The use of amphipathic maleimides to study membrane-associated proteins

A series of amphiphilic polymethylenecarboxymaleimides has been synthesized for use as sulfhydryl reagents applicable to membrane proteins. Physical properties of the compounds which are relevant to their proposed mode of action have been determined. By comparing rates of reaction in aqueous and aprotic solvents, the compounds have been shown to react exclusively with the thiolate ion. The effects of the reagents on three membrane-associated proteins are reported, and in two cases a comparative study has been made of the effects on the proteins in the absence of membranes. A mechanism is proposed whereby the reagents are anchored at the lipid/water interface by the negatively charged carboxyl group, thus siting the reactive maleimide in a plane whose depth is defined by the length of the reagent. Supporting evidence for this model is provided by the inability of the reagents to traverse membranes, and variation of their inhibitory potency with chain length when the proteins are embedded in the membrane, but not when extracted into solution. As examples of general use of the reagents to probe sulfhydryl groups in membrane proteins, the reagents have been used to (a) determine the depths in the membrane at which two populations of sulfhydryl groups occur in the mitochondrial phosphate transporter; (b) locate a single sulfhydryl associated with the active site ofD--hydroxybutyrate dehydrogenase in the inner mitochondrial membrane; (c) examine sulfhydryl groups in theD-3-glyceraldehyde phosphate dehydrogenase associated with the human red blood cell membrane.     

Biotransformation of diphenyl ether by the yeastTrichosporon beigelii SBUG 752

Trichosporon beigelii SBUG 752 was able to transform diphenyl ether. By TLC, HPLC, GC, GC-MS, NMR- and UV-spectroscopy, several oxidation products were identified. The primary attack was initiated by a monooxygenation step, resulting in the formation of 4-hydroxydiphenyl ether, 2-hydroxydiphenyl ether and 3-hydroxydiphenyl ether (48:47:5). Further oxidation led to 3,4-dihydroxydiphenyl ether. As a characteristic product resulting from the cleavage of an aromatic ring, the lactone of 2-hydroxy-4-phenoxymuconic acid was identified. The possible mechanism of ring cleavage to yield this metabolite is discussed.     

Crown alterations induced by decline: a study of relationships between growth rate and crown morphology in beech (Fagus sylvatica L.)

One of the first symptoms expressed by declining trees is reduced growth in stem diameter and length increment. The possibility of a relationship between length increment and crown thinning in beech (Fagus sylvatica L.) was investigated by developing a computer model to simulate first order branching patterns of the apical 2 m of monopodially branching beech trees, 70–100 years old, for a range of length increment rates. The model was based on values for branching angle, main axis and branch length increment, number of branches produced per year and branch mortality rates for six healthy and declining trees. Shoot growth rates in the apical 2 m of the sample trees ranged from about 5 cm/year (decline class 3) to 43 cm/ year (healthy). Simulations of branching patterns in the apical 2 m of trees growing at different rates indicated that, when growth rate exceeded about 20 cm/year, total first order branch length and area explored were independent of growth rate. When growth rates fell below this value there was a reduction in total area explored and first order branch length due primarily to the formation of fewer branches. More acute branching angles contributed to a reduction in the area explored. Growth rate-related crown thinning could increase the risk of bark necrosis and secondary pathogen infection during dry and/or hot spells.     

The xylanase introns from Cryptococcus albidus are accurately spliced in transgenic tobacco plants

The xylanase gene from Cryptococcus albidus contains seven introns. Genomic and cDNA clones under the control of the CaMV 35S promoter were transferred into tobacco plants using Agrobacterium-mediated cell transformation. The genes were transcribed and the mRNAs were amplified by the polymerase chain reaction using primers on each side of the intron region. About 90% of the amplification products from plants transformed with the genomic clone corresponded to the size of the pre-mRNA (1.2 kb) and 10% represented the spliced product (0.85 kb). The 0.85 kb fragment was cloned and sequenced and the result indicated that the introns from the xylanase gene were accurately spliced by the plant cells.     

A substance released by metamorphosing larvae and young polyps ofHydractinia echinata induces metamorphosis in conspecific larvae

Summary A metamorphosis-inducing factor was isolated from medium conditioned by either metamorphosing larvae or 3-day postmetamorphic primary polyps. The factor has a molecular weight 8 kDa and is heatlabile. It does not induce metamorphosis of isolated posterior fragments and is therefore not identical to the internal signal described by Schwoerer-Böhning et al. (1990). The biological significance of the substance is currently unclear, therefore its inducing activity may be a side effect.     

