Membrane adhesion in photosynthetic bacterial membranes. Light harvesting complex I (LHI) appears to be the main adhesion factor

We have reconstituted pigment-protein complexes isolated from Rhodopseudomonas palustris photosynthetic membranes into phospholipid liposomes. The various complexes were tested for their ability to promote adhesion of the liposome membrane in the presence and absence of Mg²⁺ ions. Samples containing a reaction center (RC)/light-harvesting I (LHI) complex appeared to stack in a manner resembling control thylakoids in 2 and 5 mM Mg²⁺. We also tested for the effects of Mg²⁺ on detergent extractablity of pigment-protein complexes from intact membranes. Mg²⁺ sharply reduced the amount of LHI solubilized from membranes, while having little effect on the extractability of the light harvesting II complex (LHII) and the RC. Based on these results we suggest that LHI is the principal adhesion factor of R. palustris thylakoids.Abbreviations LHC light harvesting complex - OG octyl glucoside - RC reaction center This paper is dedicated to Professor G. Drews on the occasion of his 60th birthday     

Circadian regulation of pineal melatonin and reproduction in the Djungarian hamster

Gonadal state, pineal melatonin rhythms, and locomotor activity rhythms were examined in juvenile male Djungarian hamsters exposed to non-24-hr light cycles ("T-cycles") or to full photoperiods. At the end of 1 month, hamsters exposed to a 1-hr pulse of light every 24.33 hr (T 24.33) exhibited small testes, whereas those receiving the same amount of light every 24.78 hr (T 24.78) displayed stimulated gonads, ten-fold larger in size. Accompanying the nonstimulatory effect of the T 24.33 cycle were nocturnal peaks in both pineal melatonin content and serum melatonin concentration which were longer by approximately 4 hr than those observed on the photostimulatory T 24.78 cycle. Exposure to an intermediate-length T-cycle (T 24.53) resulted in a mixed gonadal response and in pineal and serum melatonin peaks of intermediate duration. Wheel-running activity was entrained to the T-cycles such that light was present only near the beginning of the subjective night, its phase (relative to activity onset) differing only slightly among T-cycle groups. Hence the durational differences observed in the melatonin peaks were apparently not due to the acute suppressive or phase-advancing effects of morning light on melatonin biosynthesis, but were rather the result of differences in the endogenous control of pineal activity by the circadian pacemaker system. While no strong correlation was detected between gonadal state and the phase of locomotor activity onset relative to the light pulse, a significant correlation was observed between gonadal state and the duration of daily locomotor activity (alpha). These data were compared to similar measures obtained from hamsters exposed to long-versus short-day full photoperiods (LD 16:8 vs. LD 10:14). In summary, the results of this study indicate involvement of the circadian pacemaker system of Djungarian hamsters in the control of pineal melatonin synthesis and secretion, and in photoperiodic time measurement. Furthermore, these data strengthen the hypothesis that it is the duration of nocturnal pineal melatonin secretion that is the critical feature of this neuroendocrine gland's photoperiodic signal.     

Role of protein synthesis in the survival of carbon-starved Escherichia coli K-12.

In a typical Escherichia coli K-12 culture starved for glucose, 50% of the cells lose viability in ca. 6 days (Reeve et al., J. Bacteriol. 157:758-763, 1984). Inhibition of protein synthesis by chloramphenicol resulted in a more rapid loss of viability in glucose-starved E. coli K-12 cultures. The more chloramphenicol added (i.e., the more protein synthesis was inhibited) and the earlier during starvation it was added, the greater was its effect on culture viability. Chloramphenicol was found to have the same effect on a relA strain as on an isogenic relA+ strain of E. coli. Addition of the amino acid analogs S-2-aminoethylcysteine, 7-azatryptophan, and p-fluorophenylalanine to carbon-starved cultures to induce synthesis of abnormal proteins had an effect on viability similar to that observed when 50 micrograms of chloramphenicol per ml was added at zero time for starvation. Both chloramphenicol and the amino acid analogs had delayed effects on viability, compared with their effects on synthesis of normal proteins. The need for protein synthesis did not arise from cryptic growth, since no cryptic growth of the starving cells was observed under the conditions used. From these and previous results obtained from work with peptidase-deficient mutants of E. coli K-12 and Salmonella typhimurium LT2 (Reeve et al., J. Bacteriol. 157:758-763, 1984), we concluded that a number of survival-related proteins are synthesized by E. coli K-12 cells as a response to carbon starvation. These proteins are largely synthesized during the early hours of starvation, but their continued activity is required for long-term survival.     

