The consensus concept for thermostability engineering of proteins: further proof of concept

Previously, we calculated a consensus amino acid sequence from 13 homologous fungal phytases. A synthetic gene was constructed and recombinantly expressed. Surprisingly, consensus phytase-1 was 15-26 degrees C more thermostable than all parent phytases used in its design [Lehmann et al. (2000)Protein Eng., 13, 49-57]. In the present study, inclusion of six further phytase sequences in the amino acid sequence alignment resulted in the replacement of 38 amino acid residues in either one or both of the new consensus phytases-10 and -11. Since consensus phytase-10, again, was 7.4 degrees C more thermostable than consensus phytase-1, the thermostability effects of most of the 38 amino acid substitutions were tested by site-directed mutagenesis. Both stabilizing and destabilizing mutations were identified, but all affected the stability of the enzyme by <3 degrees C. The combination of all stabilizing amino acid exchanges in a multiple mutant of consensus phytase-1 increased the unfolding temperature from 78.0 to 88.5 degrees C. Likewise, back-mutation of four destabilizing amino acids and introduction of an additional stabilizing amino acid in consensus phytase-10 further increased the unfolding temperature from 85.4 to 90.4 degrees C. The thermostabilization achieved is the result of a combination of slight improvements from multiple amino acid exchanges rather than being the effect of a single or of just a few dominating mutations that have been introduced by chance. The present findings support the general validity of the consensus concept for thermostability engineering of proteins.     

Neoformation of clay in lateral root catchments of mallee eucalypts: a chemical perspective

Background and Aims

A previous paper (Annals of Botany 103: 673–685) described formation of clayey pavements in lateral root catchments of eucalypts colonizing a recently formed sand dune in south-west Western Australia. Here chemical and morphological aspects of their formation at the site are studied.

Methods

Chemical and physical examinations of soil cores through pavements and sand under adjacent heath assessed build-up of salts, clay and pH changes in or below pavements. Relationships of root morphology to clay deposition were examined and deposits subjected to scanning electron microscopy and energy-dispersive X-ray analysis. Xylem transport of mineral elements in eucalypt and non-eucalypt species was studied by analysis of xylem (tracheal) sap from lateral roots.

Key Results

The columns of which pavements are composed develop exclusively on lower-tier lateral roots. Such sites show intimate associations of fine roots, fungal filaments, microbiota and clay deposits rich in Si, Al and Fe. Time scales for construction of pavements by eucalypts were assessed. Cores through columns of pavemented profiles showed gross elevations of bulk density, Al, Fe and Si in columns and related increases in pH, Mg and Ca status in lower profiles. A cutting through the dune exhibited pronounced alkalinity (pH 7–10) under mallee woodland versus acidity (pH 5–6·5) under proteaceous heath. Xylem sap analyses showed unusually high concentrations of Al, Fe, Mg and Si in dry-season samples from column-bearing roots.

Conclusions

Deposition of Al–Fe–Si-rich clay is pivotal to pavement construction by eucalypts and leads to profound chemical and physical changes in relevant soil profiles. Microbial associates of roots are likely to be involved in clay genesis, with parent eucalypts supplying the required key mineral elements and carbon sources. Acquisition of the Al and Fe incorporated into clay derives principally from hydraulic uplift from ground water via deeply penetrating tap roots.Key words: Niche construction, eucalypts, root morphology, xylem transport, hydraulic lift, element mining, soil formation, biomineralization, soil pans, duplex soils     

Dimethyl sulphide and methanethiol formation in microbial mats: potential pathways for biogenic signatures

