Autoregulation of root nodule formation: signals of both symbiotic partners studied in a split-root system of Vicia sativa subsp. nigra

Inhibition of root nodule formation on leguminous plants by already induced or existing root nodules is called autoregulation of root nodule formation (AUT). Optimal conditions for AUT were determined using a split-root technique newly developed for Vicia sativa subsp. nigra. Infection of a root A with nodulating Rhizobium leguminosarum bv. viciae bacteria systemically inhibited nodulation of a spatially separated root B inoculated 2 days later with the same bacteria. This treatment gives complete AUT (total absence of nodules on root B). Only partial AUT of root B was obtained by incubation of root A with mitogenic nodulation (Nod) factors or with a noninfective strain producing normal mitogenic Nod factors. Nonmitogenic Nod factors did not evoke AUT. We identified two systemic plant signals induced by Rhizobium bacteria. Signal 1 (at weak buffering) was correlated with sink formation in root A and induced acidification of B-root medium. This signal is induced by treatment of root A with (i) nodulating rhizobia, (ii) mitogenic Nod factors, (iii) nonmitogenic Nod factors, or (iv) the cytokinin zeatin. Signal 2 (at strong buffering) could only be evoked by treatment with nodulating rhizobia or with mitogenic Nod factors. Most probably, this signal represents the specific AUT signal. Induction of complete AUT appears to require actively dividing nodule cells in nodule primordia, nodule meristems, or both of root A.     

Peptide neurotoxins, small-cell lung carcinoma andneurological paraneoplastic syndromes

Peptide neurotoxins isolated from the venom of snakes, spiders and snails have represented invaluable tools for the identification and characterisation of membrane ion channels and receptors in vertebrate cells, including human neurons. We here report on the use of these toxins for the characterisation of membrane ion channels and receptors expressed by one of the most aggressive human cancers, small-cell lung carcinoma. This tumour shares many properties with other neuro-endocrine cell types, including the ability of firing action potentials and release hormones in a calcium-dependent manner. Toxins such as alpha-bungarotoxin and omega-conotoxins, among others, have been successfully used to characterise neuronal nicotinic receptors and voltage-dependent calcium channels, respectively, in human small-cell lung carcinoma cells. These receptors and ion channels are not only crucial for the growth of this specific tumour, but also represent autoantigens against which cancer patients build an autoimmune response. Although the aim of this autoimmune response is eventually the destruction of the cancer cells, the circulating antibodies cross-react with similar ion channels and receptors present in normal neurons or other cells, causing a number of different paraneoplastic diseases, the best characterised of which is the Lambert-Eaton myasthenic syndrome. Conotoxin-based radioimmunoassays have become an invaluable tool for the diagnosis and follow up of these paraneoplastic disorders and could represent a step forward in the early diagnosis of small-cell lung carcinoma itself.     

Identification of bacterial strains by laboratories participating in the Deutsches Krebsforschungszentrum quality assurance programme

The quality assurance programme (QAP) of the Deutsches Krebsforschungszentrum (DKFZ) is a proficiency testing system developed to service the laboratory animal discipline. QAP comprises the quarterly distribution of two bacterial strains originating from various species of animals for identification to the species level and antibiotic susceptibility testing. We compared identification results reported by QAP participants over the years 1996-2004 with those obtained by the Dutch Bacterial Diagnostics reference laboratory on 68 samples comprising 71 bacterial strains and a fungus. Significant differences were found in the frequency of reported and correct identifications when bacteria were assigned to different groups based on morphology by Gram stain and on origin (animal versus environmental, rodent and rabbit versus other animal species, pathogen versus non-pathogens). Rodent and rabbit pathogens yielded 73% correct identifications, and with all bacterial strains only 60% of the identifications were correct. We assume that most QAP participants were from laboratory animal diagnostic laboratories. If this is true, the capabilities of laboratories in the laboratory animal discipline to correctly identify bacterial species are well below what are considered acceptable limits for human diagnostic laboratories. The distribution of cultured bacteria circumvents the most difficult step in the microbiological monitoring of animals, namely primary culture from clinical samples. We propose to set up a QAP that comprises the distribution of specimens mimicking clinical samples normally submitted to laboratory animal diagnostic laboratories.     

