Photoreactions of bacteriorhodopsin at acid pH.

It has been known that bacteriorhodopsin, the retinal protein in purple membrane which functions as a light-driven proton pump, undergoes reversible spectroscopic changes at acid pH. The absorption spectra of various bacteriorhodopsin species were estimated from measured spectra of the mixtures that form at low pH, in the presence of sulfate and chloride. The dependency of these on pH and the concentration of Cl- fit a model in which progressive protonation of purple membrane produces "blue membrane", which will bind, with increasing affinity as the pH is lowered, chloride ions to produce "acid purple membrane." Transient spectroscopy with a multichannel analyzer identified the intermediates of the photocycles of these altered pigments, and described their kinetics. Blue membrane produced red-shifted KL-like and L-like products, but no other photointermediates, consistent with earlier suggestions. Unlike others, however, we found that acid purple membrane exhibited a very different photocycle: its first detected intermediate was not like KL in that it was much more red-shifted, and the only other intermediate detectable resembled the O species of the bacteriorhodopsin photocycle. An M-like intermediate, with a deprotonated Schiff base, was not found in either of these photocycles. There are remarkable similarities between the photoreactions of the acid forms of bacteriorhodopsin and the chloride transport system halorhodopsin, where the Schiff base deprotonation seems to be prevented by lack of suitable aspartate residues, rather than by low pH.     

Use of pheromone chemistry to identify Grapholita lobarzewskii as an occasional pest of apple and plum

As shown by chemical analysis and field trapping, the sex pheromone of Grapholita lobarzewskii consists of a blend of (E)-8-dodecenyl acetate and (Z)-8-dodecenyl acetate. Maximum attractiveness was found at 80–95%. Dodecyl acetate and tetradecyl acetate, both present in the female gland, did not affect trap catch. G. lobarzewskii has recently gained importance as a pest of apple and plum, but has so far been referred to as Grapholita janthinana. The latter is attracted to the same two compounds, but in reversed portions (20% E).

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Zusammenfassung Chemische Analysen und Feldversuche zeigen, dass das Sexualpheromon des Kleinen Fruchtwicklers, G. lobarzewskii aus (E)-8-Dodecenylacetat (Z8-12:Ac) und (Z)-8-Dodecenylacetat (Z8-12:Ac) besteht. Die grösste Lockwirkung wurde mit einem Gemisch der beiden Substanzen bei einem E-Anteil von 80–95% erzielt. Dodecylacetat (12:Ac) und Tetradecylacetat (14:Ac) wurden ebenfalls in den weiblichen Pheromondrüsen nachgewiesen, hatten aber im Freiland keinen Einfluss auf die Lockwirkung. G. lobarzewskii tritt seit einiger Zeit in der Schweiz als Schädling von Apfel und Zwetschge auf und ist an ihrem charakteristischem Frassgang leicht erkennbar. Die Art wurde bisher irrtümlich als G. janthinana bezeichnet. G. janthinana lebt auf Rosaceen, und wird vom gleichen Substanzpaar angelockt, allerdings bei umgekehrtem Mischungsverhältnis (20% E).

     

Extracellular ATP stimulates poly(inositol phospholipid) hydrolysis and eicosanoid synthesis in mouse peritoneal macrophages in culture