Mechanisms of dimethyl sulphide (DMS) and methanethiol (MT) production and consumption were determined in moderately hypersaline mats, Guerrero Negro, Mexico. Biological pathways regulated the net flux of DMS and MT as revealed by increases in flux resulting from decreased salinity, increased temperature and the removal of oxygen. Dimethylsulphoniopropionate (DMSP) was not present in these microbial mats and DMS and MT are probably formed by the reaction of photosynthetically produced low-molecular weight organic carbon and biogenic hydrogen sulphide derived from sulphate reduction. These observations provide an alternative to the notion that DMSP or S-containing amino acids are the dominant precursors of DMS in intertidal sediment systems. The major sink for DMS in the microbial mats was biological consumption, whereas photochemical oxidation to dimethylsulphoxide was the major sink for DMS in the overlying water column. Diel flux measurements demonstrated that significantly more DMS is released from the system during the night than during the day. The major consumers of DMS in the presence of oxygen were monooxygenase-utilizing bacteria, whereas under anoxic conditions, DMS was predominantly consumed by sulphate-reducing bacteria and methanethiol was consumed by methanogenic bacteria. Aerobic and anaerobic consumption rates of DMS were nearly identical. Mass balance estimates suggest that the consumption in the water column is likely to be smaller than net the flux from the mats. Volatile organic sulphur compounds are thus indicators of high rates of carbon fixation and sulphate reduction in these laminated sediment ecosystems, and atmospheric sulphur can be generated as a biogenic signature of the microbial mat community.     

Fitness costs of induced resistance: emerging experimental support for a slippery concept

Fitness costs can explain the evolution and maintenance of induced resistance in plants. However, the methods currently used to gather evidence for such costs do not allow decreases in fitness to be cleanly attributed to individual traits responsible for induced resistance, because the studies are plagued by multiple confounding responses. Reproductive performance provides an overview of physiological performance and is thus a useful currency to understand the function of induced responses. Integrated molecular, physiological and ecological studies are needed to identify the mechanisms responsible for the decrease in fitness and to evaluate fully the usefulness of the cost paradigm.     

Immunochemical characterization of Nocardia asteroides antigens: support for a single species concept

Nocardia asteroides strains are highly heterogeneous. They show morphological, physiological, and immunological differences. In a previous study, we delineated seven immunotypes of N. asteroides. In the present study, we compared the culture filtrate antigens of these immunotypes by antigen-antibody crossed-immunoelectrophoresis and by rocket electrophoresis. We have also compared the antigen preparations by two-dimensional electrophoresis. While unique components constitute the major portion of the components, the results indicate that similar components are present in the culture filtrates of all strains. This finding supports the view of retaining all the immunotypes in the species Nocardia asteroides rather than designating different species such as N. farcinica and N. sebivorans.     

Evidence for human-mediated dispersal of exotic earthworms: support for exploring strategies to limit further spread

As potent ecosystem engineers, non-native earthworms are altering the fundamental structure and function of previously earthworm-free cold-temperate hardwood forests in North America. Discarded earthworms used for fishing bait has been presumed to be an important vector for the continued spread of non-native earthworms because epicentres of invasion often include boat landings, lakeshores and roads. However, controversy has remained about the overall importance of human-mediated spread vs. natural expansion of established earthworm populations. In this issue of Molecular Ecology , Cameron et al . explore the continued role of humans in dispersing non-native earthworms.     

Dieback of rural eucalypts: Tree phenology and damage caused by leaf-feeding insects

During three growing seasons, phenology and damage on leafy shoots of healthy eucalypts and others with dieback were monitored on pastoral properties in the Australian Capital Territory. Leaves from the dieback trees tended to be younger than those from nearby healthy trees. Leaves that were not lost prematurely lived for similar lengths of time on both dieback and healthy trees, but more leaves were initiated on the dieback trees. Dieback trees were also less active reproductively. During the study, chewing insects caused most of the damage on leaves, and there was usually more of this damage on leaves from the dieback trees. Young leaves accumulated damage at a much faster rate than older leaves, and this probably contributed to the relatively greater damage on the leaves from the dieback trees. However, the differences in the amount of damage on the leaves from healthy and dieback trees were not solely due to the differences in ages of the leaves. Generalized linear models which equalized the effects of the differences in leafing phenology produced adjusted estimates for damage to leaves that were still higher for dieback trees.     

