Linear mapping of tryptophan residues in Vesiculovirus M and N proteins by partial chemical cleavage.

Nonlimit chemical cleavage at tryptophan residues of protein labeled at the amino terminus afforded a simple procedure for generating specific fragments and for mapping tryptophan positions. A comparison of the matrix (M) and nucleocapsid (N) proteins of four members of the Vesiculovirus group by this procedure suggests considerable conservation of tryptophan number and location in the four serotypes examined.     

In vitro heterologous cytotoxicity by T effector cells from mice immunized with Sindbis virus.

An in vitro correlate of cell-mediated cross-protection among alpha-viruses was demonstrated by cytotoxicity of Sindbis-immune spleen cells from mice to both Sindbis and Semliki Forest virus (SFV)-infected target cells. This cytotoxicity was shown to be mediated by the T cell population of the spleen and was independent of the presence of macrophages or B cells. The time when the level of the lymphocyte-mediated cytotoxicity (LMC) to SFV-infected cells was maximal coincides with the time when immunity to SFV is maximal in vivo, as reported previously, and when adoptive immunity to SFV can be transferred. After one i.p. injection of Sindbis virus, the level of homologous LMC was higher than the level of heterologous LMC. However, following a second injection of Sindbis virus as immunogen, at a time when the mice are cross-protected to SFV, the heterologous LMC was considerably higher than homologous LMC. We propose that there is suppression of the effector T cells specific for Sindbis-infected cells after the second immunizing injection, probably by homologous antibody. In contrast, there appears to be an anamnestic cell-mediated response to SFV.     

Sézary's syndrome and generalized plane xanthoma.

The first case of Sézary''s syndrome associated with generalized plane xanthoma is reported, thereby extending the association of lymphoreticular proliferative disorders with plane xanthomatosis. The association of Sézary''s syndrome with plane xanthomatosis may be an in vivo example of defective cell regulation involving the major cellular components of the immune response.     

Chemical composition and ultrastructure of the epicuticular wax in three lines of Brassica napus (L)

The epicuticular wax in three lines of Brassica napus (rape) has been investigated and the detailed chemistry and ultrastructure of the waxes examined. A distinct chemical make-up has been found for all three waxes which is correlated with three distinct crystallite structures. A tentative scheme for classification of Brassica wax mutants is described in which the two newly analysed rape mutants can be placed. Mass spectral analysis of all wax components confirms and extends previous ideas about the chemistry of Brassica waxes.     

Radiosensitization in multifraction schedules. I. Evidence for an extremely low oxygen enhancement ratio

Stable monolayers of contact-inhibited C3H 10T1/2 cells were used in multifraction radiation experiments to measure the oxygen enhancement ratio (OER) at low doses/fraction under conditions where cell cycle effects (repopulation, redistribution) were minimal. Consistent with there being a dose-dependent reduction in the OER at low doses, an extremely low OER of 1.34 was measured after 20 fractions of 1.7 Gy every 12 h. The sparing effects of fractionating radiation doses were not apparent for cells irradiated under hypoxic conditions (i.e., multifraction survivals were lower than acute single-dose values) until doses exceeding 15 Gy were reached. This result suggested a deficiency in the recovery from sublethal and/or potentially lethal damage might exist after hypoxic irradiations, thereby reducing the OER. The capacity to repair potentially lethal damage was found to be nearly the same after hypoxic as compared to aerobic irradiations. However, there was an apparent absence of sublethal damage repair by 10T1/2 cells between two hypoxic irradiations which could be a major contributing factor to the extremely low OER value measured in this multifraction schedule.     

Light induces a rapid and transient increase in inositol-trisphosphate in toad rod outer segments

The sub-second time course of changes in the content of [3H]inositol-1,4,5-trisphosphate was determined in rod outer segments from very rapidly frozen Bufo retinas that had been incubated with [3H]inositol. Rod outer segments were cut off frozen specimens with a cryostat microtome and the water soluble extracts were analyzed. The content of [3H]inositol-1,4,5-trisphosphate rose after approximately 250 msec of bright illumination, but returned to the unstimulated level after 1 sec, whether the stimulus remained on or not. That is, there was rapid but transient change in the content of [3H]inositol-1,4,5-trisphosphate after the onset of stimulation.     

Intravenous endotoxin recruits a distinct subset of human neutrophils, defined by monoclonal antibody 31D8, from bone marrow to the peripheral circulation

Human neutrophils label with fluorochrome-labeled monoclonal antibody 31D8 as bright or dull. We determined the source and fate of 31D8 dull neutrophils by studying volunteers injected with endotoxin, epinephrine, or hydrocortisone, by examining bone marrow, and by examining skin blister exudate. We find that 31D8 dull neutrophils are normally not present in significant numbers in the circulation, are present in large numbers in normal marrow, and are recruited from the marrow by endotoxin, to a lesser extent by steroid, but not at all by epinephrine. 31D8 dull pattern correlates with morphologic immaturity in postendotoxin peripheral blood and bone marrow; however, blister exudate neutrophils contain only morphologically mature neutrophils, of which a significant number are 31D8 dull. We conclude that 31D8 dull neutrophils reside primarily in bone marrow and are released by agents which enhance bone marrow release of neutrophils. Their accumulation in skin blister exudate is unexplained, but suggests a special role in the inflammatory process.     

The spore coat of a fucosylation mutant in Dictyostelium discoideum

Strain HL250 of Dictyostelium discoideum cannot convert GDP-mannose to GDP-fucose, resulting in an inability to fucosylate protein. This affects a group of proteins which are normally fucosylated intracellularly and then secreted via prespore vesicles to become part of the outer lamina of the spore coat. We have found that strain HL250 nevertheless accumulates typical amounts of these proteins, stores them normally in prespore vesicles, and secretes them normally to become a part of the spore coat. However, affected proteins are proteolyzed after germination, the spore coat is more accessible to penetration by a macromolecular probe, and germination is inefficient in older spores. These findings can be explained by a dependence of the integrity of the outer layer of the spore coat on protein-linked fucose.     

Instability of orthophthalaldehyde reagent for amino acid analysis

Determination of amino acids by reversed-phase chromatography of the adduct with orthophthalaldehyde and a thiol is rapid and sensitive. The major recognized adverse feature of this method is the instability of the reaction product, which requires precise control of reaction timing and chromatographic parameters for reliable quantitative application. We report another source of major variability: reagent instability. Deterioration of reagent was noted as low peak heights and peak broadening and was predictable if the premixed reagent was left at room temperature. Restoration of sharp chromatograms was accomplished by addition of mercaptoethanol or sodium metabisulfite. Reagent which was chromatographically inert contained minimal free thiol by direct assay. Free thiol disappearance was markedly slowed by addition of a chelating agent. Excess mercaptoethanol was deleterious. We conclude that reagent deterioration represents oxidation of the thiol, may be reversed by rereduction with minimal thiol or bisulfite, and may be minimized by inclusion of a metal chelator in the reagent.