Medicago truncatula,a model plant for studying the molecular genetics of theRhizobium-legume symbiosis

Medicago truncatula has all the characteristics required for a concerted analysis of nitrogen-fixing symbiosis withRhizobium using the tools of molecular biology, cellular biology and genetics.M. truncatula is a diploid and autogamous plant has a relatively small genome, and preliminary molecular analysis suggests that allelic heterozygosity is minimal compared with the cross-fertilising tetraploid alfalfa (Medicago sativa). TheM. truncatula cultivar Jemalong is nodulated by theRhizobium meliloti strain 2011, which has already served to define many of the bacterial genes involved in symbiosis with alfalfa. A genotype of Jemalong has been identified which can be regenerated after transformation byAgrobacterium, thus allowing the analysis ofin-vitro-modified genes in an homologous transgenic system. Finally, by virtue of the diploid, self-fertilising and genetically homogeneous character ofM. truncatula, it should be relatively straightforward to screen for recessive mutations in symbiotic genes, to carry out genetic analysis, and to construct an RFLP map for this plant.     

Determination of three-dimensional structures of proteins by simulated annealing with interproton distance restraints. Application to crambin, potato carboxypeptidase inhibitor and barley serine proteinase inhibitor 2

An automated method, based on the principle of simulated annealing, is presented for determining the three-dimensional structures of proteins on the basis of short (less than 5 A) interproton distance data derived from nuclear Overhauser enhancement (NOE) measurements. The method makes use of Newton's equations of motion to increase temporarily the temperature of the system in order to search for the global minimum region of a target function comprising purely geometric restraints. These consist of interproton distances supplemented by bond lengths, bond angles, planes and soft van der Waals repulsion terms. The latter replace the dihedral, van der Waals, electrostatic and hydrogen-bonding potentials of the empirical energy function used in molecular dynamics simulations. The method presented involves the implementation of a number of innovations over our previous restrained molecular dynamics approach [Clore, G.M., Brünger, A.T., Karplus, M. and Gronenborn, A.M. (1986) J. Mol. Biol., 191, 523-551]. These include the development of a new effective potential for the interproton distance restraints whose functional form is dependent on the magnitude of the difference between calculated and target values, and the design and implementation of robust and fully automatic protocol. The method is tested on three systems: the model system crambin (46 residues) using X-ray structure derived interproton distance restraints, and potato carboxypeptidase inhibitor (CPI; 39 residues) and barley serine proteinase inhibitor 2 (BSPI-2; 64 residues) using experimentally derived interproton distance restraints. Calculations were carried out starting from the extended strands which had atomic r.m.s. differences of 57, 38 and 33 A with respect to the crystal structures of BSPI-2, crambin and CPI respectively. Unbiased sampling of the conformational space consistent with the restraints was achieved by varying the random number seed used to assign the initial velocities. This ensures that the different trajectories diverge during the early stages of the simulations and only converge later as more and more interproton distance restraints are satisfied. The average backbone atomic r.m.s. difference between the converged structures is 2.2 +/- 0.3 A for crambin (nine structures), 2.4 +/- 0.3 A for CPI (eight structures) and 2.5 +/- 0.2 A for BSPI-2 (five structures). The backbone atomic r.m.s. difference between the mean structures derived by averaging the coordinates of the converged structures and the corresponding X-ray structures is 1.2 A for crambin, 1.6 A for CPI and 1.7 A for BSPI-2.     

Influence of hirudin on the consumption of antithrombin III in experimental DIC

The effect of hirudin and heparin on thrombin-induced consumption of antithrombin III, fibrinogen and platelets was studied in a rat model. Antithrombin III is consumed by tolerated thrombin doses by about 20 per cent. Hirudin and heparin ameliorate the consumption of fibrinogen and platelets at the low thrombin dose used. At high thrombin doses, tolerated only during simultaneous administration of exogenous inhibitors, heparin leads to markedly increased consumption of anti-thrombin III, whereas hirudin does not. With either kind of treatment, the thrombin effect on fibrinogen and platelets is inhibited, however, hirudin acts independently of a cofactor in contrast to heparin.     

