Investigation of Dopamine Content, Synthesis, and Release in the Rabbit Retina In Vitro: I. Effects of Dopamine Precursors, Reserpine, Amphetamine, and l-DOPA Decarboxylase and Monoamine Oxidase Inhibitors

The basal catecholamine content of rabbit retina was determined by liquid chromatography with electrochemical detection (LC-EC) and 3,4-dihydroxyphenylethylamine (dopamine, DA) found to be the major catecholamine. The immediate DA precursor, 3,4-dihydroxyphenylalanine (L-DOPA), and the metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC), were also detected at about 2.8% and 17% of DA levels, respectively. When added exogenously, L-tyrosine did not increase the rate of DA synthesis over the basal level. In contrast, exogenous L-DOPA led to a 3.5-fold increase in DA, and to a 20-fold increase in DOPAC content. The monoamine oxidase inhibitors pargyline and (-)-deprenyl differentially affected the degradation of DA, since 100 microM pargyline was apparently more effective than 100 microM (-)-deprenyl. Reserpine and (+/-)-amphetamine each induced a Ca2+-independent decrease of DA stores. The separate actions of reserpine and (+/-)-amphetamine in lowering tissue DA levels were additive, suggesting two separate pools of DA available for release from presynaptic stores. The present study demonstrates that the LC-EC technique may be used to investigate the modulation of the synthesis and release of retinal DA in vitro, without the prior uptake of radiolabelled transmitter.     

Virulence genes are carried by a megaplasmid of the plant pathogen Pseudomonas solanacearum

Summary A class of avirulent mutants of the plant pathogenic bacterium Pseudomonas solanacearum, strain GMI1000, resistant to acridine orange (Acr^r), harbour a deletion of over 85 kb in their genome. This deletion affects, a1,000 kb megaplasmid which has previously been shown to be present in most of the strains of this species. In addition at least 11 out of 13 independent Tn5 insertions, leading to loss of virulence, are located on the megaplasmid. Nine of them are present in the region which is deleted from the Acr^r mutants. These results suggest that the majority of virulence genes identified so far are plasmid borne.     

Growth Regulators Have Rapid Effects on Photosynthate Unloading from Seed Coats of Phaseolus vulgaris L

Of nine plant growth regulators (indoleacetic acid, 1-naphthalene acetic acid, gibberellic acid, giberellin 4/7, 6-benzylaminopurine, 6-furfurylaminopurine, abscisic acid, and 1-aminocyclopropane carboxylic acid) tested, only 6-benzylaminopurine and abscisic acid affected ¹⁴C-photosynthate unloading from excised seed coats of Phaseolus vulgaris L. Unloading, in the presence of KCl, was stimulated by 25 to 40%. Stimulation occurred immediately for 6-benzylaminopurine and for abscisic acid within 10 to 12 minutes of application.     

Effect of human serum and some of its components on neutrophil adherence and migration across an epithelium

The effect of human serum and some of its components on the process of transepithelial migration of human neutrophils was investigated in an in vitro system. 10% autologous serum caused an increase in neutrophil adherence to and migration across canine kidney epithelial cells. This increase in neutrophil binding also occurred if the epithelium but not the neutrophils had been preincubated with serum. The binding was lost if the serum was either preabsorbed over the kidney epithelium before use or heat inactivated. Indirect immunofluorescence studies indicated that IgG, IgM, and a component of C3 bound to the epithelial surface, whereas IgA, IgE, or C5a were not detectable. The majority of epithelial cells were immunofluorescent, however epithelial cells with varying degrees of reactivity were also apparent and approximately 5% of the epithelial cells did not bind IgG, IgM, and C3. When epithelia were simultaneously tested for the presence of either IgG, IgM, or C3, and bound neutrophils the few epithelial cells which did not bind IgG or IgM also did not bind C3 or neutrophils. Studies with monoclonal antibodies against Fc and C3 receptors indicate that neutrophil adherence to the epithelial surface was mediated predominately by the receptors for C3b and C3bi. In response to a chemotactic gradient, bound neutrophils were able to detach and migrate across the epithelium. A separate heat-stable factor(s) in serum was able to increase neutrophil migration across the epithelial monolayer. This factor acted independently of the factors which caused the increase in neutrophil binding as the increase in neutrophil migration also occurred under conditions (preabsorption over the kidney epithelium or heat inactivation) that prevented the increase in neutrophil binding. The increase in neutrophil migration may be caused by the permeability-increasing properties of this factor as both serum and heat-inactivated serum lowered the transepithelial electrical resistance an average of 38 and 35%, respectively, in 40 min. Upon removal of serum or heat-inactivated serum, the resistance returned 100 and 81%, respectively, in 5 h.     

