Biochemical and immunocytochemical characterization of monoclonal antibody TOP 35 raised against purified tonoplast from cress root

The vacuolar membrane, the tonoplast, is a proteinrich membranehitherto only few proteins in it have been identified. As anapproach for the identification of tonoplast proteins by monoclonalantibodies (MABs), purified tonoplast from cress roots (Lepidiumsativum L.) were used for immunization and plasma membranesas a control membrane to test the absence of antigen. The MABTOP 35 identified a glycoprotein of about 35 kDa in purifiedtonoplast of cress roots. Triton X-114 phase separation showedthat it was a hydrophobic integral membrane protein. In immunocytochemistrythe MAB TOP 35 strongly labelled the vacuolar membrane. Theabsence of cell wall or plasma membrane labelling by TOP 35indicates a distinct biosynthetic pathway of this protein tothe vacuolar membrane in plants. Key words: Immnocytochemistry, Lepidium sativum, monoclonal antibody, secretion, vacuole     

MOLECULAR SYSTEMATICS OF ECTOCARPUS AND KUCKUCKIA (ECTOCARPALES, PHAEOPHYCEAE) INFERRED FROM PHYLOGENETIC ANALYSIS OF NUCLEAR- AND PIASTTD-ENCODED DNA SEQUENCES

The phylogeny of Ectocarpus and Kuckuckia strains representing widely separated populations from both hemispheres was inferred from sequence analysis of the internal transcribed spacers of the nuclear ribosomal DNA (ITS 1—5.8S-ITS 2) and the spacer region in the plastid-encoded ribulose- bis -phosphate-carboxylase (RUBISCO) cistron (partial rbc L-spacer-partial rbc S ). Both sequences resulted in matching phylogenies, with the RUBISCO spacer region being most informative at the level of genera and species and the internal transcribed spacer sequences at the level of species and populations. Three major clades were formed by strains previously described by morphology and physiology as Kuckuckia, E. fasciculatus, and E. siliculosus, confirming the validity of these taxa . Ectocarpus and Kuckuckia are regarded as sibling taxa with respect to the outgroup species Feldmannia simplex, Hincksia mitchelliae, and Pilayella littoralis. The clade formed by sexual E. siliculosus strains and most asexual Ectocarpus strains was subdivided into several clades that are consistent with geographical races within E. siliculosus. The inferred phylogeny of Ectocarpus corresponds generally with results from cross-fertilization experiments, morphology, and lipid analysis. A hypothesis on the origin and dispersal of E. siliculosus suggests several natural dispersal events during periods of global cooling as well as recent and possibly anthropogenic dispersal events .     

Two recurrent nonsense mutations and a 4 bp deletion in a quasi-symmetric element in exon 37 of the NF1 gene

We screened a total of 92 unrelated patients with neurofibromatosis type 1 (NF1) for mutations in exon 37 of the NF1 gene, by using temperature gradient gel electrophoresis. Two novel mutations were found: a 4 bp deletion in a so-called quasi-symmetric element (6789delTTAC) and a recurrent nonsense mutation, which was identified in two unrelated patients, at codon 2264 (C6792A). The independent origin of the latter mutation in two families was confirmed by haplotype analysis. The nonsense mutation and the 4 bp deletion are both predicted to lead to a truncated protein product lacking the Cterminal 20% (aproximately) of its sequence. The occurrence of three independent mutations among 92 NF1 patients at codons 2263–2264 (exon 37) suggests that a specific search for mutations in this area should be undertaken in screening programs for NF1 mutations.     

Isolation of a YAC clone covering a cluster of nine S100 genes on human chromosome 1q21: rationale for a new nomenclature of the S100 calcium-binding protein family

S100 proteins are low-molecular-weight calcium-binding proteins of the EF-hand superfamily and appear to be involved in the regulation of a number of cellular processes such as cell cycle progression and differentiation. More than 10 members of the S100 protein family have been described from human sources so far. We have now isolated a YAC clone from human chromosome 1q21, on which 9 different genes coding for S100 calcium-binding proteins could be localized. Moreover, we have mapped the gene coding for S100P to human chromosome 4p16 and thereby completed the chromosomal assignments of all known human S100 genes. The clustered organization of S100 genes in the 1q21 region allows us to introduce a new logical nomenclature for these genes, which is based on the physical arrangement on the chromosome. The new nomenclature should facilitate and further the understanding of this protein family and be easily expandable to other species.     

