Development of Cuscuta species on a partially incompatible host: induction of xylem transfer cells

Summary.  The growth of dodders, Cuscuta reflexa and Cuscuta japonica, on the partially incompatible host poinsettia (Euphorbia pulcherrima) is studied. Poinsettia responds by bark growths to the formation of the dodder haustoria and prevents dodder from obtaining normal growth. The growth instead becomes extremely branched, coral-like, and dodder lacks the ability to form haustoria. After a period of coral-like growth, long shoots sprout, resembling the normal growth. These long shoots mark an ending phase for dodder, which dies shortly after without having flowered. During the coral-like growth phase, dodder develops transfer cells in the parenchyma cells bordering the vessels of the xylem in the shoot. The transfer cells have not been observed when dodder is grown on the compatible host Pelargonium zonale. A coral-like growth phase has also been observed at the establishing phase when dodder is grown in vitro on agar; later a more normal growth form takes over. In this coral phase, xylem transfer cells are also developed. The fluorochromes carboxyfluorescein and Texas Red were loaded into the host in the phloem and xylem, respectively, and detection of these fluorochromes in the dodder stem indicated that a functional haustorial contact developed for both vascular systems. The results show that Cuscuta spp. have the genetic ability to develop xylem transfer cells and use this in response to developmental stress. Received June 12, 2002; accepted August 26, 2002; published online March 11, 2003     

Visualization of the target-membrane-inserted fusion protein of Semliki Forest virus by combined electron microscopy and crystallography

Semliki Forest virus enters cells by receptor-mediated endocytosis. The acidic environment of the endosome triggers a membrane fusion reaction that is mediated by the E1 glycoprotein. During fusion, E1 rearranges from an E1/E2 heterodimer to a highly stable, membrane-inserted E1 homotrimer (E1HT). In this study, we analyzed E1HT by a combination of electron cryomicroscopy, electron crystallography of negatively stained 2D crystals, and fitting of the available X-ray structure of the monomeric E1 ectodomain into the resulting 3D reconstruction. The visualized E1HT reveals that the ectodomain has reoriented vertically and inserted the distal tip of domain II into the lipid bilayer. Our data allow the visualization of a viral fusion protein inserted in its target membrane and demonstrate that insertion is a cooperative process, resulting in rings composed of five to six homotrimers.     

Effect of bradykinin analogues on the B1 receptor of rat ileum

The longitudinal muscle of isolated rat ileum is a sensitive bioassay suitable for testing compounds with antagonistic effects on the B(1) receptor. Bradykinin analogues with replacement of proline by alkyl-substituted phenylalanine at position 7 are effective on this receptor as entire molecules and have a stronger antagonistic effect than on the B(2) receptor. A corresponding desArg(9)-compound has a specific effect on the B(1) receptor and a very high antagonistic potency. [LNMPhe(2)]bradykinin as a compound without any replacement at position 7 or 8 shows antagonistic activity as well.     

Sequence, distribution and quantification of the motilin precursor in the cat

Due to motilin's relation to the migrating motor complex (MMC), the physiology of motilin has been mostly studied in man and dog. The cat does not have an MMC pattern, and little is known about cat motilin. Therefore we identified the cat motilin precursor (GenBank accession no. AF127917) and developed a quantitative polymerase chain reaction (PCR) to explore its distribution in the gastrointestinal tract and in the central nervous system (CNS). The precursor is closely related to the dog precursor and consists of an open reading frame of 348bp encoding the signal peptide (25 amino acids), the motilin sequence (22 amino acids) and the motilin associated peptide (69 amino acids). One amino acid of the signal peptide was subject to gene polymorphism. Quantification of motilin messenger RNA (mRNA) was for the first time achieved. It is most abundant in the gastrointestinal tract, with the highest concentration in the duodenum, the lowest in the colon and is not detectable in the corpus. However an important expression was also observed in several regions of the CNS, except the striatum and cerebral cortex. The highest level was in the hypothalamus (although 23-fold lower than in the duodenum), the lowest level in the pons. Moderate levels were found in the thyroid. These data suggest that the physiological role of motilin may extend beyond its effect on gastrointestinal motility.     

