Pseudomonas aeruginosa diaminopimelate decarboxylase: evolutionary relationship with other amino acid decarboxylases

The lysA gene encodes meso-diaminopimelate (DAP) decarboxylase (E.C.4.1.1.20), the last enzyme of the lysine biosynthetic pathway in bacteria. We have determined the nucleotide sequence of the lysA gene from Pseudomonas aeruginosa. Comparison of the deduced amino acid sequence of the lysA gene product revealed extensive similarity with the sequences of the functionally equivalent enzymes from Escherichia coli and Corynebacterium glutamicum. Even though both P. aeruginosa and E. coli are Gram-negative bacteria, sequence comparisons indicate a greater similarity between enzymes of P. aeruginosa and the Gram- positive bacterium C. glutamicum than between those of P. aeruginosa and E. coli enzymes. Comparison of DAP decarboxylase with protein sequences present in data bases revealed that bacterial DAP decarboxylases are homologous to mouse (Mus musculus) ornithine decarboxylase (E.C.4.1.1.17), the key enzyme in polyamine biosynthesis in mammals. On the other hand, no similarity was detected between DAP decarboxylases and other bacterial amino acid decarboxylases.      

Recognizing the forest for the trees: testing temporal patterns of cladogenesis using a null model of stochastic diversification

Computer simulations are developed and employed to examine the expected temporal distributions of nodes under a null model of stochastic lineage bifurcation and extinction. These Markovian models of phylogenetic process were constructed so as to permit direct comparisons against empirical phylogenetic trees generated from molecular or other information available solely from extant species. For replicate simulated phylads with n extant species, cumulative distribution functions (cdf's) of branching times were calculated, and compared (using the Kolmogorov-Smirnov test statistic D) to those from three published empirical trees. Molecular phylogenies for columbine plants and avian cranes showed statistically significant departures from the null expectations, in directions indicating recent and ancient species' radiations, respectively, whereas a molecular phylogeny for the Drosophila virilis species group showed no apparent historical clustering of branching events. Effects of outgroup choice and phylogenetic frame of reference were investigated for the columbines and found to have a predictable influence on the types of conclusions to be drawn from such analyses. To enable other investigators to statistically test for nonrandomness in temporal cladogenetic pattern in empirical trees generated from data on extant species, we present tables of mean cdf's and associated probabilities under the null model for expected branching times in phylads of varying size. The approaches developed in this report complement and extend those of other recent methods for employing null models to assess the statistical significance of pattern in evolutionary trees.      

Means for the assessment of radioligand quality and its importance in receptor-binding studies. Observations with radiolabelled formylmethionyl-leucyl-phenylalanine.

Various methods for testing the quality of radioligands were applied to two different radiolabelled forms of formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe). The purpose of the study was both to examine the value of these methods for assessing radioligand quality and to determine the suitability of these particular radioligands for studying the chemotactic formylpeptide receptors on the rabbit neutrophil. It is useful in this context to distinguish two different aspects of radioligand quality: these are purity and equivalence to the native ligand. The two methods described for measuring receptor-reactivity (or 'bindability'), by measuring binding to an increasing excess of receptors and by a re-incubation procedure, provide a reliable measure of purity that should readily be applicable to other radioligands. Equivalence to the native ligand is more difficult to establish, and any uncertainty about the specific radioactivity of the radioligand can pose serious problems with this assessment. Commercial preparations of both tritiated and 35S-labelled fMet-Leu-Phe were found to be inadequately pure for detailed receptor studies. Repurification by t.l.c., however, consistently yielded radioligand preparations of high purity and close equivalence to the native ligand. Other radioligands may often also require a suitable repurification step before use for detailed receptor studies; this is especially important whenever a complex receptor-binding pattern is envisaged.     

Production of active human glucocerebrosidase in seeds of Arabidopsis thaliana complex-glycan-deficient (cgl) plants

There is a clear need for efficient methods to produce protein therapeutics requiring mannose-termination for therapeutic efficacy. Here we report on a unique system for production of active human lysosomal acid β-glucosidase (glucocerebrosidase, GCase, EC 3.2.1.45) using seeds of the Arabidopsis thaliana complex-glycan-deficient (cgl) mutant, which are deficient in the activity of N-acetylglucosaminyl transferase I (EC 2.4.1.101). Gaucher disease is a prevalent lysosomal storage disease in which affected individuals inherit mutations in the gene (GBA1) encoding GCase. A gene cassette optimized for seed expression was used to generate the human enzyme in seeds of the cgl (C5) mutant, and the recombinant GCase was mainly accumulated in the apoplast. Importantly, the enzymatic properties including kinetic parameters, half-maximal inhibitory concentration of isofagomine and thermal stability of the cgl-derived GCase were comparable with those of imiglucerase, a commercially available recombinant human GCase used for enzyme replacement therapy in Gaucher patients. N-glycan structural analyses of recombinant cgl-GCase showed that the majority of the N-glycans (97%) were mannose terminated. Additional purification was required to remove ～15% of the plant-derived recombinant GCase that possessed potentially immunogenic (xylose- and/or fucose-containing) N-glycans. Uptake of cgl-derived GCase by mouse macrophages was similar to that of imiglucerase. The cgl seed system requires no addition of foreign (non-native) amino acids to the mature recombinant GCase protein, and the dry transgenic seeds represent a stable repository of the therapeutic protein. Other strategies that may completely prevent plant-like complex N-glycans are discussed, including the use of a null cgl mutant.     

