In vivo intermembrane transfer of phospholipids in the photosynthetic bacterium Rhodopseudomonas sphaeroides.

The kinetics of accumulation of phospholipids into the intracytoplasmic membrane of Rhodopseudomonas sphaeroides have been examined. We have previously demonstrated that accumulation of phospholipids in the intracytoplasmic membrane is discontinuous with respect to the cell cycle. In this study we demonstrated a sevenfold increase in the rate of phospholipid incorporation into the intracytoplasmic membrane concurrent with the onset of cell division. Pulse-chase labeling studies revealed that the increase in the rate of phospholipid accumulation into the intracytoplasmic membrane results from the transfer of phospholipid from a site other than the intracytoplasmic membrane, and that the transfer of phospholipid, rather than synthesis of phospholipid, is most likely subject to cell cycle-specific regulation. The rates of synthesis of the individual phospholipid species (phosphatidylethanolamine, phosphatidyglycerol, and an unknown phospholipid) remained constant with respect to one another throughout the cell cycle. Similarly, each of these phospholipid species appeared to be transferred simultaneously to the intracytoplasmic membrane. We also present preliminary kinetic evidence which suggested that phosphatidylethanolamine may be converted to phosphatidycholine within the intracytoplasmic membrane.     

Recognition of nucleophile-treated alpha 2-macroglobulin by the alveolar macrophage alpha-macroglobulin . protease complex receptor

Rabbit alveolar macrophages exhibit high affinity surface receptors which recognize alpha 2-macroglobulin . protease complexes but not native alpha 2- macroglobulin. Binding of alpha 2-macroglobulin . protease complexes to surface receptors is independent of the protease used to form the complex. In this communication, we demonstrate that treatment of human alpha 2-macroglobulin with nucleophilic agents (methyl amine, ammonium salts) converts native alpha 2-macroglobulin into a form recognized by the surface receptor for alpha 2-macroglobulin protease complexes. Analysis of the concentration dependency of ligand binding revealed that the surface receptor did not distinguish between nucleophile-treated alpha 2-macroglobulin and alpha 2-macroglobulin . protease complexes. These results are consistent with the hypothesis that proteases or nucleophilic agents effect the hydrolysis of an internal thiol-ester bond (Tack, B. F., Harrison, R. A., Janatova, J., Thomas, M. L., and Prahl, J. W. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 5764-5768), leading to an alteration in alpha 2-macroglobulin conformation. The altered conformation results in recognition of the alpha 2-macroglobulin by surface receptors.     

Cyclic guanosine 3',5'-monophosphate and phosphodiesterase activity in mitogen-stimulated human lymphocytes.

The cyclic adenosine 3′,5′-monophosphate (cyclic AMP) phosphodiesterase from human leukemic lymphocytes differes from the normal cell enzyme in having a much higher activity and a loss of inhibition by cyclic guanosine 3′,5′-monophosphate (cyclic GMP). In an effort to determine the mechanism of these alterations, we have studied this enzyme in a model system, lectin-stimulated normal human lymphocytes. Following stimulation of cells with concanavalin A (con A) the enzyme activity gradually becomes altered, until it fully resembles the phosphodiesterase found in leukemic lymphocytes. The changes in the enzyme parallel cell proliferation as measured by increases in thymidine incorporation into DNA. The addition of a guanylate cyclase inhibitor preparation from the bitter melon prevents both the changes in the phosphodiesterase and the thymidine incorporation into DNA. This blockage can be partially reversed by addition of 8-bromo cyclic guanosine 3′,5′-monophosphate (8-bromo cyclic GMP) to the con A-stimulated normal lymphocytes. These results indicate a possible role of cyclic GMP in a growth related alteration of cyclic AMP phosphodiesterase.     

Cytotoxic T-lymphocyte and spontaneous killer cell activity against human T- and B-lymphoid cell lines.

We have investigated cell-mediated immune responses to cultured human T- and B-cell lines. Two effector mechanisms were explored and found to have different capabilities for mediating cytotoxic reactions. Cytotoxic T lymphocytes were generated by stimulation with irradiated B-cell lines and demonstrated cross-reactive cytotoxicity against these lines but not against T-cell lines. Unseparated mononuclear cells showed spontaneous cytotoxicity for both T- and B-cell lines; however, T-cell lines appeared more susceptible. Cell separation procedures were employed to determine functional differences in effector cells. In contrast to cytotoxic T lymphocytes induced in vitro, spontaneous killer cells (SKC) were shown to be nylon wool adherent, non-T lymphocytes with receptors for IgG-coated sheep erythrocytes.     

Characterization of a site-specific restriction endonuclease from Rhodopseudomonas sphaeroides.

A type II restriction endonuclease, RshI, has been partially purified from photoheterotrophically grown Rhodopseudomonas sphaeroides strain 2.4.1. The enzyme preparation, after a single DE-52 column fractionation, is free of 5' exonuclease and phosphatase activities but contains a trace of 3' exonuclease activity. Based upon deoxyribonucleic acid (DNA) sequencing data in the vicinity of the enzyme-promoted cleavage of pBR322 DNA, we have concluded that RshI probably recognizes the palinodromic hexanucleotide sequence 5'-CGATCG-3' and cleaves between the T and C. lambda cI857 DNA contains three RshI sites, two of which lie in the replaceable region. The plasmid pBR322, which carries resistances to ampicillin and tetracycline, contains a single RshI site in the ampicillin resistance determinant. Insertion of DNA into the RshI site of pBR322 results in loss of ampicillin resistance but retention of tetracycline resistance, thereby providing a convenient screening procedure for recombinant plasmids.     

Microbial degradation of glycerol nitrates.

The fate of glycerol trinitrate when exposed to microbial attack has been investigated. Contrary to some earlier reports, this compound was readily biodegraded by employing batch or continuous techniques under a variety of cultural conditions. Breakdown of glycerol trinitrate took place stepwise via the dinitrate and mononitrate isomers, with each succeeding step proceeding at a slower rate. After a residence time of 8 to 15 h, none of the glycerol nitrates could be detected in the effluent from a continuous-culture apparatus (chemostat) supplied with an influent containing 30 mg of glycerol trinitrate per liter.     

Epilithic periphyton and detritus studies in a subalpine stream

The accumulation of epilithic periphyton in Ward Creek, a permanent stream within the Lake Tahoe basin, California, was measured weekly at three stations from July through September, 1972. Subsamples were analyzed for total carbon and adenosine triposphate content. The mean total carbon content at three stations over the period of investigation was 0.508 ± 0.263 mg carbon cm^–2. Live biomass, as estimated from ATP measurements, averaged 0.121 ± 0.115 mg carbon cm 2. It was estimated that approximately 76% of the organic carbon accumulating on rock substrates was present as detritus. Scanning electron microscopy of rock substrates suggested that much of this detrital accumulation may consist of diatom stalk materials.This work was supported by a grant from the National Science Foundation/RANN GI-22. C. R. Goldman, Principal Investigator.