Growth Kinetics, Cell Shape, and the Cytoskeleton of Primary Astrocyte Cultures

Abstract: We examined correlations among growth kinetics, cell shape, and cytoskeletal protein content in rat astrocytes grown in primary culture. Cell suspensions from brains of newborn rats were seeded at densities from 0.2 to 3 × 10⁵/cm². At initial densities above 1 × 10⁵ the population increased to reach confluency by 10–12 days, after which cell number remained stable for many weeks. At low initial densities, 0.2–0.4 × 10⁵/cm², cells did not increase in number. Final density increased with increasing plating densities. High-density cells had small perikarya and several long cytoplasmic processes; low-density cells appeared flat and polygonal. All cultures were almost entirely astrocytic, as judged by immunofluorescent staining with antiserum against glial fibrillary acidic protein (GFAP). Cytoskeletal proteins were analyzed by gel electrophoresis after extraction from cells with nonionic detergent. Relative amounts of the proteins differed, in that low-density cells contained large amounts of cytoskeletal actin relative to the intermediate filament (IF) proteins vimentin and GFAP, whereas high-density cells contained relatively less actin and more IF proteins. Such differences in cytoskeletal proteins between the high- and low-density cultures were mirrored in the relative rates of synthesis of the cytoskeletal proteins. In the low-density cells amino acid incorporation into cytoskeletal-associated actin was more active than that into the IFs, whereas in the high-density cells higher rates of IF protein synthesis were observed.     

A quantitative analysis of C3 binding to O-antigen capsule, lipopolysaccharide, and outer membrane protein of E. coli 0111B4

The binding of serum C3 to the O-antigen capsule (OAg Cap), lipopolysaccharide (LPS), and outer membrane proteins (OMP) of Escherichia coli 0111B4 was examined. Bacteria were intrinsically labeled with [3H] or [14C]galactose (*gal) in the OAg Cap and LPS moieties or with [14C]leucine (*leu) to label proteins. Organisms were then incubated in serum containing differentially labeled C3, the above fractions were separated, and the proportion of each binding to a column containing anti-C3 was measured. The OAg Cap fraction bound 72 to 82% of the C3, which bound to E. coli 0111B4 during incubation in absorbed 10% pooled normal human serum (10% PNHS) or absorbed 40% C8-deficient serum (C8D). This distribution did not change when the organism was presensitized with immune IgG before serum incubation. A total of 2.93% +/- 0.48 of OAg Cap and 0.52% +/- 0.16 of LPS *gal bound specifically to Sepharose-containing antibodies to C3 (A:C3-Seph) after incubation in 10% PNHS; these values increased to 10.1% +/- 4.5 and 1.8% +/- 0.3, respectively, when C3 deposition was increased fourfold by incubation in 40% C8D. When encapsulated E. coli 0111B4 was incubated in 10% PNHS containing biotinylated C3, specific attachment of OAg Cap *gal to avidin-Sepharose was demonstrated in 1% sodium dodecyl sulfate (SDS), and complete release of bound *gal but not C3 occurred with 1 M NH2OH. When a mutant of E. coli 0111B4 lacking OAg Cap was incubated in 40% C8D, the outer membrane (OM) bound 85% of C3. Five percent of OM *gal from the unencapsulated organism bound to A:C3-Seph in 0.05% SDS, indicating that the fraction of LPS molecules with bound C3 increased threefold in the absence of OAg Cap. OAg Cap does not contain protein, and no net specific binding of *leu from OAg Cap fractions to A:C3 was detectable; 2.4 to 3.6% of OM *leu bound to A:C3-Seph. Immunoprecipitation of 82.9% of OAg Cap *gal with antisera that were directed to E. coli 0111B4 was associated with co-precipitation of 69.5% of C3 in the capsular fraction. Therefore, the majority of C3 bound to E. coli 0111B4 was covalently attached to OAg Cap and LPS. As corroboration of experiments with whole bacteria, purified OAg Cap and purified LPS consumed C3 when incubated in serum in the fluid phase. These results are the first to evaluate the acceptor site for C3 deposition on a Gram-negative organism incubated in serum, and show that LPS, OAg Cap, and OMP are all major acceptor sites for C3 in nonimmune serum.     