Catalysis of peptide bond formation by histidyl-histidine in a fluctuating clay environment

Summary The condensation of glycine to form oligoglycines during wet-dry fluctuations on clay surfaces was enhanced up to threefold or greater by small amounts of histidyl-histidine. In addition, higher relative yields of the longer oligomers were produced. Other specific dipeptides tested gave no enhancement, and imidazole, histidine, and N-acetylhistidine gave only slight enhancements. Histidyl-histidine apparently acts as a true catalyst (in the sense of repeatedly catalyzing the reaction), since up to 52 nmol of additional glycine were incorporated into oligoglycine for each nmol of catalyst added. This is the first known instance of a peptide or similar molecule demonstrating a catalytic turnover number greater than unity in a prebiotic oligomer synthesis reaction, and suggests that histidyl-histidine is a model for a primitive prebiotic protoenzyme. Catalysis of peptide bond synthesis by a molecule which is itself a peptide implies that related systems may be capable of exhibiting autocatalytic growth.     

Distribution of biogenic amines in the hippocampal formation in the rabbit]

The hippocampal formation (the hippocampus and the dentate fascia) of the rabbit was studied by histochemical fluorescent method of Falk to determine localization of monoaminergic terminals containing biogenic amines: noradrenalin, dophamine and serotonin. It was shown that monoaminenergic terminals in the hippocampus were in two zones of afferent terminations: in the zone of ending of the perforating way (str. lacunosum-moleculare of fields CA1 and CA2; str. moleculare of the dentate fascia) and in the subgranular zone of the hilum where a part of septofimbrial way terminated on granular neurons of the dentate fascia, the main cellular elements of the hipocampus (pyramidal, granular and basket cells of the hippocampus) did not contain biogenic amines.     

Embryonic development of the sensory innervation of the clypeo-labral complex: further support for serially homologous appendages in the locust

The clypeo-labrum, or upper lip, of insects is intimately involved in feeding behavior and is accordingly endowed with a rich sensory apparatus. In the present study we map the temporal appearance of all major clusters of sensory cells on this structure in the locust during the first half of embryogenesis. The identities of these sensory cell clusters were defined according to the origin of the branching point of their axons from the labral sensory nerve as seen at mid-embryogenesis. The first sensory cells to differentiate from the labral epithelium do so at stereotypic sites beginning at around 32% of embryogenesis. Bilaterally symmetrical clusters of differentiated neurons rapidly appear and pioneering of the labral sensory nerve on each side is performed by a specific cell from each cluster. This cell directs its axon anteriorly towards a bilaterally symmetrical pair of cells, the frontal commissure pioneers, on either side of the developing frontal ganglion. The final trajectory of the sensory nerve within the labrum closely matches the pattern of Repo-expressing glial cells. The majority of the sensory cell clusters differentiate during embryogenesis, but the number of sensory cells in some clusters are modified significantly during postembryonic development. Comparing the innervation pattern of the clypeo-labrum with that of other mouthparts and the leg at mid-embryogenesis, we find a striking similarity in organization which we interpret as support for the homologous appendage hypothesis.     

Conularia sp. from the Pyrénées: further support for scyphozoan affinities of the Conularida

McKinney, F.K, Delvolve, 7.-J. & Sobieraj, J. 1995 11 30: Conularia sp. from the Pyrénées: further support for scyphozoan affinities of the Conularida.
Several specimens of Conularia from the mid-Carboniferous Culm Formation of the Pyrénées occur as clusters with a large central specimen and several smaller divergent individuals that may be buds from the larger central specimen. Other individual and clustered specimens exhibit complex polygonal cross-sections that are inferred to have resulted from longitudinal fission of a precursor pyramid into two or more descendant individuals. Apparent budding and transverse fission (strobilation) of conulariids have been reported previously, but we have found no earlier record of longitudinal fission. The existence of this additional mode of asexual increase in conulariids further supports their affinities with the scyphozoan Cnidaria. Conularida, Scypho-zoa, fission, Carboniferous, Pyrénées .     