Apparently normal tumor necrosis factor receptor 1 signaling in the absence of the silencer of death domains

The silencer of death domains (SODD) has been proposed to prevent constitutive signaling of tumor necrosis factor receptor 1 (TNFR1) in the absence of ligand. Besides TNFR1, death receptor 3 (DR3), Hsp70/Hsc70, and Bcl-2 have been characterized as binding partners of SODD. In order to investigate the in vivo role of SODD, we generated mice congenitally deficient in expression of the sodd gene. No spontaneous inflammatory infiltrations were observed in any organ of these mice. Consistent with this finding, in the absence of SODD no alteration in the activation patterns of nuclear factor kappaB (NF-kappaB), stress kinases, or ERK1 or -2 was observed after stimulation with tumor necrosis factor (TNF). Activation of NF-kappaB by DR3 was also unchanged. The extents of DR3- and TNF-induced apoptosis were comparable in gene-deficient and wild-type cells. Protection of cells against heat shock as mediated by the Hsp70 system and against staurosporine-induced apoptosis was independent of SODD. Furthermore, resistance to high-dose lipopolysaccharide (LPS) injections, LPS-D-GalN injections, and infection with listeriae was similar in wild-type and gene-deficient mice. In conclusion, our data do not support the concept of a unique, nonredundant role of SODD for the functions of TNFR1, Hsp70, and DR3.     

The Gap1 general amino acid permease acts as an amino acid sensor for activation of protein kinase A targets in the yeast Saccharomyces cerevisiae

Addition of a nitrogen source to yeast (Saccharomyces cerevisiae) cells starved for nitrogen on a glucose-containing medium triggers activation of protein kinase A (PKA) targets through a pathway that requires for sustained activation both a fermentable carbon source and a complete growth medium (fermentable growth medium induced or FGM pathway). Trehalase is activated, trehalose and glycogen content as well as heat resistance drop rapidly, STRE-controlled genes are repressed, and ribosomal protein genes are induced. We show that the rapid effect of amino acids on these targets specifically requires the general amino acid permease Gap1. In the gap1Delta strain, transport of high concentrations of l-citrulline occurs at a high rate but without activation of trehalase. Metabolism of the amino acids is not required. Point mutants in Gap1 with reduced or deficient transport also showed reduced or deficient signalling. However, two mutations, S391A and S397A, were identified with a differential effect on transport and signalling for l-glutamate and l-citrulline. Specific truncations of the C-terminus of Gap1 (e.g. last 14 or 26 amino acids) did not reduce transport activity but caused the same phenotype as in strains with constitutively high PKA activity also during growth with ammonium as sole nitrogen source. The overactive PKA phenotype was abolished by mutations in the Tpk1 or Tpk2 catalytic subunits. We conclude that Gap1 acts as an amino acid sensor for rapid activation of the FGM signalling pathway which controls the PKA targets, that transport through Gap1 is connected to signalling and that specific truncations of the C-terminus result in permanently activating Gap1 alleles.     

Humic substances in drinking water and the epidemiology of thyroid disease

Thyroid diseases are common in all populations but the type and frequency depends on environmental factors. In Denmark geographical differences in iodine intake are caused by different iodine contents of drinking water, which varies from < 1 to 139 microg iodine per litre. Comparative epidemiologic studies have demonstrated considerable differences in type and occurrence of thyroid disease with more goitre and hyperthyroidism in Aalborg with water iodine content around 5 microg/L, and more hypothyroidism in Copenhagen with water iodine around 20 microg/L. In Denmark, iodine in ground water is bound in humic substances, which have probably leached from marine sediments in the aquifers. Interestingly, humic substances in water from other parts of the world have goitrogenic properties, especially humic substances from coal and shale. Humic substances are heterogeneous mixtures of naturally occurring molecules, produced by decomposition of plant and animal tissues. The effect of humic substances in drinking water on the epidemiology of thyroid disease probably depends on the source of aquifer sediments.     

Analysis of the CC chemokine receptor 5 (CCR5) delta-32 polymorphism in inflammatory bowel disease

The inflammatory bowel diseases (IBD) Crohn's disease (CD) and ulcerative colitis (UC) are complex multifactorial traits involving both environmental and genetic factors. Recent studies have shown the important role of pro-inflammatory cytokines and chemokines, including RANTES, in IBD. RANTES is the natural ligand for the CC-chemokine receptor 5 (CCR5). The chromosomal location of the CCR5 gene on 3p21 coincides with an IBD-susceptibility locus identified by genome-wide scanning. A 32-bp deletion (A32) in the CCR5 gene results in a nonfunctional receptor and is found with high frequency in Caucasians. In this study, we investigated the presence of the CCR5delta32 allele in a large cohort of IBD patients and in a healthy control population. Blood samples were obtained from 538 unselected IBD cases (433 unrelated IBD patients: 289 CD, 142 UC, 2 indeterminate colitis; 105 affected first-degree relatives) and 135 unaffected first-degree family members. Of the IBD patients, 36% had familial IBD with at least two members being affected. There were no significant differences in the CCR5delta32 mutation frequency between IBD patients and healthy controls, nor between CD and UC patients. There was no correlation between the CCR5delta32 genotype and the age at IBD-diagnosis, the frequency of surgical intervention, or disease localization. Only the association between CCR5delta32 homozygosity and the presence of anal lesions in CD patients was statistically significant (P=0.007). Analysis by the transmission/disequilibrium test showed no significant transmission distortion to the probands or their clinically silent siblings. Based on these results, it is unlikely that the CCR5delta32 allele is an important marker for predisposition to IBD.     

