A comparative analysis of karyotypes of three species of Macleaya—producers of a complex of isoquinoline alkaloids

Using a set of methods (C-banding, DAPI-staining, fluorescence hybridization in situ (FISH) with probes of 26S and 5S rDNA, and analysis of meiosis), the first comparative cytogenetic study of three species of Macleaya, producers of complex isoquinoline alkaloids, cordate Macleaya cordata (Willd.) R. Br. (2n = 20), small-fruited Macleaya microcarpa (Maxim.) Fedde (2n = 20) and Macleaya kewensis Turrill (2n = 20), was first carried out. On the basis of morphometric analysis, formulas of karyotypes were made for each species. Species ideograms for M. cordata, M. microcarpa, and M. kewensis were constructed taking into account the polymorphic variants of the C-banding patterns and indicating the location of 26S and 5S rDNA sites. A comparative study revealed that the karyotypes of M. microcarpa and M. kewensis have more in common with each other than with M. cordata. Analysis of meiotic chromosomes suggests of genetic stability of Macleaya genomes. The results of chromosome analysis were used to confirm the close relationship of Macleaya and to clarify their phylogenetic relationships.     

Selection of optimal variants of Gō-like models of proteins through studies of stretching

The Gō-like models of proteins are constructed based on the knowledge of the native conformation. However, there are many possible choices of a Hamiltonian for which the ground state coincides with the native state. Here, we propose to use experimental data on protein stretching to determine what choices are most adequate physically. This criterion is motivated by the fact that stretching processes usually start with the native structure, in the vicinity of which the Gō-like models should work the best. Our selection procedure is applied to 62 different versions of the Gō model and is based on 28 proteins. We consider different potentials, contact maps, local stiffness energies, and energy scales—uniform and nonuniform. In the latter case, the strength of the nonuniformity was governed either by specificity or by properties related to positioning of the side groups. Among them is the simplest variant: uniform couplings with no i, i + 2 contacts. This choice also leads to good folding properties in most cases. We elucidate relationship between the local stiffness described by a potential which involves local chirality and the one which involves dihedral and bond angles. The latter stiffness improves folding but there is little difference between them when it comes to stretching.     

Effect of γ-Irradiation on Development of Lachrymator of Onion

A thermosensitive uracil requiring mutant of Bacillus subtilis Marburg 168 thy trp₂ ts42 was examined as to the colony forming ability at the permissive and nonpermissive temperatures. The viability of the mutant cells decreased rapidly at the restrictive temperature in the modified Woese’s (MW) medium. However, the cells retained viability when sodium succinate or potassium chloride was added to the medium at that temperature although uracil deficiency was unchanged. A little but significant incorporation of adenine-8-¹⁴C into RNA still continued even after the incorporation of N-acetyl-³H-d-glucosamine into acid insoluble fraction of the cells terminated in the MW medium at 48°C. Both incorporations as well as increase of absorbance were slowed down in the presence of sodium succinate at 48°C. This mutant, ts42, was more sensitive to deoxycholate (DOC) than the parent strain. The restoration of colony forming ability after the temperature shift back from 48 to 37°C was suppressed by the addition of DOC to the medium. However, the cell became resistant to DOC when uracil was added to the medium prior to the temperature shift.     

Mechanisms of action of the α-amylase of Micromonospora melanosporea

The -amylase of Micromonospora melanosporea was produced extracellularly during batch fermentation in a 5.0-1 fermentor. The absence of an organic nitrogen source in its growth medium facilitated subsequent purification of the enzyme by ammonium sulphate fractionation and two consecutive Superose-12 gel-filtration steps. The enzyme exhibited maxima for activity at pH 7.0 and 55° C and was 72% stable at pH 6.0–12.0 for 30 min at 40° C. It had a relative molecular mass of 45 000 and an isoelectric point at pH 7.6. The enzyme catalyses the conversion of starch to maltose (53%, w/w) as the predominant final end-product. Initial hydrolysis of this substrate, however, gave rise to the formation of maltooligosaccharides in the range maltotriose to maltohexaose. Maximum yields of these intermediate sugars accumulated to between 31 and 42% (w/w) as the reaction proceeded. The action of the M. melanosporea amylase on high concentrations of saccharides larger than maltotriose resulted in the formation of mainly maltose and maltotriose without concomitant glucose production. A combination of hydrolytic and transfer events is postulated to be responsible for this phenomenon and for the high maltose levels achieved. Correspondence to: C. T. Kelly     