l-NNA inhibits the histochemical NADPH-d reaction in rat spinal cord neurons

In nerve tissue the histochemical nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) reaction is considered a suitable marker for nitric oxide synthase (NOS) activity. We have previously shown that the NOS-specific inhibitorl-nitroarginine (l-NNA) can block NADPH-d staining in intermediolateral (IML) neurons of the rat spinal cord; such a reaction might serve as a control for the presence of a NOS-related catalytic activity, i.e.,l-NNA-dependent NO synthesis in these neurons. However,l-NNA inhibition of neuronal NADPH-d is inconsistent and is therefore disputed by others. This prompted us to reinvestigate the reaction conditions to provide a standardized protocol for inhibition experiments. In IML neurons of formaldehyde-fixed spinal cord tissue, inhibition of NADPH-d reaction was tested by preincubation of frozen sections with the flavin-binder diphenylene iodonium chloride (DPI, 10 M-1 mM) which blocked the NADPH-d reaction in a concentration-dependent way, suggesting an inverse relationship of inhibitor concentration and final reaction product generated. Preincubation with the NOS-specific inhibitorl-NNA in glycine-NaOH buffer (pH 8.5–9.5) but notl-nitroarginine methyl ester (l-NAME) revealed a concentration-dependent blocking effect on neuronal NADPH-d comparable to the effects seen with DPI, suggesting the existence of al-NNA sensitive NADPH-d activity. Blocking withl-NNA (100 M-10 mM) was prevented by excessl-arginine (10–100 mM), suggesting competitive binding sites. NADPH-d staining was not inhibited by 7-nitro indazole, another NOS inhibitor. Thus, in formaldehyde-fixed nervous tissue both DPI andl-NNA inhibit the NOS-associated catalytic NADPH-d activity, thereby preventing NADPH-dependent conversion of nitroblue tetrazolium to formazan.Presented in the Workshop Detection of NO-synthases at the XXXVI Symposium of the Society for Histochemistry on Oxy Radicals, 20–23 September 1994, Heidelberg, Germany     

A cellulase/xylanase-negative mutant of Streptomyces lividans 1326 defective in cellobiose and xylobiose uptake is mutated in a gene encoding a protein homologous to ATP-binding proteins

Centre de Recherche en Microbiologie Appliquée, Institut Armand-Frappier, Université du Québec, Ville de Laval, Québec H7N 4Z3, Canada.     

Use of microbial peptide inhibitors for characterization of the substrate specificity of thermitase,a thermostable serine protease fromThermoactinomyces vulgaris

Seven microbial peptide inhibitors—chymostatin, antipain, elastatinal, leupeptin, pepstatin, bestatin, and phosphoramidon—were tested for their efficiency to inhibit thermitase, a thermostable serine protease fromThermoactinomyces vulgaris. Chymostatin and antipain were the most effective inhibitors, with K_i values of 7×10^–8 M and 2×10^–7 M, respectively. Except for leupeptin, all inhibitors resist hydrolysis by thermitase. Leupeptin, however, is cleaved by thermitase between the two leucylresidues. Further, a close relationship in specificity between thermitase and subtilisin BPN and their distinct discrimination from elastase specificity was demonstrated by using these inhibitors.     

Isolation and visualisation of alkali stable protein/DNA complexes

Distinct polypeptides, 54,000–68,000 daltons in size, are alkali-stably bound to eukaryotic DNA. DNA fragments several hundred base pairs in length associated with these polypeptides are preferentially retained on glass fibre filters from solutions containing 1 M sodium chloride. About 50 percent of the protein/DNA complexes present in total DNA are retained on filters together with about 2 percent of the DNA. This preferential binding is demonstrated (a) by the ratio of ³H and ³⁵S radioactivity retained on filters after filtration of DNA from [³H]thymidine and L-[³⁵S]methionine labelled cells, (b) radioiodination of the material retained on filters and passing filters respectively and (c) by electron microscopical visualisation of the polypeptide component in the complexes after chemical modification with dinitrofluorobenzene (DNFB) followed by incubation with dinitrophenyl (DNP) specific antibodies.