between affected mice (C57B1/6J beige J) and controls (C57B1/6J) was significant. The CBA/N mice, which have a B-cell maturation defect, were no more resistant to infection than control mice, which was taken as further evidence that B cells were less important than other leucocytes in innate immunity to gonococcal infection. Finally, male mice were significantly more resistant than female mice to gonococcal bacteremia. Thus, in this study the two most important determinants of resistance to gonococcal infection were inflammatory cells and sex.