Chemistry of the “molecular trap” of protease-catalyzed splicing reaction of complementary segments of α-subunit of hemoglobin A

The complementary fragments of human Hb α, α_1–30, and α_31–141 are spliced together by V8 protease in the presence of 30%n-propanol to generate the full-length molecule (Hb α-semisynthetic reaction). Unlike the other protease-catalyzed protein/peptide splicing reactions of fragment complementing systems, the enzymic condensation of nonassociating segments of Hb α is facilitated by the organic cosolvent induced α-helical conformation of product acting as the “molecular trap” of the splicing reaction. The segments α_24–30 and α_31–40 are the shortest complementary segments that can be spliced by V8 protease. In the present study, the chemistry of the contiguous segment (product) α_24–40 has been manipulated by engineering the amino acid replacements to the positions α²⁷ and α³¹ to delineate the structural basis of the molecular trap. The location of Glu²⁷ and Arg³¹ residues in the contiguous segment α_24–40 (as well as in other larger segments) is ideal to generate (i, i+4) side-chain carboxylate-guanidino interaction in its α-helical conformation. The amino acid residue replacement studies have confirmed that the side chains at α²⁷ and α³¹ facilitate the semisynthetic reaction. The relative influence of the substitute at these sites on the splicing reaction depends on the chemical nature of the side chain and the location. The γ-carboxylate guanidino side-chain interaction appears to contribute up to a maximum of 85% of the thermodynamic stability of the molecular trap. The studies also demonstrate that the thermodynamic stability of the molecular trap is determined by two interdependent conformational aspects of the peptide. One is an amino acid-sequence-specific event that facilitates the induction of an α-helical conformation to the contiguous segment in the presence of organic cosolvent that imparts some amount of protease resistance to Glu³⁰-Arg³¹ peptide bond. The second structural aspect is a site-specific event, ani, i+4 side-chain interaction in the α-helical conformation of the peptide which imparts an additional thermodynamic stability to the molecular trap. The results suggest that conformationally driven “molecular traps” of protease-mediated ligation reactions of peptides could be designed into products to facilitate the modular assembly of peptides/proteins.     

Association of the subunits of the calcium-independent receptor of α-latrotoxin

CIRL-1 also called latrophilin 1 or CL belongs to the family of adhesion G protein-coupled receptors (GPCRs). As all members of adhesion GPSR family CIRL-1 consists of two heterologous subunits, extracellular hydrophilic p120 and heptahelical membrane protein p85. Both CIRL-1 subunits are encoded by one gene but as a result of intracellular proteolysis of precursor, mature receptor has two-subunit structure. It was also shown that a minor portion of the CIRL-1 receptor complexes dissociates, producing the soluble receptor ectodomain, and this dissociation is due to the second cleavage at the site between the site of primary proteolysis and the first transmembrane domain. Recently model of independent localization p120 and p85 on the cell surface was proposed. In this article we evaluated the amount of p120-p85 complex still presented on the cellular membrane and confirmed that on cell surface major amount of mature CIRL-1 presented as a p120-p85 subunit complex.     

Kinetics of renaturation and self-assembly of intermediates on the pathway of folding of the β2-subunit of Escherichia coli tryptophan-synthetase

The kinetics of renaturation of the β₂-subunit of Escherichia coli tryptophan-synthetase (l-serine hydrolyase (adding indole) E.C. 4.2.1.20) and those of its two proteolytic fragments F₁ and F₂ are studied and compared. Steps corresponding to the refolding of F₁, to the association of the folded F₁ and F₂ fragments, and to an isomerization of the associated protein are identified. These steps are ordered on the pathway of renaturation and some of their kinetic parameters are determined. This leads to a tentative kinetic model for the renaturation of nicked-β₂ starting from the denatured F₁ and F₂ fragments.The step corresponding to the refolding of the F₁ domain, as well as that corresponding to the last rate-limiting isomerization leading to the native protein, is shown to be the same in the refolding of the entire, uncleaved β₂-protein. It is concluded that the refolded F₁ fragment corresponds to a folding intermediate on the pathway of renaturation of the β₂-subunit.     

