Effect of electrolytes and surfactants on the thermoseparation of an ethylene oxide–propylene oxide random copolymer in aqueous solution

The thermoseparation of aqueous solutions of Breox 50 A 1000, an ethylene oxide–propylene oxide 50:50 (w/w) random copolymer, was studied. The cloud-point diagram for Breox in water solution and the effects of electrolytes and surfactants on the cloud-point temperature (CPT) were determined. The Breox concentration in both phases after the thermoseparation was followed with a reversed-phase HPLC method. The effects of separation temperature and additives on phase composition were evaluated.     

Oligosaccharide modification in the early secretory pathway directs the selection of a misfolded glycoprotein for degradation by the proteasome

The role of conformation-based quality control in the early secretory pathway is to eliminate misfolded polypeptides and unassembled multimeric protein complexes from the endoplasmic reticulum, ensuring the deployment of only functional molecules to distal sites. The intracellular fate of terminally misfolded human alpha1-antitrypsin was examined in hepatoma cells to identify the functional role of asparagine-linked oligosaccharide modification in the selection of glycoproteins for degradation by the cytosolic proteasome. Proteasomal degradation required physical interaction with the molecular chaperone calnexin. Altered sedimentation of intracellular complexes following treatment with the specific proteasome inhibitor lactacystin, and in combination with mannosidase inhibition, revealed that the removal of mannose from attached oligosaccharides abrogates the release of misfolded alpha1-antitrypsin from calnexin prior to proteasomal degradation. Intracellular turnover was arrested with kifunensine, implicating the participation of endoplasmic reticulum mannosidase I in the disposal process. Accelerated degradation occurred in a mannosidase-independent manner and was arrested by lactacystin, in response to the posttranslational inhibition of glucosidase II, demonstrating that the attenuated removal of glucose from attached oligosaccharides functions as the underlying rate-limiting step in the proteasome-mediated pathway. A model is proposed in which the removal of mannose from multiple attached oligosaccharides directs calnexin in the selection of misfolded alpha1-antitrypsin for degradation by the proteasome.     

Large-scale production of pharmaceutical-grade plasmid DNA for gene therapy: problems and bottlenecks

Gene therapy is a promising process for the prevention, treatment and cure of diseases such as cancer, acquired immunodeficiency syndrome (AIDS) and cystic fibrosis. One of the methods used to administer therapeutic genes is the direct injection of naked or lipid-coated plasmid DNA, but this requires considerable amounts of plasmid DNA. There are several problems and bottlenecks associated with the design and operation of large-scale processes for the production of pharmaceutical-grade plasmid DNA for gene therapy.     

Composition, temporal changes and ecological guild classification of the ichthyofaunas of large European estuaries – a comparison between the Tagus (Portugal) and the Elbe (Germany)

Fish species densities from 1025 hauls obtained with beam trawl and stow net catches from the southern Tagus and northern Elbe estuaries were analysed for two different periods of time between 1978 and 1997. The data were related with environmental factors to compare species composition and densities, contributions of different ecological guilds, structuring environmental key factors, intra‐annual cyclicity and interperiodical stability of ichthyofaunas in large European estuaries at different latitudes. Although the total number of species in both the Tagus and Elbe estuaries (Tagus: 57 species, Elbe: 58 species) was very similar, significant differences were estimated in species compositions, species densities and number of species, and individuals of the different ecological guilds between both estuaries. A total of 15 species occurred in both estuaries but only two of these species, the catadromous Anguilla anguilla and the marine estuarine opportunist Pleuronectes flesus, contributed substantially to the total abundance. Marine species together contributed more than 5% of the total number of species in the Tagus and Elbe estuaries, whereas their contribution in number of individuals amounted to 22% in the Tagus and only 9% in the Elbe. In terms of number of individuals, the ichthyofauna of the Tagus estuary was dominated by estuarine species (especially Pomatoschistus spp.) contributing 68% of the total abundance. In contrast, the fish fauna of the Elbe estuary was dominated by high numbers of anadromous species (88%) individuals, especially Osmerus eperlanus. Canonical corresponding analyses (CCA) indicated that, besides intra‐annual variables, salinity and mouth distance were two of the most important environmental factors structuring the fish communities in both estuaries. Moreover, water depth was found to be an important environmental factor in the Tagus estuary, whereas the water temperature substantially affected the ichthyofaunal composition in the Elbe estuary. In both estuaries, contributions by number of species and number of individuals of the different ecological guilds were not statistically different between the two periods considered, 1978–80 and 1995–97 for the Tagus, and 1981–86 and 1989–95 for the Elbe. Nevertheless, from 1981–86 until 1989–95 a remarkable increase of about 4.5 times in the densities of O. eperlanus was observed in the Elbe estuary, probably related to improved water quality, especially of oxygen conditions in the nurseries since 1990. This was also supported by the results of CCA, suggesting important influences of the years 1989 and 1990 on the Elbe estuary fish assemblage. Climate change as a result of global warming may be evident for the observed changes of the densities of P. flesus which decreased in the southern Tagus estuary and, in contrast, increased in the northern Elbe estuary during comparable periods of time. It is recommended to use the ecological guild classification scheme which was applied for the Tagus and Elbe estuaries for further studies of European estuarine fish assemblages.     

