Modification of two peptides of bacteriorhodopsin with a pentaamminecobalt (III) complex

Bacteriorhodopsin (bR) was regenerated from the cation-depleted blue membrane with pentaammineaquocobalt(III) tetrafluoroborate [( Co(NH3)5H2O]3+[BF4-]3). Illumination of the sample with orange light decreased the extinction at 568 nm concomitantly with a hypsochromic shift of the absorption maximum. The photocycle of this sample was inhibited, and the rate of proton pumping was reduced. Chymotryptic cleavage of the corresponding apomembrane into the two fragments C1 and C2 and their subsequent separation revealed that cobalt label is only attached to C1. The maximal incorporation of Co into this peptide was 0.3 Co/C1. After cleavage of C1 with cyanogen bromide and subsequent proteolysis with trypsin and chymotrypsin, this modification could be associated with peptides from cyanogen bromide fragments 6 and 9. The sequences were determined to be 101Val-Asp-Ala-Asp-Gln and 228Ala-Ile-Phe-Gly-Glu-Ala-Glu-Ala. These peptides contain the sequences Asp-Ala-Asp and Glu-Ala-Glu, respectively, which might be constituents of the same cation binding site. The observation that the incorporation of Co into bacteriorhodopsin is enhanced under illumination with orange light indicates that this site might be involved in the proton uptake.     

Changes in the auditory system of locusts (Locusta migratoria and Schistocerca gregaria) after deafferentation

Deafferentation experiments during postembryonic development show morphological and/or physiological changes of receptor fibers and of identified auditory interneurons in the CNS of the locusts Locusta migratoria and Schistocerca gregaria after unilateral ablation of one tympanic organ either in the larva or the adult animal.

Control of bovine placental progesterone synthesis: roles of cholesterol availability and calcium-activated systems

It was previously reported that dispersed bovine placentome secretes progesterone and that the steroidogenic activity of these cells is stimulated by a calcium-mediated, cyclic nucleotide independent mechanism. In the present study, the influence of substrate availability was explored and the roles of calmodulin and protein kinase C in progestin production examined. Incubation of dispersed fetal cotyledon cells with 25-hydroxycholesterol (25-OH-C), a soluble sterol which readily enters cells and is metabolized to steroid hormones, increased progesterone secretion in a dose-dependent manner. The response to 25-OH-C was dependent on the extracellular calcium concentration. Methyl isobutyl xanthine (MIX) alone also increased pregnenolone as well as progesterone secretion, and the combination of 25-OH-C and MIX stimulated progesterone secretion was inhibited by trifluoperazine. The phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), caused no major effects on steroidogenesis but the stimulatory effects of MIX or the ionophore A23187 were enhanced in its presence. These findings suggest that (1) basal progesterone secretion by fetal cotyledon cells is limited by cholesterol availability; (2) MIX increases steroidogenesis in part by increasing the synthesis of pregnenolone, but its actions are expressed independently of cholesterol availability; (3) both calmodulin and protein kinase C may participate in the modulation of bovine placental steroidogenesis.     

Species-specific structural differences in the alpha 1 + alpha 2 domains determine the frequency of murine cytotoxic T cell precursors stimulated by human and murine class I molecules

The frequency of murine CTL precursors (CTLp) that recognize the human histocompatibility Ag HLA-A2 and HLA-B7 was measured and found to be approximately two orders of magnitude lower than the frequency of CTLp that recognize murine H-2 alloantigens. The possible contribution of other cell surface molecules to this difference in response was addressed by expression of the H-2Ld molecule on a human cell and the HLA-B7 molecule on a murine cell. It was found that both human and murine H-2Ld expressing cells elicited comparable levels of H-2Ld specific CTL. Although murine HLA-B7 positive cells stimulated a higher frequency of HLA-B7-specific CTLp than did human cells, this appeared to be largely due to stimulation of CTLp that recognized HLA-B7 in the context of H-2 molecules; consequently, it was concluded that the difference in the frequency of murine CTLp elicited by human and murine class I Ag is due to species specific structural differences in these molecules. The regions of the class I molecule that were responsible for this difference were mapped using chimeric class I molecules constructed to replace domains of the human molecule with their murine counterparts. It was found that the frequency of CTLp is controlled by structures within the alpha 1 and alpha 2 domains of the molecule. These results are discussed in the light of models for T cell recognition of class I Ag and the diversification of the T cell receptor repertoire.     

