Temperature jump study of charge translocation during the bacteriorhodopsin photocycle

Temperature jump experiments were carried out on purple membranes oriented and fixed in polyacrylamide gel. With green background illumination a relaxation of the photocurrent after an infrared laser pulse could be observed. To simulate the temperature jump signals different models of the bacteriorhodopsin photocycle were tested. The parameters of these models were obtained by measuring absorbance changes and photocurrent after excitation with a 575-nm laser flash.

A model with a temperature-dependent branching before the M state turned out to be satisfying. Other models, especially those with a late branching or without branching, could not reproduce the temperature jump measurements.

     

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.     

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.     

Ha-ras-1 alleles in Norwegian lung cancer patients

Summary We have examined DNA restriction fragment length polymorphisms (RFLP) of the Ha-ras-1 gene in DNA from 118 lung cancer patients and 123 unaffected controls. When DNA samples were digested with MspI/ HpaII restriction endonucleases. Southern blot analysis demonstrated 4 common, 4 intermediate and 7 different rare alleles in the combined population after hybridization to the pGDa1 probe. Six of the rare alleles were unique for the lung cancer group and 1 rare allele for the control group. The frequency of rare alleles in lung cancer patients (10/236) was significantly different (P<0.01) from the control group (1/246). The lung cancer group also had a significantly lower frequency of the common 2.57 kb fragment than the controls (P<0.02). The results thus indicate that Ha-ras genotyping may be of value in lung cancer risk assessment.     

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.