Formation of the Neurotransmitter Glycine from the Anticonvulsant Milacemide Is Mediated by Brain Monoamine Oxidase B

Milacemide (2-n-pentylaminoacetamide) is a secondary monoamine that in the brain is converted to glycinamide and glycine. This oxidative reaction was suspected to involve the reaction of monoamine oxidase (MAO). Using mitochondrial preparations from tissues that contain MAO-A and -B (rat brain and liver), MAO-A (human placenta), and MAO-B (human platelet and bovine adrenal chromaffin cell), it has been established that mitochondria containing MAO-B rather than MAO-A oxidize (H2O2 production and glycinamide formation) milacemide. The apparent Km (30-90 microM) for milacemide oxidation by mitochondrial MAO-B preparations is significantly lower than that for milacemide oxidation by mitochondrial MAO-A (approximately 1,300 microM). In vitro MAO-B (l-deprenyl and AGN 1135) rather than MAO-A (clorgyline) selectively inhibited the oxidation of milacemide. These in vitro data are matched by ex vivo experiments where milacemide oxidation was compared to oxidation of serotonin (MAO-A) and beta-phenylethylamine (MAO-B) by brain mitochondria prepared from rats pretreated with clorgyline (0.5-10 mg/kg) and l-deprenyl (0.5-10 mg/kg). Furthermore, in vivo experiment demonstrated that l-deprenyl selectively increased the urinary excretion of [14C]milacemide and the total radioactivity with a concomitant decrease of [14C]glycinamide. Such changes were not observed after clorgyline treatment, but were evident only at doses beyond clorgyline selectivity. The present data therefore demonstrate that milacemide is a substrate for brain MAO-B, and its conversion to glycinamide, further transformed to the inhibitory neurotransmitter, glycine, mediated by this enzyme may contribute to its pharmacological activities.     

Effect of transferrin on amphibian limb regeneration: a blastema cell culture study

Summary In order to study mitogenic control during axolotl limb regeneration, we have developed a primary blastema cell culture as a very sensitive bioassay for blastema mitogens. Transferrin, an iron-binding glycoprotein which has been shown to be the neurotrophic factor for muscle cells, is the mitogen which has been analysed in the present report. Addition of approximately 2 g human transferrin/ ml of serum-free culture medium enhances blastema cell proliferation 11-fold over control levels and 2-fold over that produced by the addition of nerve extracts or purified growth factors extracted from nerve tissues (basic and acidic fetal growth factor, FGF). At a higher concentration (20 g/ml), transferrin alone has no mitogenic effect unless the medium is also supplemented with FeCl₃ (100 M). The results are discussed with regard to the sensitivity of the blastema cell culture bioassay and in the context of the neurotrophic theory of urodele limb regeneration.     

Restriction map construction using a 'complete sentences compatibility' algorithm

We have developed a new algorithm ‘Complete sentencescompatibility’ (CSC) which uses single and double digestionfragments to rapidly determine restriction maps of circularDNA. From possible combinations of fragments of each simpledigestion, which we call ‘sentences of decomposition’,we construct a restriction map which combines the sentenceswhile taking into account compatibility rules. The algorithmcan also deal with experimental errors of fragment weight andcan suggest solutions that account for non-readable bands (fragmentsof zero length or multiple bands) on the gel. Because experimentsusing pairs of restrictive enzymes often result in multiplesolutions, a complementary algorithm tries to reduce the numberof proposed solutions by establishing consensus maps. The restrictionmap construction algorithm was tested on real cases, some containingmore than fifteen fragments. Execution times range from 1 –10 s on an IBM PC compatible microcomputer. Received on July 21, 1987; accepted on December 31, 1987