The rolB Gene of Agrobacterium rhizogenes Does Not Increase the Auxin Sensitivity of Tobacco Protoplasts by Modifying the Intracellular Auxin Concentration

Phenotypical alterations observed in rolB-transformed plants have been proposed to result from a rise in intracellular free auxin due to a RolB-catalyzed hydrolysis of auxin conjugates(J.J. Estruch, J. Schell, A. Spena [1991] EMBO J 10: 3125-3128).We have investigated this hypothesis in detail using tobacco (Nicotiana tabacum) mesophyll protoplasts isolated from plants transformed with the rolB gene under the control of its own promoter (BBGUS 6 clone) or the cauliflower mosaic virus 35S promoter (CaMVBT 3 clone). Protoplasts expressing rolB showed an increased sensitivity to the auxin-induced hyperpolarization of the plasma membrane when triggered with exogenous auxin. Because this phenotypical trait was homogeneously displayed over the entire population, protoplasts were judged to be a more reliable test system than the tissue fragments used in previous studies to monitor rolB gene effects on cellular auxin levels. Accumulation of free 1-[3H]-naphthaleneacetic acid (NAA) was equivalent in CaMVBT 3, BBGUS 6, and wild-type protoplasts, Naphthyl-[beta]-glucose ester, the major NAA metabolite in protoplasts, reached similar levels in CaMVBT 3 protoplasts, reached similar levels in CaMVBT 3 and normal protoplasts and was hydrolyzed at the same rate in BBGUS 6 and normal protoplasts. Furthermore, NAA accumulation and metabolism in BBGUS 6 protoplasts were independent of the rolB gene expression level. Essentially similar results were obtained with indoleacetic acid. Thus, it was concluded that the rolB-dependent behavior of transgenic tobacco protoplasts is not a consequence of modifying the intracellular auxin concentration but likely results from changes in the auxin perception pathway.     

Comparison of the intermediate complexes of human growth hormone bound to the human growth hormone and prolactin receptors.

The crystal structures of complexes of human growth hormone (hGH) with the growth hormone and prolactin receptors (hGHR and hPRLR, respectively), together with the mutational data available for these systems, suggest that an extraordinary combination of conformational adaptability, together with finely tuned specificity, governs the molecular recognition processes operative in these systems. On the one hand, in the active 1:2 ligand-receptor complexes, 2 copies of the same receptor use the identical set of binding determinants to recognize topographically different surfaces on the hormone. On the other hand, comparing the 1:1 hGH-hGHR and hGH-hPRLR complexes, 2 distinct receptors use this same set of binding determinants to interact with the identical binding site on the ligand, even though few residues among the binding determinants are conserved. The structural evidence demonstrates that this versatility is accomplished by local conformational flexibility of the binding loops, allowing adaptation to different binding environments, together with rigid-body movements of the receptor domains, necessary for the creation of specific interactions with the same binding site.     

The essential mitotic target of calmodulin is the 110-kilodalton component of the spindle pole body in Saccharomyces cerevisiae.

Two independent methods identified the spindle pole body component Nuf1p/Spc110p as the essential mitotic target of calmodulin. Extragenic suppressors of cmd1-1 were isolated and found to define three loci, XCM1, XCM2, and XCM3 (extragenic suppressor of cmd1-1). The gene encoding a dominant suppressor allele of XCM1 was cloned. On the basis of DNA sequence analysis, genetic cosegregation, and mutational analysis, XCM1 was identified as NUF1/SPC110. Independently, a C-terminal portion of Nuf1p/Spc110p, amino acid residues 828 to 944, was isolated as a calmodulin-binding protein by the two-hybrid system. As assayed by the two-hybrid system, Nuf1p/Spc110p interacts with wild-type calmodulin and triple-mutant calmodulins defective in binding Ca2+ but not with two mutant calmodulins that confer a temperature-sensitive phenotype. Deletion analysis by the two-hybrid system mapped the calmodulin-binding site of Nuf1p/Spc110p to amino acid residues 900 to 927. Direct binding between calmodulin and Nuf1p/Spc110p was demonstrated by a modified gel overlay assay. Furthermore, indirect immunofluorescence with fixation procedures known to aid visualization of spindle pole body components localized calmodulin to the spindle pole body. Sequence analysis of five suppressor alleles of NUF1/SPC110 indicated that suppression of cmd1-1 occurs by C-terminal truncation of Nuf1p/Spc110p at amino acid residues 856, 863, or 881, thereby removing the calmodulin-binding site.     

