Optimization and maintenance of soluble methane monooxygenase activity inMethylosinus trichosporium OB3b

Soluble methane monooxygenase (sMMO) maximization studies were carried out as part of a larger effort directed towards the development and optimization of an aqueous phase, multistage, membrane bioreactor system for treatment of polluted groundwater. A modified version of the naphthalene oxidation assay was utilized to determine the effects of methane:oxygen ratio, nutrient supply, and supplementary carbon sources on maximizing and maintaining sMMO activity inMethylosinus trichosporium OB3b.Methylosinus trichosporium OB3b attained peak sMMO activity (275–300 nmol of naphthol formed h^–1 mg of protein^–1 at 25°C) in early stationary growth phase when grown in nitrate mineral salts (NMS) medium. With the onset of methane limitation however, sMMO activity rapidly declined. It was possible to define a simplified nitrate mineral salts (NMS) medium, containing nitrate, phosphate and a source of iron and magnesium, which allowed reasonably high growth rates (_max 0.08 h^–1) and growth yields (0.4–0.5 g cells/g CH₄) and near maximal activities of sMMO. In long term batch culture incubations sMMO activity reached a stable plateau at approximately 45–50% of the initial peak level and this was maintained over several weeks. The addition of d-biotin, pyridoxine, and vitamin B₁₂ (cyanocobalamin) increased the activity level of sMMO in actively growing methanotrophs by 25–75%. The addition of these growth factors to the simplified NMS medium was found to increase the plateau sMMO level in long term batch cultures up to 70% of the original peak activity.Abbreviations sMMO soluble methane monooxygenase - pMMO particulate methane monooxygenase - NMS nitrate mineral salts - TCE trichloroethene - NADH reduced nicotinamide adenine dinucleotide     

The evolution of pathways for aromatic hydrocarbon oxidation inPseudomonas

The organisation and nucleotide sequences coding for the catabolism of benzene, toluene (and xylenes), naphthalene and biphenylvia catechol and the extradiol (meta) cleavage pathway inPseudomonas are reviewed and the various factors which may have played a part in their evolution are considered. The data suggests that the complete pathways have evolved in a modular way probably from at least three elements. The commonmeta pathway operons, downstream from the ferredoxin-like protein adjacent to the gene for catechol 2,3-dioxygenase, are highly homologous and clearly share a common ancestry. This common module may have become fused to a gene or genes the product(s) of which could convert a stable chemical (benzoate, salicylate, toluene, benzene, phenol) to catechol, thus forming the lower pathway operons found in modern strains. The upper pathway operons might then have been acquired as a third module at a later stage thus increasing the catabolic versatility of the host strains.     

A cladistic analysis of Nassauvia Comm. ex Juss. (Asteraceae, Mutisieae) and related genera

FREIRE, S. E., CRISCI, J. V. & KATINAS, L., 1993. A cladistic analysis of Nassauvia Comm. ex Juss. (Asteraceae, Mutisieae) and related genera. Nassauvia and the most closely related genera Calopappus and Triptilion from the southern Andes and Patagonia of South America, form a monophyletic group diagnosed by the following synapomorphies: cypsela trichomes single two-celled, cypsela testa with strengthened cells, pollen grains spheroidal to spheroidal-oblate, colpi membrane with sexine processes, pappus bristles two to six, and pappus deciduous. Furthermore, Nassauvia, Triptilion, and Calopappus form a group with two other Andean genera, Moscharia and Polyachyrus, diagnosed by occurrence of pseudocephalia and a reduction in the number of flowers to five, three or one. A cladistic analysis of the group was undertaken using 35 characters from morphology, anatomy, and palynology. The monophyletic terminal taxa were the 38 species of Nassauvia, the genus Triptilion, the monotypic genus Calopappus, the genus Polyachyrus, and the genus Moscharia. Character polarity was based on outgroup comparison using Cephalopappus. The analysis resulted in 223 equally parsimonious cladograms, each with 70 steps and a consistency index of 0.57. A successive weighting procedure was applied, resulting in 15 cladograms with a consistency index of 0.82. Results of the cladistic analysis support most of the current systematic classification of Nassauvia, with three exceptions: (1) Nassauvia (excluding Calopappus) is paraphyletic; (2) section Masligophorus appears to be a polyphyletic group (N. pygmaea does not cluster with the remaining species of the section); (3) section Panargyrum (without N. lagascae= section Caloptilium) appears to be a paraphyletic group. The capitula arranged in cymose conflorescences in Triptilion are regarded as a primitive condition which gave rise to all stages present in Nassauvia (conflorescence spicate, pseudocephalium, capitula solitary). The capitula arranged in pseudocephalia in Moscharia and Polyachyrus are regarded as a parallel development to the pseudocephalium found in Nassauvia. Nassauvia, subgenus Strongyloma appears as the most primitive taxon, with its spicate conflorescence, whereas section Masligophorus with its solitary capitulum is thought-derived. These results correspond well with cytological data where species of the subgenus Strongyloma have n= 11 and the species of section Masligophorus are tetraploids (n = 22).     