Dipyridamole-insensitive nucleoside transport in mutant murine T lymphoma cells

From a mutagenized population of S49 murine T lymphoma cells, a mutant cell line, JPA4, was selected that expressed an altered nucleoside transport capability. JPA4 cells transported low concentrations of purine nucleosides and uridine more rapidly than the parental S49 cell line. The transport of these nucleosides by mutant cells was insensitive to inhibition by either dipyridamole (DPA) or 4-nitrobenzylthioinosine (NBMPR), two potent inhibitors of nucleoside transport in mammalian cells. Kinetic analyses revealed that the apparent Km values for the transport of uridine, adenosine, and inosine were 3-4-fold lower in JPA4 cells compared to wild type cells. In contrast, the transport of both thymidine and cytidine by JPA4 cells was similar to that of parental cells, and transport of these pyrimidine nucleosides remained sensitive to inhibition by both NBMPR and DPA. Furthermore, thymidine was a 10-12-fold weaker inhibitor of inosine transport in JPA4 cells than in wild type cells. Thus, JPA4 cells appeared to express two types of nucleoside transport activities; a novel (mutant) type that was insensitive to inhibition by DPA and NBMPR and transported purine nucleosides and uridine, and a parental type that retained sensitivity to inhibitors and transported cytidine and thymidine. The phenotype of the JPA4 cell line suggests that the sensitivity of mammalian nucleoside transporters to both NBMPR and DPA can be genetically uncoupled from its ability to transport certain nucleoside substrates and that the determinants on the nucleoside transporter that interact with each nucleoside are not necessarily identical.     

Isoleucyl-tRNA synthetase from bakers' yeast: multistep proofreading in discrimination between isoleucine and valine with modulated accuracy, a scheme for molecular recognition by energy dissipation

For discrimination between isoleucine and valine by isoleucyl-tRNA synthetase from yeast, a multistep sequence is established. The initial discrimination of the substrates is followed by a pretransfer and a posttransfer hydrolytic proofreading process. The overall discrimination factor D was determined from kcat and Km values observed in aminoacylation of tRNAIle-C-C-A with isoleucine and valine. From aminoacylation of the modified tRNA species tRNAIle-C-C-3'dA and tRNAIle-C-C-A (3'NH2), the initial discrimination factor I (valid for the reversible substrate binding) and the proofreading factor P1 (valid for the aminoacyl adenylate formation) could be determined. Factor I was computed from ATP consumption and D1, the overall discrimination factor for this partial reaction which can be obtained from kinetic constants, and P1 was calculated from AMP formation rates. Proofreading factor P2 (valid for aminoacyl transfer reaction) was determined from AMP formation rates observed in aminoacylation of tRNAIle-C-C-A and tRNAIle-C-C-3'dA. From the initial discrimination factor I and the AMP formation rates, discrimination factor DAMP in aminoacylation of tRNAIle-C-C-A can be calculated. These values deviate by a factor II from factor D obtained by kinetics which may be due to the fact that for acylation of tRNAIle-C-C-A an initial discrimination factor I' = III is valid. The observed overall discrimination varies up to a factor of 16 according to conditions. Under optimal conditions, 38 000 correct aminoacyl-tRNAs are produced per 1 error while the energy of 5.5 ATPs is dissipated. With the determined energetic and molecular flows for the various steps of the enzymatic reaction, a coherent picture of this new type of "far away from equilibrium enzyme" emerges.     