Stability of salt tolerance at the cell level after regeneration of plants from a salt tolerant tobacco cell line

Plants were regenerated from both the wild type and a stable NaCI-tolerant line of tobacco cells ( Nicotiana tabacum/gossii ). The regeneration process was much more difficult in the case of the NaCI-tolerant line and was only successful in the absence of NaCI. These plants differed morphologically from those regenerated from the wild type cell line, exhibiting abnormally short internodes, small leaves and reduced growth. Cell suspension cultures derived from plants regenerated from the stable NaCI-tolerant line retained a high level of tolerance to salt. The NaCI-concentration required to reduce fresh and dry weight gain by 50% was about twice that observed in the case of the cells obtained from wild type plants.
The results presented here, together with those of Watad et al. (1985), indicate that resistance to salt is operating and stable at the cellular level before and after plant regeneration. When the regenerated plants were grown in increasing levels of salt their growth response was not clearly different from that of the plants regenerated from the wild type cell line. However, the survival of plants on high concentrations of NaCI tended to be higher in the case of plants regenerated from the NaCI-tolerant cell line.     

Isolation of third, fourth, and fifth instar larval midgut epithelia of the moth, Trichoplusia ni

A new tissue isolation technique was used to create intact midgut epithelial wholemounts from three Trichoplusia ni (Lepidoptera: Noctuidae) larval instars. The protease, dispase, removed the basal lamina and associated connective tissue and allowed for high resolution light microscopy of entire epithelia. Columnar, goblet, differentiating, and stem cells were characterized by double fluorescent labelling of f-actin and nuclei. A comparison of cell populations by digital image analysis revealed significant regional and temporal changes in the density and number of differentiating and stem cells. Growth of the midgut epithelium from third to fourth instar, and from fourth to fifth instar, was accomplished by both cell differentiation and cell division. Cell division however, was greatly reduced from fourth to fifth instar with a concomitant sharp decrease in the stem cell population.     

Derivation of cytotoxic T cell lines that express T cell receptor but exhibit reversible loss of stable antigen binding

Stable cell lines lacking cytotoxic activity against specific target cells were derived from highly active murine CTL clones by the omission of antigen from the culture for several weeks. Several independent CTL clones cultured in the absence of antigen showed a gradual decline in cytotoxic activity, resulting in complete loss by 5 to 10 wk. Such noncytotoxic (NC) cells lacked the ability to form stable conjugates with specific target cells, but were able to kill all target cells tested in the presence of Con A. It was shown by subcloning at limiting dilution that all cells in the starting population were cytolytically active, and that all cells in the NC population derived from such a clone were cytolytically inactive against target cells bearing an appropriate antigen under normal assay conditions. By using the monoclonal antibody F23.1, which reacted with the antigen receptors of two of the CTL clones, it was shown that the NC cells derived from these clones continued to express the receptor at normal levels. Levels of expression of Thy-1.2, Lyt-2.2, and LFA-1 were also similar in all cytotoxic cell lines and their noncytotoxic derivatives. The F23.1 antibody induced an increase in cytoplasmic free Ca2+ in both CTL and NC cells, and NC cells lysed F23.1 hybridoma cells in the absence of Con A. When cells expressing appropriate target cell antigen were added back to cultures of NC cells, cytotoxic activity of appropriate specificity was fully recovered in 2 wk. These results indicate that expression of an apparently functional antigen receptor alone is insufficient for stable binding of CTL to specific target cells, and that other factors dependent upon antigen stimulation may be involved in the recognition process. A difference in affinity for antigen between CTL and NC cells is suggested as a possible explanation for these observations.     

Construction of novel class I histocompatibility antigens by interspecies exon shuffling

Human and mouse class I histocompatibility antigens share considerable structural homology at both the protein and DNA sequence level. This homology has allowed the production of hybrid class I molecules by the reciprocal exchange of DNA sequences corresponding to equivalent domains of HLA-B7 and either H-2Ld or H-2Dd. It is shown that these genes give rise to protein products that are stably expressed on the surface of murine L cells after DNA-mediated gene transfer. These proteins express only those monoclonal antibody-defined H-2 determinants that are expected based on their genetic construction. The molecules have allowed the localization of a number of polymorphic and monomorphic HLA-specific epitopes. In all but one case, expression of an epitope on a domain does not appear to be influenced by the replacement of adjacent human domains with their murine equivalents, suggesting a considerable degree of structural independence of the domains. Cells expressing the hybrid molecules have also been tested as targets for a panel of HLA-B7-specific cytotoxic T cell clones. The results show that the polymorphic determinants recognized by these clones map to the alpha 1 and alpha 2 domains of the HLA-B7 molecule. No evidence for an influence of species-related amino acid sequence differences in the third extracellular domain on T cell recognition was seen. The results are discussed in light of the proposed domain structure of the class I proteins and the potential use of such molecules for further functional studies.