Diatom assemblages in superficial sediments from the Gulf of Riga,eastern Baltic Sea

Twenty one superficial sediment samples from areas of high and moderate eutrophication in the Gulf of Riga were studied with respect to siliceous microfossils, mainly diatoms.The results seem to imply that the number of taxa and the abundance of the frustules are affected by runoff from rivers and the degree of eutrophication in different parts of the Gulf. Areas with high eutrophication, e.g. the river estuaries, have diatom assemblages of varying composition, while in areas with moderate eutrophication the composition is almost constant and the influx of freshwater diatoms and littoral periphyton small. The high abundance and low diversity of brackish-marine and brackish, planktonic diatoms seem to be a result of an influx of nutrients and pollutants in the water and bottom sediments. The sea ice diatoms occurring in the Baltic waters and also in the Gulf of Riga tend to be resistant to eutrophication or are even favoured by it.     

Biochemical characterization and crystallization of porin from Rhodopseudomonas blastica

Oligomeric porin of the phototrophic bacterium Rhodopseudomonas blastica DSM 2131 was obtained from cell envelopes by differential temperature extraction in the presence of detergent and salt. The isolated porin exhibited strong porin activity after reconstitution into lipid bilayer membranes. The effective channel diameter for the trimer was estimated as 1.5 nm from single channel conductance measurements in the presence of 1 M KCl. Moderate cation-selectivity was observed. Oligomeric porin migrated as a single band (apparent molecular weight 81 kDa) on sodium dodecyl sulfate polyacrylamide gelelectrophoresis when solubilized below 70 °C. The oligomers were converted into monomers on heating to 70 °C or above forming two bands with apparent molecular weight of 36 kDa and 35 kDa. The oligomer was not sensitive to EDTA. Its molecular weight was determined to be 119.3 kDa by analytical ultracentrifugation. The isoelectric point was 5.7. Circular dichroism data indicated a high content of -sheet structure. Gasphase sequencing of the N-terminal residues revealed the sequence: NH₂-Glu-Ile-Ser-Leu-Asn-Gly-Tyr-Gly-Arg-Phe. Crystals with a maximal side length of 300 m and diffracting to 0.32 nm resolution were obtained with the porin oligomer in the presence of C8E4 and 1,2,3-heptanetriol by using the vapor phase equilibration technique.Abbreviations C8E4 n-octyl tetraoxyethylene - M_r apparent molecular weight - Octyl-POE n-octyl polyoxyethylene - LDAO N,N-dimethyl dodecyl aminoxide - LPS lipopolysaccharide - PAGE polyacrylamide gel-electrophoresis - PEG polyethylene glycol     

Behaviour of suspended and sedimentary organic matter in the deltaic areas of the Gulf of Lions (Mediterranean Sea)

The behaviour of organic matter has been approached in two fluvio-marine areas of the Gulf of Lions in the Northwestern Mediterranean Sea: deltas of the Rhône and Têt rivers. Elemental analysis, pyrolysis-gas chromatography-mass spectrometry (main classes of organic compounds) and high performance liquid chromatography (phenolic compounds) enabled to show the fractioning processes and the differentiation of water layers from the river mouth to the open sea. Transitory horizontal and vertical geochemical gradients appeared in the water column. They were more marked in surficial sediments. They underlined the effects of the river inputs which were characterized by high phenol contents. The increase of the marine character towards the open sea was shown by increasing nitrogen-containing compounds. In the Rhône delta, the distribution of the suspended material and organic compounds emphasized the occurrence of a multi-layered water system. In the surficial layers the suspended organic matter was in a little degraded state. On the contrary, the bottom nepheloid contained altered organo-mineral complexes enriched in resistant organic compounds. In the deeper areas of the Lacaze-Duthiers Canyon, in front of the Têt prodelta, the abundance of phytoplanktonic material in the euphotic zone was emphasized by the increase of both nitrogen-containing compounds and aminosugars. In the deeper layers, the suspended material transported by advective currents showed a detrital characteri.e. enriched in clays and in degraded organic matter. The observations carried out in the Rhône delta, the Têt prodelta and the Lacaze-Duthiers Canyon underlined the importance of the general transfer system of suspended material from East to West enhanced by the Liguro-Provençal current.     

