Conserved DNA Motifs,Including the CENP-B Box-like,Are Possible Promoters of Satellite DNA Array Rearrangements in Nematodes

Tandemly arrayed non-coding sequences or satellite DNAs (satDNAs) are rapidly evolving segments of eukaryotic genomes, including the centromere, and may raise a genetic barrier that leads to speciation. However, determinants and mechanisms of satDNA sequence dynamics are only partially understood. Sequence analyses of a library of five satDNAs common to the root-knot nematodes Meloidogyne chitwoodi and M. fallax together with a satDNA, which is specific for M. chitwoodi only revealed low sequence identity (32–64%) among them. However, despite sequence differences, two conserved motifs were recovered. One of them turned out to be highly similar to the CENP-B box of human alpha satDNA, identical in 10–12 out of 17 nucleotides. In addition, organization of nematode satDNAs was comparable to that found in alpha satDNA of human and primates, characterized by monomers concurrently arranged in simple and higher-order repeat (HOR) arrays. In contrast to alpha satDNA, phylogenetic clustering of nematode satDNA monomers extracted either from simple or from HOR array indicated frequent shuffling between these two organizational forms. Comparison of homogeneous simple arrays and complex HORs composed of different satDNAs, enabled, for the first time, the identification of conserved motifs as obligatory components of monomer junctions. This observation highlights the role of short motifs in rearrangements, even among highly divergent sequences. Two mechanisms are proposed to be involved in this process, i.e., putative transposition-related cut-and-paste insertions and/or illegitimate recombination. Possibility for involvement of the nematode CENP-B box-like sequence in the transposition-related mechanism and together with previously established similarity of the human CENP-B protein and pogo-like transposases implicate a novel role of the CENP-B box and related sequence motifs in addition to the known function in centromere protein binding.     

Tetanus Toxin and Botulinum A and C Neurotoxins Inhibit Noradrenaline Release from Cultured Mouse Brain

The specific binding protein for substance P (SP) was solubilized in an active form from the crude mitochondrial (P2) fraction of bovine brainstem. After incubation with 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) and 0.1 M NaCl at 0 degrees C for 30 min, the SP binding to the supernatant fraction (100,000 g, 60 min) was determined by the glass fiber filtration method reported by Bruns et al. (1983). The specific [3H]SP binding to the solubilized fraction was highly specific for SP and was displaced by nanomolar concentrations of SP and physalaemin, but only by micromolar concentrations of eledoisin. In addition, the binding was inhibited by GTP (approximately 40% of the specific binding decreased by 10 microM GTP) in both preparations. These results were virtually identical to those of P2 membrane preparations and suggested that this high-affinity SP binding site belongs to the SP-P type. Scatchard analyses of SP binding to the solubilized fraction revealed a single saturable component with a Bmax of 22.0 +/- 5.10 fmol/mg protein and a KD of 0.79 nM, and these values are almost the same as those obtained in the P2 fraction (Bmax = 31.3 +/- 3.56 fmol/mg protein, KD = 0.82 nM). Gel filtration analysis showed that the detergent-SP binding protein complex has two calculated molecular weights of greater than 1,000,000 and 55,000-60,000 (a corresponding Stokes radius of 35.5 nm).     

Kinetic evidence of a surface membraneous step during the endocytosic process of 3H-labelled asialoorosomucoid and its alteration in diabetic mellitus rats

The apparent internalization rate constant of asialoorosomucoid in normal and diabetic hepatocytes was determined using different experimental processes, either following a synchronous wave of prebound ligand or a continuous flux ligand endocytosis, either alone or simultaneously. In continuous flux conditions, no difference between normal and diabetic hepatocytes appeared (k = 0.15 +/- 0.04 and 0.11 +/- 0.02 min-1, respectively). In contrast, in the one-turn endocytosis of prebound ligand, k was lower for diabetic hepatocytes than for normal ones whether it was measured alone (0.20 +/- 0.03 and 0.59 +/- 0.09 min-1, respectively) or simultaneously with a continuous flux of unlabelled ligand (0.25 +/- 0.03 and 0.70 +/- 0.08 min-1, respectively). These differences are attributed to an impediment or a delay in the preclustering of receptors in coated pits at the cell surface of diabetic cells.     