Amino acid size, charge, hydropathy indices and matrices for protein structure analysis

Background  

Prediction of protein folding and specific interactions from only the sequence (ab initio) is a major challenge in bioinformatics. It is believed that such prediction will prove possible if Anfinsen's thermodynamic principle is correct for all kinds of proteins, and all the information necessary to form a concrete 3D structure is indeed present in the sequence.     

Analyses to determine the role of the megagametophyte and other seed tissues in dormancy maintenance of yellow cedar (Chamaecyparis nootkatensis) seeds; morphological, cellular and physiological changes following moist chilling and during germination

Yellow cedar (Chamaecyparis nootkatensis) seeds exhibit prolonged dormancy following their dispersal from the parent plant. Embryos excised fully from their enclosing seed tissues exhibit 100% germination, indicating that the seed tissues enclosing the embryo (the testa, remnants of the nucellus and the megagametophyte) play an inhibitory role and prevent radicle emergence. As part of an assessment of the role of seed tissues in the dormancy mechanism of yellow cedar seeds, light microscopy was used to examine changes within the major structures of the seed following a 90 d war (26C)/cold (4C) moist treatment ('stratification') and during germination. In the micropylar tip of the seed, the nucellus forms a hard nucellar cap covering the radicle. The nucellar cap is composed primarily of degenerated cells; histological staining with ruthenium red revealed a predominance of pectins. There were no obvious cellular or morphological differences (detected by light microscopy) between mature seeds subjected to a 3 d soak and seeds subjected to a 3 d soak and the 90 d dormancy-breaking treatment. However, just prior to germination there was an outward projection of the nucellar cap through the micropyle, which appeared to be caused by the extension of highly folded proteinaceous strands lying immediately in front of the radicle. When the testa was removed, the embryo enclosed within the intact megagametophyte was incapable of germination. If, however, the megagametophyte surrounding the embryo was slit or the embryo surrounded by an intact megagametophyte was subjected to a 3d rinse in water, some germination occurred, perhaps as a result of an enhanced release of inhibitors from the megagametophyte. After stratification, dormancy of yellow cedar seeds is broken; concurrent with dormancy breakage, there was a mechanical weakening of the megagametophyte. The embryo also underwent changes that included an increase in turgor and a reduced sensitivity to highly negative osmotic potential. It is concluded that coat-imposed dormancy of yellow cedar seeds is enforced by mechanical restraint of the megagametophyte as well as a leachable chemical inhibitor (most probably ABA).     

Measuring the impact of Leptoglossus occidentalis (Heteroptera: Coreidae) on seed production in lodgepole pine using an antibody-based assay

We measured the impact of Leptoglossus occidentalis on seed production in lodgepole pine, Pinus contorta variety latifolia Engelmann, using an antibody marker developed to detect residual saliva in fed-on seeds. Nymphs, adult females, and adult males were caged on cones during early, mid- and late season cone development. Individual analysis of 12,887 seeds extracted from 365 cones revealed that 37.3% seeds tested positive for seed bug saliva. The antibody assay was 38 times more effective than radiography at detecting seed bug damage. Radiography can detect partially emptied seed but cannot discriminate between aborted seeds and those emptied by seed bugs. The antibody marker was least sensitive in detecting early season damage compared with mid- and late season damage. We hypothesize that residual saliva in seeds fed on early in the season was either absorbed by the damaged seed or degraded over time. Early season feeding resulted in the greatest number of seeds fused to cone scales and the extraction efficiency for cones exposed to feeding during this time was reduced by 64% compared with control cones. Adding fused seeds to antibody-positive seeds raised the proportion of damaged seeds to 48.3%. At all stages of cone development, adult females were the most destructive life stage, damaging up to two seeds per day late in the season. When seed losses were adjusted to damage per degree-day, female damage was greatest early in the season, while males caused the same amount of damage regardless of cone development period. The results of the antibody assay provide baseline data for developing damage prediction formulae, and establish L. occidentalis as a potentially serious pest in lodgepole pine seed orchards.