Partial Purification and Immunocytocharacterization of the Plasma Membrane ATPase of Jerusalem artichoke (Helianthus tuberosus L.) Tubers in Relation to Dormancy

Plasmalemma ATPase from Jerusalem artichoke tubers was studiedin relation to the dormancy of tubers. After partial purification,one peptide of 110 kDa appeared on SDS PAGE electrophoresisfrom dormant and non-dormant materials. ATPase specific activitywas twice higher on dormant material in the crude and solubilizedfractions, but was the same in both materials after partialpurification. Immunolabeling of this enzyme was made using aspecific antibody raised against the C terminal portion of theH⁺-ATPase from Arabidopsis thaliana. Immunolabeling was morepronounced in dormant material, in vitro and in situ. Severalworks had shown that the C terminal part of the enzyme couldbe involved in its regulation. The results presented are discussedin relation to the hypothesis according to which an internaleffector could modulated the plasmalemma ATPase activity, duringdormancy breaking. (Received October 25, 1993; Accepted September 6, 1994)     

EPR characterization of oxide supported transition metal ions: Relevance to catalysis

EPR spectroscopy is a powerful tool to identify at a molecular level, the different steps of catalyst preparation, and of catalytic reactions:

1. Deposition of paramagnetic transition metal ions onto a support is monitored, and the coordination sphere of the metallic center is characterized by EPR.
2. The catalyst is also characterized after activation (thermal oxidation or reduction):

- the distribution among the different sites in zeolites can be determined;

- the dispersion of the active phase may be appreciated;

- the unsaturation degree of the active site may be evaluated using probe molecules such as water or¹³C enriched carbon monoxide.

1. The catalytic mechanisms can be investigated by studying the elementary steps of the catalytic reaction, as illustrated for methanol oxidation over Mo/SiO₂ catalysts whose EPR results have extended the reaction mechanism proposed on the basis of kinetic data. In addition, reaction intermediates may be isolated inquasi-in situ conditions as in the case of olefin oligomerization catalyzed by Ni/SiO₂ systems.

     

Multiplicity of genes encoding secreted aspartic proteinases in Candida species

The secreted aspartic proteinases (SAP) of Candida sp. are presumed to be potential virulence factors. In the opportunistic pathogen Candida albicans the proteinase genes identified to date, SAP1, SAP2, SAP3 and SAP4, constitute a multigene family. Before addressing the possible role of each proteinase in virulence, we sought to isolate all the members of this multigene family by screening a genomic library with a SAP1 probe for additional C. albicans SAP genes using low-stringency hybridization conditions. Three putative new members, SAP5, SAP6 and SAP7 were isolated and sequenced. The N-terminal segments of the deduced amino acid sequences of SAP5 and SAP6 contained secretion signal sequences similar to those of other Candida SAPs. Upon comparison and alignment with the other reported SAP amino acid sequences, SAP7 is not only the most divergent protein but also exhibits a much longer putative pro-sequence with a single Lys-Lys putative processing site. Using SAP1 to SAP7 as probes, the overall number of SAP genes in C. albicans was tentatively estimated by low-stringency hybridization to EcoRI-digested genomic DNA. While each isolated SAP gene could be assigned to distinct EcoRI bands, the existence of two additional genes not isolated after screening of the C. albicans gene library was inferred. Furthermore, evidence was obtained for the existence of SAP muttigene families in other Candida species such as C. tropicalis, C. parapsilosis and C. guiller-mondii.     

A two-component signal-transducing system is involved in competence and penicillin susceptibility in laboratory mutants of Streptococcus pneumoniae

Penicillin resistance in Streptococcus pneumoniae has been attributed so far to the production of penicillin-binding protein (PBP) variants with decreased affinities for β-lactam antibiotics. Cefotaxime-resistant laboratory mutants, selected after several steps on increasing concentrations of this β-lactam, become deficient in transformation as well. A DNA fragment conferring both cefotaxime resistance and transformation deficiency was isolated and cloned from the mutant C306. The cefotaxime resistance associated with this resistance determinant was not accompanied with apparent changes in PBP properties, and it mapped on the chromosome distinct from the known resistance determinants, genes encoding PBP2x, PBP1a or PBP2b. Determination of a 2265 bp DNA sequence of the resistance determinant revealed two open reading frames, claR and claH, whose deduced amino acid sequence identified the corresponding proteins as the response regulator and histidine kinase receptor, respectively (members of the two families of bacterial signal-transducing proteins). Two hydrophobic peptide regions divided the histidine kinase ClaH into two putative domains: an N-terminal extracelluiar sensor part, and an intracelluiar C-terminal domain with the conserved His-226 residue, the presumed phosphorylation site. The single point mutations responsible for cefotaxime-resistance and transformation deficiency of C306 and of another two independently isolated cefotaxime-resistant mutants were each located in the C-terminal half of ClaH. A small extracellular protein, the competence factor, is required for induction of competence. Neither C306 nor the transformants obtained with the mutated claH gene produced competence factor, and exogenous competence factor could not complement the transformation deficiency, indicating that the signal-transducing system cia is involved in early steps of competence regulation.     