Promoters of auxin-induced genes from tobacco can lead to auxin-inducible and root tip-specific expression

In previous studies we have identified several mRNAs which accumulate after addition of 2,4-dichlorophenoxyacetic-acid (2,4-D) to auxin-starved tobacco cells [45, 46]. The mRNAs corresponding to cDNA clone pCNT103 were found to accumulate transiently prior to the cell division response due to auxin treatment. In this study we determined the sequences of three 103-like cDNAs and two 103-like genes, GNT1 and GNT35. To further study the regulation of the expression of these genes their 5 regions were translationally fused with the -D-glucuronidase reporter gene (GUS). The GNT1 5 region led to GUS expression only in the root tips of transgenic plants. By using transgenic hairy-root cultures and transformed cell suspension cultures it was shown that the 5 regions of both GNT1 and GNT35 lead to 2,4-D-inducible expression of GUS activity. The homology of the 103-like genes with other auxin-regulated genes is evaluated.Department of Plant Molecular Biology, Leiden University     

Identification of DNA sequence variation in Campylobacter jejuni strains associated with the Guillain-Barré syndrome by high-throughput AFLP analysis

Background  

Campylobacter jejuni is the predominant cause of antecedent infection in post-infectious neuropathies such as the Guillain-Barré (GBS) and Miller Fisher syndromes (MFS). GBS and MFS are probably induced by molecular mimicry between human gangliosides and bacterial lipo-oligosaccharides (LOS). This study describes a new C. jejuni-specific high-throughput AFLP (htAFLP) approach for detection and identification of DNA polymorphism, in general, and of putative GBS/MFS-markers, in particular.     

Engineering dehydrated amino acid residues in the antimicrobial peptide nisin.

The small antimicrobial peptide nisin, produced by Lactococcus lactis, contains the uncommon amino acid residues dehydroalanine and dehydrobutyrine and five thio ether bridges. Since these structures are posttranslationally formed from Ser, Thr, and Cys residues, it is feasible to study their role in nisin function and biosynthesis by protein engineering. Here we report the development of an expression system for mutated nisin Z (nisZ) genes, using nisin A producing L. lactis as a host. Replacement by site-directed mutagenesis of the Ser-5 codon in nisZ by a Thr codon, led to a mutant with a dehydrobutyrine instead of a dehydroalanine residue at position 5, as shown by NMR. Its antimicrobial activity was 2-10-fold lower relative to wild-type nisin Z, depending on the indicator strain used. In another mutagenesis study a double mutation was introduced in the nisZ gene by replacing the codons for Met-17 and Gly-18 by codons for Gln and Thr, respectively, as in the third lanthionine ring of the related antimicrobial peptide subtilin from Bacillus subtilis. This resulted in the simultaneous production of two mutant species, one containing a Thr residue and the other containing a dehydrobutyrine residue at position 18, both having different bacteriocidal properties.     

Opportunistic infections in hysterectomy-derived, barrier-maintained guinea pigs

Guineapigs were rederived by hysterectomy and associated with a colonization-resistant enteric microflora (CRF) of mouse origin to establish a strict barrier-maintained breeding colony. Infections were suspected in 55 of 209 animals autopsied. From 65 of 67 tissue samples selected for bacteriological examination 167 isolates, belonging to 16 usually non-pathogenic bacterial species or groups, were isolated mostly from cases of mastitis, typhlitis, enteritis, otitis media and pneumonia. Faecal streptococci, Pseudomonas aeruginosa, Clostridium perfringens, Citrobacter spp., C. freundii, Escherichia coli, Klebsiella oxytoca and Enterobacter cloacae accounted for 86.8% of all isolates. Variations in the recovery rate of these 8 species during the study and a gradual decline in the incidence of infections were observed. Elimination by rederivation of the autochthonous bacterial flora seemed to have predisposed to opportunistic infections by aerobic and facultatively anaerobic bacteria. Enteric colonization resistance to the growth of these bacteria was presumably insufficiently provided by the CRF of mouse origin and might be remedied by using a flora of guineapig origin.