Inhibition of colony-spreading activity of Staphylococcus aureus by secretion of δ-hemolysin

Staphylococcus aureus spreads on the surface of soft agar, a phenomenon we termed "colony spreading." Here, we found that S. aureus culture supernatant inhibited colony spreading. We purified δ-hemolysin (Hld, δ-toxin), a major protein secreted from S. aureus, as a compound that inhibits colony spreading. The culture supernatants of hld-disrupted mutants had 30-fold lower colony-spreading inhibitory activity than those of the parent strain. Furthermore, hld-disrupted mutants had higher colony-spreading ability than the parent strain. These results suggest that S. aureus negatively regulates colony spreading by secreting δ-hemolysin.     

Sulfated flavonoids and leaf morphology of the Halophila ovalis—H. minor complex (hydrocharitaceae) in the Pacific Islands and Australia

Sulfated flavonoids showed a systematic pattern among Halophila populations of the Pacific and Australia. Large-leaved plants, typical of H. ovalis (R.Br.) Hook. f., in the Palau Islands (Belau) and in Australia contained sulfated flavonoids, but small-leaved plants that have received various treatments under H. ovalis, including ssp. bullosa (Setchell) den Hartog and ssp. hawaiiana (Doty & Stone) den Hartog, and under H. minor (Zoll.) den Hartog or H. ovata Gaud. in American Samoa, Western Samoa, Tonga, Fiji, Wallis I., New Caledonia, Yap, Guam, Hawaii and Australia lacked sulfated flavonoids. Plants collected in their natural habitats as well as those grown in experimental cultures showed the characteristic patterns of these secondary compounds. Although the large-leaved plants have the potential for producing the largest leaves in the complex, they are not separable from small-leaved plants in leaf dimensions or in number of pairs of cross veins under all environmental conditions. The production of sulfated flavonoids and the potential for greater leaf length are the characteristics that can be used to separate typical H. ovalis and ssp. australis (Doty & Stone) den Hartog from other members of the complex.     

Ecology and survival of Nigritella nigra, a threatened orchid species in Scandinavia

Nigritella nigra ssp. nigra (Orchidaceae) is the only taxon of the genus in Scandinavia, where it occurs as an endemic with a disjunct distribution, mostly in upland areas of central Norway and Sweden. Nigritella nigra is a boreal-alpine, basiophilous species of open grasslands and dry fens. Both the number of localities and the number of individuals in a majority of the localities, including the study area at Sølendet, Røros, central Norway, have declined in recent decades. The most important threat is the change in agricultural practices followed by overgrowing of land earlier harvested for hay or intensively grazed. The total population of N. nigra at Sølendet is estimated to be about 3000 individuals, the largest known. Flowering individuals have been counted annually and all 225 individuals in nine permanent plots have been recorded for twenty years. The population has a large proportion of long-lived individuals with a half-life of up to 8 years in particular cohorts. Flowering behaviour varies greatly and flowering rarely occurs several years in succession. Together with the reduced size of individuals after flowering, this indicates a high cost of flowering. Correlation analyses indicate that high precipitation in autumn and winter negatively affects flowering (in July) on dry grasslands. On damp grasslands, warmth in autumn and in May of the flowering year correlates positively with flowering density. Many individuals are killed in years with heavy microtine rodent grazing. Experimental mowing as a management measure, and apparently also cattle grazing, have positive effects on the population. Some small, shallow valleys with a good supply of base-rich water on its flat, sandy floor is the main N. nigra biotopes at Sølendet.     

