β-Adrenergic receptors of brain cells. Membrane integrity implies apparent positive cooperativity and higher affinity

β-Adrenergic receptors were studied in intact cells of chick, rat and mouse embryo brain in primary cultures, by the specific binding of [³H]dihydro-l-alprenolol ([³H]DHA). The results were compared to the receptor binding of broken cell preparations derived from the cell cultures or from the forebrain tissues used for the preparation of the cultures. Detailed analysis of [³H]DHA binding to living chick brain cells revealed a high-affinity, stereoselective, β-adrenergic-type binding site. Equilibrium measurements indicated the apparent positive cooperativity of the binding reaction. By direct fitting of the Hill equation to the measured data, values of B_max = 12.01 fmol/10⁶ cells (7200 sites/cell), K_d = 60.23 pM and the Hill coefficient n = 2.78 were found. The apparent cooperative character of the binding was confirmed by the kinetics of competition with l-alprenolol, resulting in maximum curves at low ligand concentrations. The rate constants of the binding reaction were estimated as k₊ = 8.31·10⁷ M^?1 · min^?1 and k_? = 0.28 min^?1 from the association results, and k_? = 0.24 min^?1 from the dissociation data. The association kinetics supported the cooperativity of the binding, providing a Hill coefficient n = 1.76; K_d, as $\text{(k}{}_{\mathrm{?}}\text{/k}{}_{\mathrm{+}}\text{)}{}^{\mathrm{<mtext>1</mtext><mtext>n</mtext>}}$ was found to be 101 pM. Analysis of the equilibrium binding of [³H]DHA to rat and mouse living brain cells resulted in values of B_max = 13.04 fmol/10⁶ cells (7800 sites/cell), K_d = 43.85 pM and n = 2.52, and B_max = 8.08 fmol/10⁶ cells (4800 sites/cell), K_d = 46.70 pM and n = 1.63, respectively, confirming the apparent cooperativity of the β-receptor in mammalian objects, too. The [³H]DHA equilibrium binding to broken cell preparations of either chick, rat or mouse brain cultures or forebrain tissues was found to be non-cooperative, with a Hill coefficient n = 1, K_d in the range 1–2 nM, and a B_max of 10³–10⁴ sites/cell. Our findings demonstrate that cell disruption causes marked changes in the kinetics of the β-receptor binding and in the affinity of the binding site, although the number of receptors remains unchanged.     

Dna amplification polymorphisms of Mucor piriformis

Mucor piriformis can cause postharvest decay in various fruits and vegetables stored at low temperatures. Thirty isolates of this fungus, collected from infected fruit, were subjected to random amplified polymorphic DNA (RAPD) analysis. Seven different 10-bp primers were used to determine the type and extent of intraspecific genetic polymorphisms. Nineteen composite amplification types were identified, indicating a higher degree of variability than found in previous isoenzyme studies. Numerical analysis with the UPGMA technique revealed three clusters, which correlated with the mating competency of the isolates or their place of origin. These results demonstrate that RAPD analysis can identify isolates and subspecific populations of M. piriformis.     

Investigations of the Blue-green Fluorescence Emission of Plant Leaves

The UV light (337 nm) induced blue-green fluorescence emission of green leaves is characterized at room temperature (298 K) by a maximum near 450 nm (blue region) and a shoulder near 525 nm (green region) and was here also studied at 77 K. At liquid nitrogen temperature (77 K) the blue (F450) and green fluorescence (F525) are much enhanced as is the red chlorophyll fluorescence near 735 nm. During development of green tobacco leaves the blue fluorescence F450 (77 K) is shifted towards longer wavelengths from about 410 nm to 450 nm. The isolated leaf epidermis of tobacco showed only slight fluorescence emission with a maximum near 410 nm. The green fluorescence F525 was found to mainly originate from the mesophyll of the leaf, its intensity increased when the epidermis was removed. The red chlorophyll fluorescence emission was also enhanced when the epidermis was stripped off; this considerably changed the blue/red fluorescence ratios F450/F690 and F450/F735. The epidermis, with its cell wall and UV-light-absorbing substances in its vacuole, plays the role of a barrier for the exciting UV-light. In contrast to intact and homogenized leaves, isolated intact chloroplasts and thylakoid membranes did not exhibit a blue-green fluorescence emission.     

Location and quantification of metal ions in enzymes combining polyacrylamide gel electrophoresis and particle-induced X-ray emission

A method is presented to identify and determine the relative amounts of protein-bound metal ionsin situ. Proteins or their subunits are directly scanned by a collimated proton beam of 3 MeV energy, and the characteristic X-rays produced are detected. The determination of Fe content of an iron-sulfur protein (HiPiP), as well as the Fe and Ni analysis of the hydrogenese fromThiocapsa roseopersina, have shown the feasibility of this technique.     