Evolutionary aspects of accuracy of phenylalanyl-tRNA synthetase. Accuracy of the cytoplasmic and chloroplastic enzymes of a higher plant (Phaseolus vulgaris)

The phenylalanyl-tRNA synthetases from cytoplasm and chloroplasts of bean (Phaseolus vulgaris) leaves employ different strategies with respect to accuracy. The chloroplastic enzyme that is coded for by the nuclear genome follows the pathway of posttransfer proofreading, also characteristic for enzymes from eubacteria and cytoplasm and mitochondria of lower eukaryotic organisms. In contrast, the cytoplasmic enzyme uses pretransfer proofreading in the case of noncognate natural amino acids, characteristic for higher eukaryotic organisms and archaebacteria. Dependent on the nature of the noncognate amino acid, pretransfer proofreading in this case occurs without tRNA stimulation or with tRNA stimulated with no or little effect of the nonaccepting 3'-OH group of the terminal adenosine. The fundamental mechanistic difference in proofreading between the heterotopic intracellular isoenzymes of the plant cell supports the idea of the origin of the chloroplastic gene by gene transfer from a eubacterial endosymbiont to the nucleus. Origin by duplication of the nuclear gene, as indicated for mitochondrial phenylalanyl-tRNA synthetases [Gabius, H.-J., Engelhardt, R., Schroeder, F.R., & Cramer, F. (1983) Biochemistry 22, 5306-5315], appears unlikely. Further analyses of the ATP/PPi pyrophosphate exchange and aminoacylation of tRNAPhe-C-C-A(3'NH2), using 11 phenylalanine analogues, reveal intraspecies and interspecies variability of the architecture of the amino acid binding part within the active site.     

Photochemical demonstration of stacked C.C+ base pairs in a novel DNA secondary structure

The secondary structure of the alternating polydeoxynucleotide sequence poly[d(C-T)] was studied as a function of pH by ultraviolet absorbance and circular dichroism spectroscopy and by the analysis of UV-induced photoproducts. As the pH was lowered, poly[d(C-T)] underwent a conformational transition that was characterized by changes in the long-wavelength region (280-320 nm) of the CD spectrum. These changes have previously been interpreted as evidence for the formation of a core of stacked, protonated C X C+ base pairs in a double-helical complex of poly[d(C-T)], with the thymidyl residues being looped out into the solvent [Gray, D. M., Vaughan, M., Ratliff, R. L., & Hayes, F. N. (1980) Nucleic Acids Res. 8, 3695-3707]. In the present work, poly[d(C-T)] was labeled with [U-14C]cytosine and [methyl-3H]thymine and irradiated at pH values both above and below the conformational transition point (monitored by CD spectroscopy). The distribution of radioactivity in uracil means value of uracil dimers, uracil means value of thymine dimers (the deamination products of cytosine means value of cytosine and cytosine means value of thymine dimers, respectively), and thymine-means value of thymine dimers was then determined. As the pH was decreased, we found an increase in the yield of uracil means value of uracil dimers and a decrease in the yield of uracil means value of thymine dimers, which occurred concomitantly with the change in the CD spectrum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on the Inoculation and Competitiveness of a Rhizobium leguminosarum Strain in Soils Containing Indigenous Rhizobia

The competitiveness of a Rhizobium leguminosarum strain was investigated at two separate locations in field inoculation studies on commercially grown peas. The soil at each location (sites I and II) contained an indigenous R. leguminosarum population of ca. 3 × 10⁴ rhizobia per g of soil. At site I it was necessary to use an inoculum concentration as large as 4 × 10⁷ CFU ml⁻¹ (2 × 10⁶ bacteria seed⁻¹) to establish the inoculum strain in the majority of nodules (73%). However, at site II the inoculum strain formed only 33% of nodules when applied at this (10⁷ CFU ml⁻¹) level. Establishment could not be further improved by increasing the inoculum concentration even as high as 10⁹ CFU ml⁻¹ (9.6 × 10⁷ bacteria seed⁻¹). The inoculum strain could be detected at both sites 19 months after inoculation. Analysis by intrinsic antibiotic resistance patterns and plasmid DNA profiles indicated that a dominant strain(s) and plasmid pool existed among the indigenous population at site II. Competition experiments were carried out under laboratory conditions between a dominant indigenous isolate and the inoculum strain. Both strains were shown to be equally competitive.     

Catalysis by heteropentalenes of electron transfer to oxygen from ferredoxin-NADP oxidoreductase reduced by NADPH

A range of heteropentalene and bipyridinium compounds have been tested as catalysts of electron transfer to oxygen from spinach ferredoxin-NADP⁺ oxidoreductase reduced by NADPH. For a particular class of compound, the rate of oxygen reduction increased with increasing midpoint potential of the compound under conditions in which reduction of the compound was rate-limiting. Compounds with similar midpoint potentials from different structural classes showed marked differences in rate, attributed to specificity in the interaction with ferredoxin-NADP⁺ oxidoreductase.