Analysis of morphological characters of Chara baltica, C. hispida, C. horrida, and C. rudis from Europe

Morphological characters of Chara baltica, C. hispida, C. horrida, and C. rudis from different localities in Central Europe and Scandinavia were analyzed. The plants were preliminarily classified using specific qualitative features into four species: C. baltica, C. hispida, C. horrida, and C. rudis. Of the 14 characters, all were used in principal-component and cluster analyses, and 12 were applied in a discriminant analysis. In the discriminant analysis, the most useful characters for distinguishing particular species were selected. The methods (canonical discriminant and canonical analyses) used herein offered good differentiation between brackish water species C. horrida and C. baltica and fresh water species C. hispida and C. rudis. The analysis showed that the two morphologically very similar species C. hispida and C. rudis are only partially distinguished.     

Hierochloë odorata (L.) Beauv. confermata per la flora italiana

Abstract

On the presence of Hierochloë odorata (L.) Beauv. in Italy.—H. odorata has been found in an oligotrophic marsh in the Italian part of the Eastern Alps and therefore definitively confirmed for the flora of Italy. An extended population occurs at 1800 m above sea level near Misurina, in the Dolomites (province of Belluno). All preceding records of H. odorata for the Italian flora must be referred to H. australis (Schrader) R.&S. Differential characters and distribution of the two species are shortly discussed.     

Posidonia australis seed predation in seagrass habitats of Two Peoples Bay,Western Australia

Posidonia australis seed predation experiments conducted in three seagrass habitats (P. australis, Posidonia sinuosa, Halophila ovalis) and bare sand in Two Peoples Bay, Western Australia, showed higher predation rates in seagrass than bare sand, supporting general conclusions from two previous predation studies in Western Australia. However, much higher rates were noted in H. ovalis, compared to previous observations of very low rates in H. ovalis on Rottnest Island, Western Australia. We attribute these differences to gammaridean amphipods (family Lysianassidae) that were present in a detrital layer within the H. ovalis in Two Peoples Bay. Our data from Two Peoples Bay continues to add to the growing body of information showing high seed predation rates in most seagrass habitats by a diverse group of crustacean species.     

Diversity of management practices required to ensure conservation of rare and locally threatened plant species in grasslands: a case study at a regional scale (Lorraine, France)

The Lorraine region, in northeast France, harbours 77 protected plantspecies whose main localities are in grasslands. About 80% of them are very rareor rare (less than 15 localities) in that territory and are threatened by changes inagriculture. These species occur in a large range of grassland habitats,reflecting variability of climatic, hydrological and edaphic parameters, butalso of agricultural practices. A more precise analysis of managementrequirements was carried out for seven of these species, Euphorbiapalustris, Aster amellus, Pulsatillaalba, Carex hordeistichos, Thalictrumminus ssp. majus, Botrychiummatricariifolium and Ophioglossum vulgatum, whichwere represented on a triangle whose corners correspond to grazing, cutting andno or very low exploitation. It confirmed the large diversity of managementpractices required to ensure conservation of rare and threatened grassland plantspecies, even at a local level. These appropriate conservation measures havetherefore to be carried out or, at least, monitored by specialized natureconservation organizations.     

Morphology and taxonomy of the genus Cochlearia (Brassicaceae) in Northern Scandinavia

The morphological variation between populations of Cochlearia in Northern Scandinavia has been analysed. The variation is correlated to ecology, and four ecotypes are recognized. Based on the morphological differentiation of the ecotypes and the results of crossing experiments, the populations are referred to the following three taxa: C. officinalis ssp. officinalis which includes the common beach ecotype and the bird cliff ecotype; C. officinalis ssp. integrifolia stat. nov. which includes the spring ecotype growing in inland localities up to about 600 m a.s.l. in cold more or less eutrophic springs, and C. officinalis ssp. norvegica ssp. nov. which includes the estuary ecotype, growing in poor, often inundated habitats near river outlets at the fjord ends. All the differentiation has taken place at the tetraploid level (2n = 24). The subspecies are kept more or less distinct due to ecological and seasonal isolating mechanisms rather than internal, reproductive barriers. A key to the subspecies and distribution maps for ssp. integrifolia and ssp. norvegica are presented.     

