Autoamplification of a two-component regulatory system results in "learning" behavior

We have tested the hypothesis that the autoamplification of two-component regulatory systems results in "learning" behavior, i.e., that bacteria respond faster or more extensively to a signal when a similar signal has been perceived in the past. Indeed, the induction of alkaline phosphatase activity upon phosphate limitation was faster if the cultures had been limited for phosphate previously, and this faster response correlated with the autoamplification of the cognate two-component system.     

The xanthopsins: a new family of eubacterial blue-light photoreceptors.

Photoactive yellow protein (PYP) is a photoreceptor that has been isolated from three halophilic phototrophic purple bacteria. The PYP from Ectothiorhodospira halophila BN9626 is the only member for which the sequence has been reported at the DNA level. Here we describe the cloning and sequencing of the genes encoding the PYPs from E.halophila SL-1 (type strain) and Rhodospirillum salexigens. The latter protein contains, like the E.halophila PYP, the chromophore trans p-coumaric acid, as we show here with high performance capillary zone electrophoresis. Additionally, we present evidence for the presence of a gene encoding a PYP homolog in Rhodobacter sphaeroides, the first genetically well-characterized bacterium in which this photoreceptor has been identified. An ORF downstream of the pyp gene from E.halophila encodes an enzyme, which is proposed to be involved in the biosynthesis of the chromophore of PYP. The pyp gene from E.halophila was used for heterologous overexpression in both Escherichia coli and R.sphaeroides, aimed at the development of a holoPYP overexpression system (an intact PYP, containing the p-coumaric acid chromophore and displaying the 446 nm absorbance band). In both organisms the protein could be detected immunologically, but its yellow color was not observed. Molecular genetic construction of a histidine-tagged version of PYP led to its 2500-fold overproduction in E.coli and simplified purification of the heterologously produced apoprotein. HoloPYP could be reconstituted by the addition of p-coumaric anhydride to the histidine-tagged apoPYP (PYP lacking its chromophore). We propose to call the family of photoactive yellow proteins the xanthopsins, in analogy with the rhodopsins.     

Measurement of branched chain amino acids in blood plasma by high performance liquid chromatography

A simple and rapid high performance liquid chromatographic technique is described for the separation and quantitation of plasma branched chain amino acids. After addition of a norleucine internal standard, plasma samples are acidified with acetic acid, and amino acids are separated from proteins and other plasma components by passage of the acidified plasma through an ion exchange resin. The ammonium hydroxide eluate from the resin is dried, phenylisothiocyanate derivatives are prepared, and the amino acids are separated on a Waters reverse-phase "Pico-Tag" column with an ultraviolet detector set at 254 nm. In addition to the branched chain amino acids (leucine, valine, and isoleucine), aspartate, glutamate, serine, threonine, alanine, and methionine are quantitated with high precision and accuracy, as verified by quantitative recovery and comparison with an automatic amino acid analyzer. The advantages of the method are its simplicity, speed, stability of derivatives, high reproducibility, low per-sample cost, and the use of a simple fixed-wavelength ultraviolet detector.     

Nerve fiber formation and catecholamine content in adult rat adrenal medullary transplants after treatment with NGF, NT-3, NT-4/5, bFGF, CNTF, and GDNF

Adrenal chromaffin cells have been characterized by the ability to change the phenotype in response to neurotrophic factor stimulation. The adrenal gland expresses numerous trophic factors endogenously, but there is still a lack of knowledge as to how the adrenal medullary cells respond to these factors. Accordingly, we evaluated nerve fiber outgrowth and cell morphology, and measured catecholamine content in adult rat adrenal medullary tissue transplanted to the anterior chamber of the eye after exposure to neurotrophin-3 (NT-3), neurotrophin-4/5 (NT-4/5), basic fibroblast growth factor (bFGF), ciliary neurotrophic factor (CNTF), or glial cell line-derived neurotrophic factor (GDNF) compared with the effects after exposure to recombinant human nerve growth factor (rhNGF). The results show that rhNGF was the most potent factor in inducing neurite outgrowth from the grafted chromaffin cells. CNTF was also a powerful inducer of nerve fiber formation, while NT-4/5, GDNF, and bFGF were less potent. NT-3 did not produce neurite outgrowth above that seen in vehicle-treated eyes. Combining two neurotrophins, rhNGF and NT-3, reduced nerve fiber formation. Tyrosine hydroxylase (TH) immunohistochemistry revealed good cell survival in all grafts, and no morphological differences were detected with the different treatments. The adrenaline: noradrenaline: dopamine ratio was approximately 49%: 49%: 2%, independent of treatment, and the catecholamine content was equal irrespective of treatment. In conclusion, all neurotrophic factors used, except for NT-3, promoted neurite outgrowth from adult rat chromaffin transplants. Differences in outgrowth induced by the various trophic factors did not, however, change the catecholamine content in grafts when analyzed together with the graft-derived nerve plexus.     

Effect of calcium removal on monoamine-elicited depressions of cultured tuberal neurons

The role of extracellular calcium in monoamine responses of central neurons was investigated using explant cultures of tuberal hypothalamus. The spontaneous activity of neurons in cultures was recorded in balanced salt and calcium-deficient salt solutions. The firing rate was reversibly augmented during perfusion with calcium-free salt solutions. This increased firing rate was counteracted by the addition of magnesium. Addition of magnesium also regularized the pattern of firing. Iontophoretic application of putative monoamine neurotransmitters reversibly decreased the rate of firing in both normal and calcium-deficient salt solutions. These results suggest that monoamine inhibitions are not primarily mediated by transmembrane calcium fluxes.