Isolated hepatocytes fixed on collagen, an accurate model for the the secretion of proteins in a minimal medium. Response to inflammation

The albumin, orosomuco?d and alpha 2-macroglobulin secretion by isolated hepatocytes of normal and suffering from Turpentine-induced inflammation rats, is investigated for 4 hr. The model, stable over the whole duration of incubation, is a true reflect of hepatic secretion in vivo and can be used to measure it.     

Suppression of fungal β-glucan-induced plant defence in soybean (Glycine max L.) by cyclic 1,3-1,6-β-glucans from the symbiont Bradyrhizobium japonicum

The production of antimicrobial phytoalexins is one of the best-known inducible defence responses following microbial infection of plants or treatment with elicitors. In the legume soybean (Glycine max L.), 1,3-1,6--glucans derived from the fungal pathogen Phytophthora sojae have been identified as potent elicitors of the synthesis of the phytoalexin, glyceollin. Recently it has been reported that during symbiotic interaction between soybean and the nitrogen-fixing bacterium Bradyrhizobium japonicum USDA 110 the bacteria synthesize cyclic 1,3-1,6--glucans. Here we demonstrate that both the fungal and the bacterial -glucans are ligands of -glucan-binding sites which are putative receptors for the elicitor signal compounds in soybean roots. Whereas the fungal -glucans stimulate phytoalexin synthesis at low concentrations, the bacterial cyclic 1,3-1,6--glucans appear to be inactive even at relatively high concentrations. Competition studies indicate that increasing concentrations of the bacterial 1,3-1,6--glucans progressively inhibit stimulation of phytoalexin synthesis in a bioassay induced by the fungal 1,3-1,6--glucans. Another type of cyclic -glucan, a 1,2--glucan from Rhizobium meliloti, that does not nodulate on soybean, seems to be inactive as elicitor and as ligand of the -glucan-binding sites. These results may indicate a novel mechanism for a successful plant-symbiont interaction by suppressing the plant's defence response.Abbreviations HG-APEA 1-[2-(4-aminophenyl)ethyl]amino-l-[hexaglucosyl]deoxyglucitol - HG-AzPEA l-[2-(4-azidophenyl)-ethyl]amino-l-[hexaglucosyl]deoxyglucitol - IC₅₀ concentration for half-maximal displacement We thank Ines Arlt for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 369), the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie, Fonds der Chemischen Industrie (J.E.), and USDA CSRS NRI Competitive Research grant 93373059233 (A.A.B.).     

Stress and Age Related Spots with Immunoreactivity to Ubiquitin-Antibody at Protoplast Surfaces

Mesophyll protoplasts from primary leaves of 2, 3, and 4 weekold Viciafaba L. plants and from not expanded leaves of 2 weekold plants were incubated with rabbit anti-ubiquitin antibodyand FITC labeled goat anti-rabbit IgG. Dependent on age of theplant material, an increase in size and number of immunoreactivespots at protoplast surfaces were observed, when incubationswere performed after 16 h storage to allow protoplast to recover.A relationship between isolation stress and the intensity ofimmunolabeling was demonstrated for protoplasts from not expandedleaves. Furthermore, the surface of isolation stressed protoplastsshowed an increasing number of immunoreactive spots when plantswere previously exposed to water deficiency conditions for 1,2 or 4 days. Water deficiency conditions and isolation stressare therefore thought to induce ubiquitination of surface locatedproteins. A phenomenon, which seemed to be normally correlatedwith early events of senescence. (Received October 28, 1993; Accepted February 21, 1994)