Sedimentary records of accelerated nutrient loading in Florida lakes

Transfer functions relating trophic state (Carlson's TSI_{chlorophyll-a}) to present day accumulation rate of (1) nutrients, (2) cations, and (3) organic sediment, are computed using Binford's ²¹⁰Pb-dilution method. As computed from surficial sediments of 27 lakes, former trophic states are reconstructed for recent (²¹⁰Pb-dated) sedimentary histories of 14 lakes. Of the three kinds of models potentially available, model (3) (TSI vs. organic accumulation) is the weakest statistically, and may be unduly influenced by exceptional deposition and/or preservation of allochthonous organic matter. At present, however, it is the only model applicable to all 14 of the histories tested. Results are encouraging in that model accurately predicts observed TSI's in several mesotrophic and eutrophic lakes. Clearly significant increases (accelerations) are inferred only for a of the most eutrophic lakes of the set, while the model consistently overpredicts TSI's of the 7 most oligotrophic lakes. As Whitmore's diatom-assemblage index is a better predictor of TSI than is model (3) in the one eutrophic lake in which it has been tested, we expect more persuasive results when models (1) and (2) can be tested within a more complete set of analytical data. We were surprised to find 3 severely disturbed lakes among the 12 that show little or no acceleration in rate of eutrophication in recent decades, but we defer attempts at explanation until former nutrient loading can be tested by model (1).     

Rhodobacteraceae on the marine brown alga Fucus spiralis are abundant and show physiological adaptation to an epiphytic lifestyle

Macroalgae harbour specific microbial communities on their surface that have functions related to host health and defence. In this study, the bacterial biofilm of the marine brown alga Fucus spiralis was investigated using 16S rRNA gene amplicon-based analysis and isolation of bacteria. Rhodobacteraceae (Alphaproteobacteria) were the predominant family constituting 23% of the epibacterial community. At the genus level, Sulfitobacter, Loktanella, Octadecabacter and a previously undescribed cluster were most abundant, and together they comprised 89% of the Rhodobacteraceae. Supported by a specific PCR approach, 23 different Rhodobacteraceae-affiliated strains were isolated from the surface of F. spiralis, which belonged to 12 established and three new genera. For seven strains, closely related sequences were detected in the 16S rRNA gene dataset. Growth experiments with substrates known to be produced by Fucus spp. showed that all of them were consumed by at least three strains, and vitamin B₁₂ was produced by 70% of the isolates. Since growth of F. spiralis depends on B₁₂ supplementation, bacteria may provide the alga with this vitamin. Most strains produced siderophores, which can enhance algal growth under iron-deficient conditions. Inhibiting properties against other bacteria were only observed when F. spiralis material was present in the medium. Thus, the physiological properties of the isolates indicated adaption to an epiphytic lifestyle.     

Specific and unspecific responses of plants to cold and drought stress

Different environmental stresses to a plant may result in similar responses at the cellular and molecular level. This is due to the fact that the impacts of the stressors trigger similar strains and downstream signal transduction chains. A good example for an unspecific response is the reaction to stressors which induce water deficiency e.g. drought, salinity and cold, especially frost. The stabilizing effect of liquid water on the membrane bilayer can be supported by compatible solutes and special proteins. At the metabolic level, osmotic adjustment by synthesis of low-molecular osmolytes (carbohydrates, betains, proline) can counteract cellular dehydration and turgor loss. Taking the example of Pinus sylvestris, changes at the level of membrane composition, and concomitantly of photosynthetic capacity during frost hardening is shown. Additionally the effect of photoperiod as measured via the phytochrome system and the effect of subfreezing temperatures on the incidence of frost hardening is discussed. Extremely hydrophilic proteins such as dehydrins are common products protecting not only the biomembranes in ripening seeds (late embryogenesis abundant proteins) but accumulate also in the shoots and roots during cold adaptation, especially in drought tolerant plants. Dehydrins are characterized by conserved amino acid motifs, called the K-, Y-or S-segments. Accumulation of dehydrins can be induced not only by drought, but also by cold, salinity, treatment with abscisic acid and methyl jasmonate. Positive effects of the overexpression of a wild chickpea (Cicer pinnatifidum) dehydrin in tobacco plants on the dehydration tolerance is shown. The presentation discusses the perception of cold and drought, the subsequent signal transduction and expression of genes and their products. Differences and similarities between the plant responses to both stressors are also discussed.