Molecular electroporation: a unifying concept for the description of membrane pore formation by antibacterial peptides, exemplified with NK-lysin

The antibacterial activity of many small, positively charged peptides and proteins is based on pore formation in lipid bilayers. It is here proposed to arise from an electroporation effect. This hypothesis is supported by calculations of the electrostatic potential of NK-lysin associated to a membrane. For a significant area of the protein-membrane interface, the electrostatic potential is found to be above the minimum threshold for electroporation. A single highly charged alpha-helical segment of NK-lysin is mainly responsible for this effect. It is experimentally demonstrated that a peptide comprising this helix has antibacterial activity. We propose that superficial association to membranes suffices to trigger electroporation, provided the peptide is sufficiently charged. The effect is referred to as molecular electroporation.     

Glacial refugia and reticulate evolution: the case of the Tasmanian eucalypts

Tasmania is a natural laboratory for investigating the evolutionary processes of the Quaternary. It is a large island lying 40-44 degrees S, which was repeatedly glaciated and linked to southeastern continental Australia during the Quaternary. Climate change promoted both the isolation of species in glacial refugia, and an exchange between Tasmanian and mainland floras. Eucalyptus is a complex and diverse genus, which has increased in abundance in Australia over the past 100 kyr, probably in response to higher fire frequency. Morphological evidence suggests that gene flow may have occurred between many eucalypt species after changes in their distribution during the Quaternary. This paper summarizes recent genetic evidence for migration and introgressive hybridization in Tasmanian Eucalyptus. Maternally inherited chloroplast DNA reveals a long-term persistence of eucalypts in southeastern Tasmanian refugia, coupled with introgressive hybridization involving many species. Detailed analysis of the widespread species Eucalyptus globulus suggests that migration from mainland Australia was followed by introgression involving a rare Tasmanian endemic. The data support the hypothesis that changes in distribution of interfertile species during the Quaternary have promoted reticulate evolution in Eucalyptus.     

Use of positive pressures to establish vulnerability curves : further support for the air-seeding hypothesis and implications for pressure-volume analysis

Loss of hydraulic conductivity occurs in stems when the water in xylem conduits is subjected to sufficiently negative pressure. According to the air-seeding hypothesis, this loss of conductivity occurs when air bubbles are sucked into water-filled conduits through micropores adjacent to air spaces in the stem. Results in this study showed that loss of hydraulic conductivity occurred in stem segments pressurized in a pressure chamber while the xylem water was under positive pressure. Vulnerability curves can be defined as a plot of percentage loss of hydraulic conductivity versus the pressure difference between xylem water and the outside air inducing the loss of conductivity. Vulnerability curves were similar whether loss of conductivity was induced by lowering the xylem water pressure or by raising the external air pressure. These results are consistent with the air-seeding hypothesis of how embolisms are nucleated, but not with the nucleation of embolisms at hydrophobic cracks because the latter requires negative xylem water pressure. The results also call into question some basic underlying assumptions used in the determination of components of tissue water potential using “pressure-volume” analysis.     

Enhancement of peptide bond formation by polyribonucleotides on clay surfaces in fluctuating environments