The effects of extracellular ATP on inositol phospholipid breakdown and synthesis of eicosanoids were studied in mouse peritoneal macrophages. Addition of ATP to intact cells labelled with [3H]inositol stimulated a rapid (within 10 s) formation of inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate. In parallel there was also a substantial accumulation of inositol 1,3,4-trisphosphate and the monophosphate and bisphosphate derivatives of inositol. Within 10 s after the addition of 30 microM ATP there was a twofold increase in inositol trisphosphate (InsP3), which declined over 2 min. The ED50 for ATP-stimulated generation of InsP3 was approximately 12 microM. ADP and GTP showed only weak effects on InsP3 formation, while AMP and adenosine were completely ineffective at 30 microM. Furthermore, the rank order of potency of ATP analogues was ATP greater than ATP[S] greater than AdoPP[NH]P = AdoPP[CH2]P greater than AdoP[CH2]PP thus, indicating the presence of a P2y-purinergic receptor. Cells labelled with [3H]arachidonic acid showed a 50% increase of label in 1,2-diacylglycerol after 15 s upon stimulation with ATP. In parallel to the stimulation of inositol phospholipid hydrolysis, ATP also caused a marked synthesis of prostaglandin E2 (PGE2) and leukotriene C4 (LTC4) in mouse peritoneal macrophages. The rank order of potency of ATP analogues was identical with that of InsP3 generation. The effect on eicosanoid synthesis could be mimicked by the calcium ionophore A23187 and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate. These results suggest that ATP-induced activation of P2y-purinergic receptors in mouse peritoneal macrophages triggers inositol phospholipid breakdown and eicosanoid synthesis.     

Evidence for a novel growth factor in Xenopus oocytes

Xenopus oocytes contain a novel growth factor, as determined by its effect on the anchorage-dependent and -independent growth of various rat cells and on a Xenopus cell line; it is destroyed by trypsin, acidification (pH 2.0), heating (100 degrees C, 3 min), 8 M urea but not by dithiothreitol. Gel filtration of this activity in nondissociating conditions suggests the existence of aggregates and the presence of a very high (approximately 2000 Kd) and a much lower (approximately 30 Kd) form.     

Monoclonal antibodies to the pituitary growth-hormone receptor by the anti-idiotypic approach. Production and initial characterization.

We obtained 10/192 and 3/384 antibody-secreting hybrids after immunization of Balb/c mice with either human growth hormone or affinity-purified rabbit anti-(human growth hormone) respectively. Radiolabelled rabbit anti-(human growth hormone) antibodies, but not human growth hormone, were specifically bound by supernatants from the 13 hybrids. The binding was completely inhibited by human-growth-hormone serum binding protein. However, anti-(human growth hormone antibodies) were detected in the sera of all the mice immunized with human growth hormone. In an independent fusion, which was carried out after immunization with fewer doses of human growth hormone, anti-(human growth hormone) antibodies were also obtained. Five hybrids, where the starting antigen was human growth hormone, were selected for ascites production, and the corresponding monoclonal antibodies were partially purified and characterized with respect to their immunoglobulin isotype and their interaction with human-growth-hormone receptors. These antibodies were found to enhance the binding of radioiodinated human growth hormone to human-growth-hormone serum binding protein from human and rabbit plasma by 40%. Scatchard analysis of the effect of one of the monoclonal antibodies showed that this enhancement was due to an increased number of binding sites. All of the partially purified antibodies but one (F12) inhibited the binding of human growth hormone to rat but not rabbit, liver microsomes to various extents, as well as to H-4-II-E rat hepatoma cells. Monoclonal antibody F12 enhanced the binding of radiolabelled human growth hormone to rat liver microsomes and H-4-II-E hepatoma cells. This enhancement was found to be due to an increase in the number of binding sites.     

Morphology of neurons and axon terminals associated with descending and ascending pathways of the lateral forebrain bundle in Rana esculenta

Summary The cells of origin of afferent and efferent pathways of the lateral forebrain bundle were studied with the aid of the cobalt-filling technique. Ascending afferents originated from the lateral thalamic nucleus, central thalamic nucleus, posterior tuberculum and the cerebellar nucleus. They terminated in the anterior entopeduncular nucleus, amygdala and the striatum. Telencephalic projection neurons, which are related to the lateral forebrain bundle, were located mainly in the ventral striatum and the anterior entopeduncular nucleus, but were not so numerous in the dorsal striatum. Irrespective of their location, most of the neurons projecting axons into the lateral forebrain bundle had piriform or pyramidal perikarya. Long apical dendrites usually arborized in a narrow space, whereas widely arborizing secondary dendrites originated from short dendritic trunks. The other neurons that contributed to the lateral forebrain bundle were fusiform or multipolar cells. Striatal efferents terminated in the pretectal area and in the anterodorsal, anteroventral and posteroventral tegmental nuclei.     