Analysis of phytochrome- and ABA-deficient mutants suggests that ABA degradation is controlled by light in Nicotiana plumbaginifolia

The phytochrome chromophore-deficient mutant, pew1, of Nicotiana plumbaginifolia exhibited decreased germination and slower dehydration of detached leaves during water stress as compared with the wild-type. These physiological processes are controlled by abscisic acid (ABA) and we examined, therefore, whether phytochrome plays a specific role in the regulation of ABA metabolism using the pew1 mutant. The ABA contents of mature seeds and young leaves were analysed and in both cases mutant material was found to contain higher amounts of ABA as compared with the wild-type. This indicates that the phytochrome activation can lead to a decrease of the ABA level in the wild-type plant. The role of phytochromes was investigated in greater detail using the ABA-deficient mutant aba1 of N. plumbaginifolia exhibiting an early and synchronous germination. This mutant accumulates at very high levels a metabolite derived from a precursor (ABA-aldehyde) in the ABA biosynthetic pathway. The first biochemical characterization of this molecule, which corresponds to the glucose-conjugated ABA-alcohol (ABA-AG) is described. A pew1-aba1 double mutant exhibiting both an etiolated growth and early germination was also obtained. The comparable accumulation of ABA-AG in the pew1-aba1 double mutant as compared with the aba1 mutant allowed the proposition that, in a wild-type plant, the phytochrome-mediated light signal enhances ABA degradation rather than inhibits its biosynthesis.     

Molecular characterization of Arabidopsis thaliana cDNAs encoding three purine biosynthetic enzymes

Glycinamide ribonucleotide (GAR) synthetase, GAR transformylase and aminoimidazole ribonucleotide (AIR) synthetase are the second, third and fifth enzymes in the 10-step de novo purine biosynthetic pathway. From a cDNA library of Arabidopsis thaliana, cDNAs encoding the above three enzymes were cloned by functional complementation of corresponding Escherichia coli mutants. Each of the cDNAs encode peptides comprising the complete enzymatic domain of either GAR synthetase, GAR transformylase or AIR synthetase. Comparisons of the three Arabidopsis purine biosynthetic enzymes with corresponding enzymes/polypeptide-fragments from procaryotic and eucaryotic sources indicate a high degree of conserved homology at the amino acid level, in particular with procaryotic enzymes. Assays from extracts of E. coli expressing the complementing clones verified the specific enzymatic activity of Arabidopsis GAR synthetase and GAR transformylase. Sequence analysis, as well as Northern blot analysis indicate that Arabidopsis has single and monofunctional enzymes. In this respect the organization of these three plant purine biosynthesis genes is fundamentally different from the multifunctional purine biosynthesis enzymes characteristic of other eucaryotes and instead resembles the one gene, one enzyme relationship found in procaryotes.     

Neurocircuitry of the basal ganglia studied by monitoring neurotransmitter release

The neurocircuitries of the basal ganglia are studied with in vivo microdialysis, with special consideration to dopamine transmission and its interaction with other neurotransmitter systems. The aim is to develop experimental models to study the pathophysiology and therapy of neurodegenerative disorders of the basal ganglia, as well as to develop models to study the short- and long-term consequences of perinatal asphyctic lesions. A main goal of these studies is to find and to characterize new treatments for these disorders.     

Cloning and expression of distinct nitrite reductases in tobacco leaves and roots

Summary Three tobacco nitrite reductase (NiR) cDNA clones were isolated using spinach NiR cDNA as a probe. Sequence analysis and Southern blot hybridization revealed four genes in tobacco. Two of these genes presumably derived from the ancestral species Nicotiana tomentosiformis, the other two from the ancestor N. sylvestris. Northern blot analysis showed that one gene from each ancestral genome was expressed predominantly in leaves, whilst RNA from the other was detected mostly in roots. The accumulation of both leaf and root NiR mRNAs was induced by nitrate and repressed by nitrate- or ammonium-derived metabolites. In addition, the expression of the root NiR gene was detectable in leaves of a tobacco nitrate reductase (NR)-deficient mutant. Thus, the regulation of expression of tobacco NiR genes is comparable to the regulation of expression of barley NR genes.     

Effect of exogeneous methyl oleate on the time course of some parameters of Streptomyces hygroscopicus NRRL B-1865 culture

Addition of methyl oleate to a Streptomyces hygroscopicus NRRL B-1865 culture modified the metabolic properties of this strain. This addition decreased the pH of the medium, increased the valine uptake of the cells and reduced their consumption of glucose until the beginning of antibiotic biosynthesis, which was delayed. At the same time, an increase in growth (× 1.8) and a marked improvement in antibiotic production (× 20) could be observed. The use of labelled methyl oleate showed that methyl oleate was not a precursor of antibiotics produced by S. hygroscopicus NRRL B-1865. It is suggested that methyl oleate addition may cause some alteration in membrane permeability, inducing an increase in H⁺ extrusion and stimulating the accumulation of branched amino acids, known to be direct precursors of polyether antibiotics. Correspondence to: L. David