Direct high-performance liquid chromatographic resolution of the enantiomers of tiaprofenic acid using immobilized human serum albumin

Resolution of racemic tiaprofenic acid (TA) has been performed using immobilized human serum albumin as the stationary phase. The eluent was phosphate buffer—acetonitrile—n-octanoic acid (90:10:0.015, v/v). Detection was achieved at 305 nm. The pharmacokinetics of the enantiomers were studied following oral administration into humans and after subcutaneous injection in rats. Plasma concentrations of (+)-TA were much greater than those of (−)-TA. For the rat, the pharmacokinetic parameters between (−)-TA and (+)-TA were all statistically different (p < 0.005).     

CROSSING EXPERIMENTS,LIPID COMPOSITION,AND THE SPECIES CONCEPT IN ECTOCARPUS SILICULOSUS AND E. FASCICULATUS (PHAEOPHYCEAE,ECTOCARPALES)1

Sporophytes of Ectocarpus siliculosus (Dillwyn) Lyngbye and E. fasciculatus Harvey were collected in the vicinity of Roscoff Brittany, France. Gametophytes derived from meiospores were used for intra- and interspecific crosses. Intraspecific gamete combinations gave viable zygotes, which developed into fertile sporophytes. Interspecific crosses were unsuccessful. Gamete fusions did not occur between female gametes of E. fasciculatus and male gametes of E. siliculosus. Hybrid zygotes were formed in the reciprocal combination but died soon after germination. We conclude that the two species of Ectocarpus at Roscoff represent distinct taxonomic entities, which are separated by pre- and postzygotic compatibility barriers. These biological findings are confirmed by the differential occurrence of the chemotaxonomic marker betaine-lipid diacetylglycerylhydroxymethyltrimethyl-β-alanine, which is present in our cultures of E. fasciculatus but absent in E. siliculosus.     

Use of histone antibodies for studying chromatin topography and the phosphorylation of chromatin subunits

Polyclonal and monoclonal antibodies specific for histones as well as sera directed against synthetic peptides of histones were used to probe the topography of chromatin subunits. In native chromatin, the regions corresponding to residues 130-135 of H3 and 6-18 of H2B were found to be exposed and able to interact with antibodies whereas the regions 26-35 and 36-43 of H2B and 80-89 and 85-102 of H4 were not. In vitro phosphorylation of H3 and H5 in native chromatin or of H3 in H1/H5-depleted chromatin led to a marked drop in the binding of antibodies specific for residues 130-135 of H3 and 6-18 of H2B. Phosphorylation of H1/H5-depleted chromatin also altered the degree of exposure of certain H2A epitopes but it did not affect the surface accessibility of residues 1-11 of H2B.     

Preparation of analogues of NAD+ as substrates for a sensitive fluorimetric assay of nucleotide pyrophosphatase.

1,N6-Etheno derivatives of pyridine analogues of NAD+ were synthesized, characterized and tested as substrates for a fluorimetric assay of nucleotide pyrophosphatase (EC 3.6.1.9). Upon cleavage of their pyrophosphate bond, the fluorescence of pyridine-1,N6-ethenoadenine dinucleotide (epsilon PdAD+) and of 4-hydrazinocarbonyl-pyridine-1,N6-ethenoadenine dinucleotide (epsilon hy4PdAD+) increased respectively 15-and 73-fold, at pH 7.4. This property allows a convenient steady-state assay of nucleotide pyrophosphatase by continuous monitoring of reaction progress. Both compounds were good substrates of this class of enzyme. The relative insensitivity of the fluorescence of epsilon PdAD+ and epsilon hy4PdAD+ to pH changes allowed assays under conditions preserving cellular integrity. epsilon PdAD+ is useful as a substrate for measuring nucleotide pyrophosphatase activity on the outside of mammalian cells because it is not a substrate for the external NAD+ glycohydrolase. epsilon Hy4PdAD+ proved useful when high sensitivity was needed.