Biogeography of the octocorallian coelenterate fauna of southern Africa

The geographical and bathymetric distribution of southern African octocorals is analysed. Of the over 200 estimated species of regional octocorals, 81 confirmed and adequately described species are studied using a radial sector method. Two primary faunal components are recognized–endemic (53.3% of the fauna by numbers of species) and Indo-Pacific (39.4%). An Atlantic component contributes only minimally (about 1.7%), while the remaining fauna is made up of cosmopolites (2.8%) and scattered species (2.8%). A subantarctic component is not evident for the present-day, although evidence for previous contact is presented. A sister-group analysis using genera as a guide to sister species, shows the biogeographic affinities for the present-day fauna as a whole to be 45% Indo-Pacific, 31% cosmopolitan, 10% endemic, 10% Atlantic and 4% southern oceans (subantarctic). Applying the same method to only those genera with endemic species shows the affinities of the present-day endemic fauna to be 27.5% Indo-Pacific, 27.5% endemic, 24% cosmopolitan, 14% Atlantic and 7% subantarctic. Clearly defined boundaries for west, south, and east coast faunas (as recognized by previous authors in describing various intertidal faunas) are found not be present with regard to the octocoral fauna (largely due to its overwhelmingly subtidal nature). Instead two primary zoogeographic provinces are recognized–the Cape Endemic Province (extending from Liideritz to Inhaca Island) and the south-western fringe of the Indo-Pacific Province from East London north-eastwards. An overlap zone between these two is recognized between East London and Inhaca Island, with the region in the vicinity of Richards Bay having an essentially evenly mixed fauna (roughly 50% Cape Endemic Province and 50% Indo-Pacific). Of the 84 octocoral genera recorded for the region, seven (or 8.3%) are endemic, and of these, five are monotypic while two are ditypic. The fauna is shown to be predominantly sublittoral (about 95% by numbers of species), the shallow sublittoral (< 100 m in depth) being the region with highest species richness. Pennatulaceans are eurybathic (intertidal to 4756 m) and clearly show a high proportion of cosmopolites (20% of presently identified species). Soft corals are stenobathic and restricted to the intertidal, continental shelf and uppermost portion of the continental slope (<500m), while gorgonians are intermediate in depth distributon (intertidal to 1200 m). No cosmopolitan alcyonaceans are presently recorded. The centre of the Cape Endemic Province is the Agulhas Bank–an extensive region of shallow continential shelf (< 200 m in depth) between Cape Town and East London. Two regions of octocoral radiation for southern Africa are postulated–the Agulhas Bank and the western Indian Ocean.     

Characterization of New Highly Virulent German Isolates of Bremia lactucae and Efficiency of Resistance in Wild Lactuca spp. Germplasm

Four German isolates (FS1, SR2, SAW1 and DEG2) of Bremia lactucae originating from lettuce cultivars with R‐factors R18 and Dm6 + R36 were used for detailed characterization of virulence factors (v‐factors) and for the study of the resistance efficiency in wild Lactuca spp. germplasm. The highest complexity of v‐phenotype was recognized in isolate DEG2, which overcomes resistance in cv. Mariska (R18) and line CS‐RL (L. serriola × L. sativa, R18 + ?), until now known as resistant to all known races of B. lactucae in Europe. However, some sparse sporulation also occurred on cv. Titan (Dm6 + R36). The isolates SR2 and SAW1 overcome the resistance based on the gene R36, but are avirulent to R18. The v‐phenotype of SR2 is highly complex with the most important v‐factors being present except for v14 and v18. The isolate FS1 is the first in Germany originating from a cultivar with R18 (cv. Samourai). The search for efficient sources of resistance in 64 accessions of 11 wild Lactuca spp. and primitive forms of L. sativa showed broad variation in accession–isolate interactions. Expression of race‐specific resistance in wild Lactuca spp. (L. serriola, L. viminea, L. virosa) was recorded frequently. Lactuca indica and L. saligna could be considered as the most efficient sources of resistance against isolates FS1, SR2 and SAW1. The isolate DEG2 showed the highest level of virulence. On seedlings of L. saligna, which is considered as a most important source of resistance against B. lactucae, there was frequently recorded limited sporulation, however this response cannot be considered as a susceptible. Except for some L. saligna accessions (CGN 05310 and CGN 05315), the resistance to all studied isolates was only observed in one accession of L. serriola (PI 253467).     

RT－cDNA克隆－PCR法获取犬瘟热病毒融合蛋白基因（F）

纯化鸡胚成纤维细胞培养的犬瘟热病毒（ＣａｎｉｎｅＤｉｓｔｅｍｐｅｒＶｉｒｕｓ,ＣＤＶ）,获得病毒基因组ＲＮＡ后,反转录合成双链病毒Ｆ基因ｃＤＮＡ。将此双链ｃＤＮＡ平端插入ＰＵＣ１９质粒ＳａｍⅠ位点构建重组质粒,进行ｃＤＮＡ克隆。以重组克隆质粒为模板ＰＣＲ扩增,获得ＣＤＶ全长Ｆ基因。将此Ｆ基因插入表达载体ＰＢＶ２２０,在大肠杆菌中表达,通过对表达产物的最终鉴定,可确认所获片段为ＣＤＶ全长Ｆ基因．