Invasion Success in a Marginal Habitat: An Experimental Test of Competitive Ability and Drought Tolerance in Chromolaena odorata

Climatic niche models based on native-range climatic data accurately predict invasive-range distributions in the majority of species. However, these models often do not account for ecological and evolutionary processes, which limit the ability to predict future range expansion. This might be particularly problematic in the case of invaders that occupy environments that would be considered marginal relative to the climatic niche in the native range of the species. Here, we assess the potential for future range expansion in the shrub Chromolaena odorata that is currently invading mesic savannas (>650 mm MAP) in South Africa that are colder and drier than most habitats in its native range. In a greenhouse experiment we tested whether its current distribution in South Africa can be explained by increased competitive ability and/or differentiation in drought tolerance relative to the native population. We compared aboveground biomass, biomass allocation, water use efficiency and relative yields of native and invasive C. odorata and the resident grass Panicum maximum in wet and dry conditions. Surprisingly, we found little differentiation between ranges. Invasive C. odorata showed no increased competitive ability or superior drought tolerance compared to native C. odorata. Moreover we found that P. maximum was a better competitor than either native or invasive C. odorata. These results imply that C. odorata is unlikely to expand its future range towards more extreme, drier, habitats beyond the limits of its current climatic niche and that the species’ invasiveness most likely depends on superior light interception when temporarily released from competition by disturbance. Our study highlights the fact that species can successfully invade habitats that are at the extreme end of their ranges and thereby contributes towards a better understanding of range expansion during species invasions.     

Morphological Characters Useful for the Delimitation of Taxa Within Viola Subsect. Viola (Violaceae): A Morphometric Study from the West Carpathians

Forty-nine morphological characters were scored or measured on 44 populations (376 individuals) of Viola subsect. Viola from the West Carpathians and adjacent areas (Slovakia, Czech Republic, Austria and Hungary). The presence of six species, namely V. alba (represented by subsp. alba), V. ambigua, V. collina, V. hirta, V. odorata and V. suavis s.l. was revealed based on pollen fertility, cytological and morphometric analyses. The morphological characters traditionally used to delimit taxa within the subsection and those revealed by our study as most reliable are widely discussed. A key for identifying the taxa and most common hybrids of subsection Viola occurring in the West Carpathians is presented. Chromosome counting and flow cytometry were used to determine the ploidy levels of the populations studied. All individuals of V. alba subsp. alba, V. collina, V. hirta and V. odorata were tetraploid, while those of V. ambigua and V. suavis s.l. were octoploid.     

Microarray and Functional Gene Analyses of Sulfate-Reducing Prokaryotes in Low-Sulfate, Acidic Fens Reveal Cooccurrence of Recognized Genera and Novel Lineages

Low-sulfate, acidic (approximately pH 4) fens in the Lehstenbach catchment in the Fichtelgebirge mountains in Germany are unusual habitats for sulfate-reducing prokaryotes (SRPs) that have been postulated to facilitate the retention of sulfur and protons in these ecosystems. Despite the low in situ availability of sulfate (concentration in the soil solution, 20 to 200 μM) and the acidic conditions (soil and soil solution pHs, approximately 4 and 5, respectively), the upper peat layers of the soils from two fens (Schlöppnerbrunnen I and II) of this catchment displayed significant sulfate-reducing capacities. 16S rRNA gene-based oligonucleotide microarray analyses revealed stable diversity patterns for recognized SRPs in the upper 30 cm of both fens. Members of the family “Syntrophobacteraceae” were detected in both fens, while signals specific for the genus Desulfomonile were observed only in soils from Schlöppnerbrunnen I. These results were confirmed and extended by comparative analyses of environmentally retrieved 16S rRNA and dissimilatory (bi)sulfite reductase (dsrAB) gene sequences; dsrAB sequences from Desulfobacca-like SRPs, which were not identified by microarray analysis, were obtained from both fens. Hypotheses concerning the ecophysiological role of these three SRP groups in the fens were formulated based on the known physiological properties of their cultured relatives. In addition to these recognized SRP lineages, six novel dsrAB types that were phylogenetically unrelated to all known SRPs were detected in the fens. These dsrAB sequences had no features indicative of pseudogenes and likely represent novel, deeply branching, sulfate- or sulfite-reducing prokaryotes that are specialized colonists of low-sulfate habitats.