Summary The condensation of glycine to form oligoglycine during temperature and moisture fluctuations on clay surfaces was enhanced up to fourfold by polyribonucleotides. Polydeoxyribonucleotides gave no enhancement. Yields were greatly reduced in the absence of clay. A small preference was observed among the polyribonucleotide bases with enhancements in the order of Poly G > Poly A = Poly U > Poly C at high density of polynucleotide on the clay surface and Poly G > Poly U > Poly C > Poly A at low density. This and other experiments seem to indicate a codonic bias in the interaction of polynucleotides with amino acids reacting to form peptide bonds. A mechanism is proposed which involves (1) activation of glycine on the clay surface, (2) formation of esters between glycine and the 2-OH groups of the polyribonucleotide, and (3) formation of peptide bonds between adjacent amino acyl esters. If this mechanism is correct, it may provide the basis for a simple, direct-template translation process.Abbreviations Poly A Polyadenylic acid - Poly C Polycytidylic acid - Poly G Polyguanylic acid - Poly U Polyuridylic acid - Poly dA Polydeoxyadenylic acid     

Cross-adaptation and bitterness inhibition of L-tryptophan, L-phenylalanine and urea: further support for shared peripheral physiology

A previous study investigating individuals' bitterness sensitivities found a close association among three compounds: L-tryptophan (L-trp), L-phenylalanine (L-phe) and urea (Delwiche et al., 2001, Percept. Psychophys. 63, 761-776). In the present experiment, psychophysical cross-adaptation and bitterness inhibition experiments were performed on these three compounds to determine whether the bitterness could be differentially affected by either technique. If the two experimental approaches failed to differentiate L-trp, L-phe and urea's bitterness, then we may infer they share peripheral physiological mechanisms involved in bitter taste. All compounds were intensity matched in each of 13 subjects, so the judgments of adaptation or bitterness inhibition would be based on equal initial magnitudes and, therefore, directly comparable. In the first experiment, cross-adaptation of bitterness between the amino acids was high (>80%) and reciprocal. Urea and quinine-HCl (control) did not cross-adapt with the amino acids symmetrically. In a second experiment, the sodium salts, NaCl and Na gluconate, did not differentially inhibit the bitterness of L-trp, L-phe and urea, but the control compound, MgSO(4), was differentially affected. The bitter inhibition experiment supports the hypothesis that L-trp, L-phe and urea share peripheral bitter taste mechanisms, while the adaptation experiment revealed subtle differences between urea and the amino acids indicating that urea and the amino acids activate only partially overlapping bitter taste mechanisms.     

Gonadotropin-releasing hormone analog structural determinants of selectivity for inhibition of cell growth: support for the concept of ligand-induced selective signaling

GnRH and its receptor are expressed in human reproductive tract cancers, and direct antiproliferative effects of GnRH analogs have been demonstrated in cancer cell lines. The intracellular signaling responsible for this effect differs from that mediating pituitary gonadotropin secretion. The GnRH structure-activity relationship is different for the two effects. Here we report a structure-activity relationship study of GnRH agonist antiproliferative action in model cell systems of rat and human GnRH receptors stably expressed in HEK293 cells. GnRH II was more potent than GnRH I in inhibiting cell growth in the cell lines. In contrast, GnRH I was more potent than GnRH II in stimulating inositol phosphate production, the signaling pathway in gonadotropes. The different residues in GnRH II (His(5), Trp(7), Tyr(8)) were introduced singly or in pairs into GnRH I. Tyr(5) replacement by His(5) produced the highest increase in the antiproliferative potency of GnRH I. Tyr(8) substitution of Arg(8) produced the most selective analog, with very poor inositol phosphate generation but high antiproliferative potency. In nude mice bearing tumors of the HEK293 cell line, GnRH II and an antagonist administration was ineffective in inhibiting tumor growth, but D-amino acid stabilized analogs (D-Lys(6) and D-Arg(6)) ablated tumor growth. Docking of GnRH I and GnRH II to the human GnRH receptor molecular model revealed that Arg(8) of GnRH I makes contact with Asp(302), whereas Tyr(8) of GnRH II appears to make different contacts, suggesting these residues stabilize different receptor conformations mediating differential intracellular signaling and effects on gonadotropin and cell growth. These findings provide the basis for the development of selective GnRH analog cancer therapeutics that directly target tumor cells or inhibit pituitary gonadotropins or do both.