Two pregnancies after preimplantation genetic diagnosis for osteogenesis imperfecta type I and type IV

Osteogenesis imperfecta (OI) is an autosomal dominant genetic disorder characterized by the presence of brittle bones and decreased bone mass (osteopenia), as a result of mutations in the genes that encode the chains of type I collagen, the major protein of bone. The clinical features of the disease range from death in the perinatal period to normal life span with minimal increase in fractures. The present report describes two polymerase chain reaction (PCR)-based assays allowing preimplantation genetic diagnosis (PGD) on the one hand for OI type I, the mildest form, and on the other hand for OI type IV, which is intermediate in severity between OI type I and OI type III. In the couple referred for PGD for OI type I, the female partner carried a 1-bp deletion in exon 43 of the COL1A1 gene, resulting in a premature stop codon in exon 46. The synthesis of too little type I procollagen results from such a non-functional or COL1A1 null allele. In the other couple, referred for PGD for OI type IV, the male partner carried a G to A substitution in exon 19 of the COL1A2 gene, which results in an abnormal gene product due to an alphaGly247 (GGT) to Ser (AGT) substitution (G247S). Both mutations result in the loss of a specific restriction enzyme recognition site and can therefore be detected by PCR amplification followed by restriction fragment analysis. PCR amplification of genomic DNA of the parents-to-be with one of the two primers fluorescently labelled, followed by automated laser fluorescence (ALF) gel electrophoresis of the amplified and restricted fragments, allowed a distinction between the healthy and affected genotypes. PCR on single Epstein-Barr-virus (EBV)-transformed lymphoblasts resulted in acceptable amplification efficiencies (87% and 85% for OI type I and OI type IV respectively) and the allele drop-out (ADO) rate was assessed at 11.5% and 11.1% for OI type I and OI type IV respectively. With research blastomeres, 100% amplification rates were obtained and no contamination was observed in the blank controls, which validated the tests for clinical application. Embryos obtained after intracytoplasmic sperm injection (ICSI) were evaluated for the presence of the normal genotype of the non-affected parent. For OI type I, two frozen-thawed ICSI-PGD cycles and two fresh ICSI-PGD cycles were carried out for the same couple. The transfer of two unaffected embryos in the last cycle resulted in a twin pregnancy. A twin pregnancy was also achieved in one clinical ICSI-PGD cycle for OI type IV.     

The Rhizobium etli rpoN Locus: DNA Sequence Analysis and Phenotypical Characterization of rpoN, ptsN, and ptsA Mutants

The rpoN region of Rhizobium etli was isolated by using the Bradyrhizobium japonicum rpoN1 gene as a probe. Nucleotide sequence analysis of a 5,600-bp DNA fragment of this region revealed the presence of four complete open reading frames (ORFs), ORF258, rpoN, ORF191, and ptsN, coding for proteins of 258, 520, 191, and 154 amino acids, respectively. The gene product of ORF258 is homologous to members of the ATP-binding cassette-type permeases. ORF191 and ptsN are homologous to conserved ORFs found downstream from rpoN genes in other bacterial species. Unlike in most other microorganisms, rpoN and ORF191 are separated by approximately 1.6 kb. The R. etli rpoN gene was shown to control in free-living conditions the production of melanin, the activation of nifH, and the metabolism of C₄-dicarboxylic acids and several nitrogen sources (ammonium, nitrate, alanine, and serine). Expression of the rpoN gene was negatively autoregulated and occurred independently of the nitrogen source. Inactivation of the ptsN gene resulted in a decrease of melanin synthesis and nifH expression. In a search for additional genes controlling the synthesis of melanin, an R. etli mutant carrying a Tn5 insertion in ptsA, a gene homologous to the Escherichia coli gene coding for enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system, was obtained. The R. etli ptsA mutant also displayed reduced expression of nifH. The ptsN and ptsA mutants also displayed increased sensitivity to the toxic effects of malate and succinate. Growth of both mutants was inhibited by these C₄-dicarboxylates at 20 mM at pH 7.0, while wild-type cells grow normally under these conditions. The effect of malate occurred independently of the nitrogen source used. Growth inhibition was decreased by lowering the pH of the growth medium. These results suggest that ptsN and ptsA are part of the same regulatory cascade, the inactivation of which renders the cells sensitive to toxic effects of elevated concentrations of malate or succinate.