A comparison of the ability of opioid peptides and opiates to affect active avoidance conditioning in rats

Enkephalins reduce acquisition of an active avoidance response when administered intraperitoneally shortly before training. The present study examined whether microgram or delta opiate receptors are involved in this enkephalin effect. This was done by comparing the efficacy of micro- and delta-receptor agonists; by attempting to block the enkephalin effect with micro- and delta-receptor antagonists; and by comparing the characteristics of the effects of Met-enkephalin and Leu-enkephalin. In addition, the efficacy of kappa-agonists in reducing acquisition was assessed. It was found that micro-agonists are inactive in this assay; several delta- and kappa-agonists are active. However, not all of the data are consistent with the adequacy of this receptor classification. The micro-receptor antagonist naloxone did not readily block the effect of Met- or Leu-enkephalin but neither did the micro/delta-antagonist, diprenorphine. An additional complexity is the emergence of differences in behavioral activity of Met- snd Leu-enkephalin.     

Detection of T-DNA transfer to plant cells by A. tumefaciens virulence mutants using agroinfection

Summary To test whether virulence mutants of Agrobacterium tumefaciens are capable of promoting T-DNA transfer into plant cells, a tandem array of Cauliflower Mosaic Virus (CaMV) DNA was cloned between T-region border sequences on a wide host range plasmid and introduced into various virulence mutants. The resulting strains were used to infect Brassica rapa cv. Just Right. This assay, recently referred to as agroinfection, is based on the appearance of viral symptoms following transfer of T-DNA to plant cells, and is shown to be at least 100 times more sensitive in detecting T-DNA transfer than tumour formation. Mutants in the loci vir A, B and G, which were avirulent on turnip, failed to induce virus symptoms. Of the two vir D mutants tested, neither induced tumours, but one was capable of inducing virus symptoms. Mutants in vir E, C and F, which induced respectively no, small and normal tumours on turnip, all induced virus symptoms.     

Induced Resistance in Hypersensitive Tobacco Against Tobacco Mosaic Virus by Injection of Intercellular Fluid from Tobacco Plants with Systemic Acquired Resistance

Acquired resistance in hypersensitive tobacco plants against tobacco mosaic virus (TMV) was induced by components occurring in the intercellular fluid (IV) obtained from virus-infected plants or by plant cell wall components. Induced resistance could be transmitted through seed to the progeny. Lesion size and number were reduced significantly when the progeny was tested by TMV-inoculation. IV was extracted from the upper uninoculated leaves of four times TMV-inoculated Nicotiana tabacum cv. ‘Xanthi’ nc plants. Injection of IV from induction-inoculated plants (SAR-IV) into leaves of healthy plants followed by TMV-infection reduced lesion size significantly. A concentration of 5 × 10^?7 g SAR-IV/ml was still active. IV from healthy plants was inactive. The IV's were partly purified by gel filtration on a Sephadex G-50 column. Some fractions were active in inducing resistance as expressed in reduction of lesion size. Fractions of control-IV were inactive. It is still unknown whether the active substances in SAR-IV are in fact cell wall fragments acting as regulatory molecules in disease resistance.     

In vitro folding, purification and characterization of Escherichia coli outer membrane protease ompT.

OmpT is a protease present in the outer membrane of Escherichia coli. The enzyme was overexpressed without its signal sequence in E. coli using a T7 system, resulting in the accumulation of OmpT as inclusion bodies. After solubilization of the inclusion bodies in urea, the protein could be folded in vitro by dilution in the presence of detergent n-dodecyl-N, N-dimethyl-1-ammonio-3-propanesulphonate. The addition of lipopolysaccharide to the protein was essential to obtain active enzyme. The correctly folded protein was purified to homogeneity by ion exchange chromatography with a 57% overall yield. Autoproteolysis between Lys217-Arg218 was a major problem during purification, but degradation could be abolished by introducing the mutations G216K and K217G. A novel fluorimetric assay using the internally quenched substrate Abz-Ala-Arg-Arg-Ala-Tyr(NO2)-NH2 (where Abz is o-aminobenzoyl and Tyr(NO2) is 3-nitrotyrosine) enabled the determination of the kinetic parameters. The wild-type enzyme has an affinity Km of 0.4 microM for the substrate and a turnover number kcat of 40 s-1. The Km and kcat for the double variant were 1.1 microM and 1.6 s-1, respectively. The pH profiles of the wild type and variant were identical, showing optimal activity at pH 6.5 and pKa values of 5.6 and 7.5, respectively. Circular dichroism spectra of both enzymes indicated a high content of beta-strand conformation, and on that basis a beta-barrel topology model is proposed.     

Study of permeation and blocker binding in TMEM16A calcium-activated chloride channels

We studied the effects of mutations of positively charged amino acid residues in the pore of X. tropicalis TMEM16A calcium-activated chloride channels: K613E, K628E, K630E; R646E and R761E. The activation and deactivation kinetics were not affected, and only K613E showed a lower current density. K628E and R761E affect anion selectivity without affecting Na⁺ permeation, whereas K613E, R646E and the double mutant K613E + R646E affect anion selectivity and permeability to Na⁺. Furthermore, altered blockade by the chloride channel blockers anthracene-9-carboxylic acid (A-9-C), 4, 4''-Diisothiocyano-2,2''-stilbenedisulfonic acid (DIDS) and T16inh-A01 was observed. These results suggest the existence of 2 binding sites for anions within the pore at electrical distances of 0.3 and 0.5. These sites are also relevant for anion permeation and blockade.     

FREE AMINO ACID POOLS, 15N2 FIXATION AND FIXED N2 ASSIMILATION IN LEUCAENA LEUCOCEPHALA VAR. K-8

Various organs of Leucaena leucocephala (Lam.) de Wit were analyzed for their levels of total nitrogen and free amino acids as well as for changes in free amino acid pools from the time of germination through nodulation. Also an assessment was made of the sink of fixed N₂ (transport product) in the nodules using ¹⁵N methodology. L. leucocephala organs showed total nitrogen levels similar to those of other legumes. Asparagine was the most prevalent amino acid in the nodules and roots followed by glutamate and mimosine. Asparagine was the second most common amino acid in the leaves and stems, with mimosine being the most abundant. Strong correlations were found between the total plant levels of aspartate and glutamate, asparagine and NH₄⁺, acetylene reduction and glutamate, and asparagine and plant age. Asparagine amino- and amide-N accounted for over 75% of the fixed ¹⁵N₂ in nodules. It was concluded that L. leucocephala is an asparagine transporter of fixed N₂ in the nodule.