Expression of interleukin-2 receptor on blood lymphocytes stimulated with allogeneic lymphocytes or autologous tumor cells

Summary The kinetics of interleukin-2 receptor (IL-2R) expression and the [³H]dT incorporation of blood lymphocytes after the first and the second stimulation with allogeneic leukocytes (primary and secondary MLC) or with the autologous tumor cells (primary and secondary MLTC) were compared. The expression of IL-2R paralleled the induction of DNA synthesis. The proportion of IL-2R⁺ cells of the unprimed donors peaked earlier in the secondary MLC as compared to the primary MLC (on days 3 and 5 respectively). In MLC of alloimmunized healthy individuals and in the MLTC of cancer patients the highest proportions of IL-2R⁺ cells were detected between days 2 and 3 after both the first and second stimulations. Thus the first in vitro stimulation in the MLTC showed similar kinetics to those of the secondary MLC of unprimed individuals and to the primary MLC response of the allo-immunized individuals. The findings in the MLTC substantiate the hypothesis that cancer patients can be sensitized to their own tumors. The kinetics of the appearance of the IL-2R together with the characteristics of the IL-2-propagated cultures provide useful information for the strategy of expansion of auto-tumor reactive lymphocyte populations.     

Crystal structure of the cytokine interleukin-1 beta.

The crystal structure of human recombinant interleukin-1 beta has been determined at 3.0 A resolution by the isomorphous replacement method in conjunction with solvent flattening techniques. The model prior to refinement has a crystallographic R-factor of 42.3%. The structure is composed of 12 beta-strands forming a complex network of hydrogen bonds. The core of the structure can best be described as a tetrahedron whose edges are each formed by two antiparallel beta-strands. The interior of this structure is filled with hydrophobic side chains. There is a 3-fold repeat in the folding of the polypeptide chain. Although this folding pattern suggests gene triplication, no strong internal sequence homology between topologically corresponding residues exists. The folding topology of interleukin-1 beta is very similar to that described by McLachlan (1979) J. Mol. Biol., 133, 557-563, for soybean trypsin inhibitor.     

Immunocytochemical localization of lipophorins in the flight muscles of the migratory locust (Locusts migratoria) at rest and during flight

Summary Locust lipoproteins (lipophorins) were localized by indirect immunofluorescence- and immunogold labelling in cryosections of dorsolongitudinal flight muscles. Immunolabelling was performed with monoclonal antibodies against apolipoprotein epitopes that are exposed at the surfaces of the lipophorin particles. Both at rest and during flight, lipophorins were located only in the wider spaces of the extracellular matrix, in the basement membranes of the individual muscle fibers and in the extracellular spaces that surround interfibrillar tracheoles. No internalization of lipophorins by the flight muscle cells was observed. Our results indicate that the unloading of lipophorins at the flight muscles is an extracellular event. Similarities with the vertebrate system of chylomicron and very-low-density lipoprotein degradation are discussed.     

The core region of human glutaminyl-tRNA synthetase homologies with the Escherichia coli and yeast enzymes.

We have isolated from a Lambda-gt 11 library a human cDNA clone with one open reading frame of about 2400 bases. A stretch of about 350 amino acids in the deduced amino acid sequence is up to 40 percent identical with parts of the known amino acid sequences of E. coli and yeast glutaminyl (Gln)-tRNA synthetase. The isolated cDNA sequence corresponds to an internal section of a 5500 bases long mRNA that codes for a 170 kDa polypeptide associated with Gln-tRNA synthetase. Thus, the human enzyme is about three times larger than the E. coli and two times larger than the yeast Gln-tRNA synthetase. The three enzymes share an evolutionarily conserved core but differ in amino acid sequences linked to the N-terminal and C-terminal side of the core.