DNA base sequence changes induced by diethyl sulfate in postmeiotic male germ cells of Drosophila melanogaster

Summary The DNA base sequence changes induced by diethyl sulfate (DES) were analyzed in postmeiotic male germ cells of Drosophila melanogaster. 31 transmissible vermilion mutants were recovered in F₁ and F₂ generations, with a frequency of 2.6 × 10^–4 for the F₁, and of 1.8–13 × 10^–4 for the F₂. The results show that DES induces both base pair substitutions (93%) and deletions (7%). In accord with its relatively high ability to alkylate oxygens in DNA, the most frequent type of sequence alteration among the basepair changes are GC-AT transitions, accounting for 73% of mutations, followed by transversions AT-TA (10%). DES also induced AT-GC transitions and AT-CG transversions. Both induced deletions were intralocus deletions, not occurring between basepair repeats. No influence of neighboring bases on the mutation position was found.     

Nucleotide sequence of the D-loop region of the sperm whale (Physeter macrocephalus) mitochondrial genome

We have amplified, by the polymerase chain reaction, and have sequenced the D-loop region of the mitochondrial DNA from the sperm whale (Physeter macrocephalus). The sperm whale D-loop was aligned with D- loop sequences from four other cetaceans (Commerson's dolphin, orca, fin whale, and minke whale) and an out-group (cow). This alignment showed the sperm whale sequence to be larger than that of other cetaceans. In addition, some sequence blocks were highly conserved among all six species, suggesting roles in the functioning of mitochondrial DNA. Other blocks that were previously reported to be well conserved among cetaceans showed little sequence conservation with the sperm whale D-loop, which argues against the functional importance of these sequence blocks in cetaceans.      

Continuous lipolysis in a reversed micellar membrane bioreactor

The enzymatic hydrolysis of olive oil using Chromobacterium viscosum lipase B encapsulated in reversed micelles of AOT in isooctane was carried out in a continous reversed micellar membrane bioreactor. A tubular ceramic membrane installed in an ultrafiltration module was used to retain the lipase and separate the products from the reaction media. Water filled micelles were supplemented to the reactor together with the substrate/solvent solution to compensate for the permeation of reversed micelles. The influence of substrate concentration, residence time and water content in the productivity and conversion of the system were investigated. A linear relationship between productivity and conversion degree was found for each substrate concentration tested. Operational stability of the bioreactor was tested in a long term operation confirming the high stability of this catalytic system.List of Symbols a(S ₀ ), b(S ₀ ) parameters of Eq. (3) - [AOT] mM AOT concentration - C _c mM concentration in the concentrate - C _p mM concentration in the permeate - E _t mg total amount of lipase - [lipase] mg/cm³ overall lipase concentration - [OIL] mM olive oil concentration - [OLEIC] mM oleic acid concentration in the permeate - P mol/(min · mg) oleic acid productivity - Q _in cm³/min inlet flow rate - Q _out cm³/min outlet flow rate (equal to permeate flow rate) - Q _r cm³/min recirculation flow rate - W _o ratio of water to AOT molar concentrations - X steady state conversion degree in the permeate stream - T °C temperature - rejection coefficient