Effect of insulin imprinting on the 3H-amino acid uptake of the Tetrahymena

Insulin imprinting given to the unicellular Tetrahymena considerably increases the uptake and intracellular storage of amino acids even many generations after the actual contact with the hormone. On the other hand, both the first and the second contacts with insulin increase the rate of the excretion of the stored amino acids. On the basis of the results obtained it seems to be possible that both protein synthesis and exocytosis of the Tetrahymena change as an effect of imprinting, either in general or specifically due to the formation of new hormone receptors.     

Influence of enkephalins on the ACTH-induced hormonal imprinting in tetrahymena

Both adrenocorticotrop hormone (ACTH) and the synthetic enkephalins investigated evoked imprinting in Tetrahymena and led to increased hormone binding at further contact with ACTH. Neither molecule evoked, however, imprinting for the enkephalins. The pentapeptide enkephalin containing also proline had the most pronounced imprinting effect and, when given together with ACTH, it increased the imprintatory effect of ACTH considerably. In all the situations investigated the enkephalin tetrapeptide inhibited the positive effect of the enkephalin pentapeptide, whereas it did not influence the imprintatory effect of ACTH. Similarities can be found between the pharmacological and imprinting effects of enkephalin in mammals, and the effects seen in the present investigations.     

Steroid hormone (hydrocortisone, oestradiol and testosterone) uptake, storage or induced synthesis in tetrahymena

After cyclodextrin-coated 10(-6) m steroid hormone treatment for 3 days (hormonal imprinting), Tetrahymena cells and their media were analysed by radioimmunoassay for the same hormone and for the presence of the other two. In the absence of hormone treatment, the cells contained no detectable levels of the three steroids. By 2 days in fresh medium following exposure of cells to a 72 h pretreatment of each specific hormone, correspondingly high quantities of hydrocortisone and oestradiol, but lesser quantities of testosterone, were found in both the media and the cells. One week after treatment only traces of hydrocortisone were found, exclusively within the cells themselves. Oestradiol was present in measurable quantities in both cells and media, whereas testosterone was only present in the medium. The presence of the other two hormones to the one used in the pretreatment were not usually present, except that when testosterone had been given, some oestradiol was also detected at 48 h, suggesting Tetrahymena has a functional cytochrome P(450)aromatase.     

Effect of digoxin imprinting in adolescence on the sexual behavior of adult rats

Four-time 3 micrograms digoxin treatment of male rats at puberty (in six weeks old rats) significantly increased the libido of rats (number of intromissions) and reduced the number of ejaculations, two months after the treatments (in three and a half months old rats). In female rats the Meyerson index and lordosis quotient were not significantly decreased. The experiment calls attention to the wide-ranging imprinting effect of digoxin which was also demonstrated earlier after prenatal (maternal) treatment. The experiment also supports the male sexual potency influencing effect of digoxin treatment, previously supposed in men.     

Induction of melatonin synthesis in Tetrahymena pyriformis by hormonal imprinting--a unicellular "factory" of the indoleamine.

Melatonin is present in Tetrahymena and its synthesis can be enhanced by pretreatment (imprinting) with melatonin. Two days after imprinting melatonin level is elevated in the cells and more elevated in the supematant. Such a minute quantity, as 10(-12) M melatonin for 1 hour is able to provoke imprinting, however the effect is more expressed using 10(-6) M. Maintenance in light conditions further elevated the amount of melatonin in the cells and supematant alike, related to the melatonin content of cells kept in darkness. The experiments call attention to the light-sensitivity of imprinting-provoked melatonin production in Tetrahymena and to the possibility of using this property for important physiological functions in higher grades of phylogeny.     

Alteration of plant growth and development by Rhizobium nodA and nodB genes involved in the synthesis of oligosaccharide signal molecules

The highly conserved Rhizobium nodulation genes nodABC are required to produce lipid-linked chitooligosaccharide signal molecules which elicit nodule organogenesis in roots of leguminous plants. Recently, it has been shown that NodB deacetylates chitooligosaccharides at the non-reducing terminus, so that the free amino group of the chitooligosaccharide backbone can then be acylated by a specific fatty acid. The Rhizobium NodA protein together with the nodB encoded chitooligosaccharide deacetylase are involved in generating small, heat-stable compounds that stimulate mitosis in protoplasts derived from either legumes or other plant species. To test whether these gene products could play a role in regulation of plant development, we introduced and expressed the Rhizobium meliloti nodA and nodB genes singly or in combination under the control of diverse promoters in tobacco. Altered phenotypes correlating with nodA and nodB gene expression in transgenic plants indicate that tobacco contains the necessary substrates for the NodA and NodB proteins to produce signal molecules modulating plant growth and organ development.