Relationship between intermediate filaments and microfilaments in cultured fibroblasts: evidence for common foci during cell spreading

Spreading and fully spread chick embryo fibroblasts (CEF) were examined by double-label fluorescence microscopy using the actin-specific probe rhodamine-phalloidin and an antibody directed against CEF intermediate filaments (IF). During midspreading, a striking relationship became discernible: statistical analysis showed that approximately half of the cell population exhibited one or more phase-dense, phalloidin-binding nodules that appeared to act as foci from which IF diverged. Coincidence between actin-containing structures and IF was not limited to these centers; IF could also frequently be seen running in close parallel arrays with stress fibers. Ultrastructural analysis confirmed the presence of non-membrane-bound out-pocketings along the length of stress fibers from which 10-nm IF diverged. These structures varied in size and shape, and displayed a dense, fine fibrillar appearance. IF and microfilaments (MF) were distinguished by size and by decoration of MF with myosin subfragment-1. Other IF-MF interactions were seen in cells of all stages: IF were observed to loop through stress fibers, most frequently at the cell margins. In colchicine-treated cells, IF became redistributed into cables that often ran parallel and appeared to merge with stress fibers. Cytochalasin D-treated CEF exhibited loose aggregates of actin-containing material that appeared to be associated with IF. These results suggest the possibility of an interaction between actin-containing structures and IF, particularly during cell spreading in cultured fibroblasts.     

Protein Patterns of Growing and Starved Cells of a Marine Vibrio sp.

Fingerprint protein patterns were produced by two-dimensional polyacrylamide electrophoresis on lysed cells of a Vibrio sp., Ant-300, which were prepared from growing and starved cultures. The cells were labeled with [³⁵S]methionine during growth and subsequently starved for up to 30 days. Samples were taken at selected time points representing stages in the starvation-survival process. Unlabeled starved cells were allowed to recover in the presence of [³⁵S]methionine to determine protein changes associated with the recovery from starvation. All growth conditions produced similar protein fingerprints; however, some protein spots disappeared, whereas others were seen only during starvation.     

A rapid procedure for preparing fluorescein-labeled specific antibodies from whole antiserum: its use in analyzing cytoskeletal architecture

A rapid method for the direct conjugation of affinity-purified antibodies with fluorescein (termed DCAPA) is described. This procedure involves the immobilization of antibodies as antigen-antibody complexes on nitrocellulose blots, and subsequently the bound antibodies are reacted with fluorescein isothiocyanate. An enriched sample of smooth muscle tropomysin transferred to nitrocellulose paper by the Western blotting procedure has been used as the affinity medium for purification of specific tropomyosin antibody from whole rabbit antiserum. Direct conjugation of the antibody with fluorescein was carried out following the binding of antibody to antigen. Direct conjugation and affinity purification of antibodies directed against tropomyosin was accomplished in 2-3 d using an enriched tropomyosin sample and whole antiserum directed against tropomyosin. The immunofluorescence images obtained with this procedure exhibit distinct advantages with regard to background fluorescence and overall specificity of antibody binding. The usefulness of this direct conjugation method in various experimental protocols is discussed.