An improved protein bioreactor: efficient product isolation during in vitro protein biosynthesis via affinity tag

In vitro protein biosynthesis became a powerful technology for biochemical research. Beside the determination of structure and function in vitro selection of proteins is also of great interest. In most cases the use of a synthesized protein for further applications depends on its purity. For this purpose the in vitro production and purification of proteins with short affinity tails was established. A cell-free protein synthesis system was employed to produce bovine heart fatty acid-binding protein and bacterial chloramphenicol acetyltransferase with and without fusion of the Strep-tag affinity peptide. The quantitative removal of fusion protein during cell-free synthesis from a batch reaction and a semicontinuous flow cell-free reactor were achieved. No significant influence of the Strep-tag and the conditions during the affinity chromatography on maturation or activity of the proteins were observed. The product removal from the continuous flow cell-free reactor is still an only partially solved problem, because the use of ultrafiltration membranes has some limitations. The results document that it should be possible to avoid these limitations by introducing an affinity system.     

Characterization of the Maleylacetate Reductase MacA of Rhodococcus opacus 1CP and Evidence for the Presence of an Isofunctional Enzyme

Maleylacetate reductases (EC 1.3.1.32) have been shown to contribute not only to the bacterial catabolism of some usual aromatic compounds like quinol or resorcinol but also to the degradation of aromatic compounds carrying unusual substituents, such as halogen atoms or nitro groups. Genes coding for maleylacetate reductases so far have been analyzed mainly in chloroaromatic compound-utilizing proteobacteria, in which they were found to belong to specialized gene clusters for the turnover of chlorocatechols or 5-chlorohydroxyquinol. We have now cloned the gene macA, which codes for one of apparently (at least) two maleylacetate reductases in the gram-positive, chlorophenol-degrading strain Rhodococcus opacus 1CP. Sequencing of macA showed the gene product to be relatively distantly related to its proteobacterial counterparts (ca. 42 to 44% identical positions). Nevertheless, like the known enzymes from proteobacteria, the cloned Rhodococcus maleylacetate reductase was able to convert 2-chloromaleylacetate, an intermediate in the degradation of dichloroaromatic compounds, relatively fast and with reductive dehalogenation to maleylacetate. Among the genes ca. 3 kb up- and downstream of macA, none was found to code for an intradiol dioxygenase, a cycloisomerase, or a dienelactone hydrolase. Instead, the only gene which is likely to be cotranscribed with macA encodes a protein of the short-chain dehydrogenase/reductase family. Thus, the R. opacus maleylacetate reductase gene macA clearly is not part of a specialized chlorocatechol gene cluster.Maleylacetate reductases (EC 1.3.1.32) have long been known to be involved in the degradation of chloroaromatic compounds via chlorocatechols as intermediates (10, 31). By reduction of a carbon-carbon double bond they form 3-oxoadipate, a metabolite also of catechol catabolism, and thus compensate for the different oxidation states of chlorinated and nonchlorinated compounds. 2-Chloromaleylacetate, which is formed during turnover of several dichlorocatechols, is initially reductively dechlorinated and then reduced to 3-oxoadipate in a second reaction (22, 47).Corresponding to the biochemical function in chlorocatechol degradation, the following maleylacetate reductase genes have been shown to be associated with dioxygenase, cycloisomerase, and dienelactone hydrolase genes as components of specialized chlorocatechol catabolic operons: tfdF and tfdF_II on pJP4 from the 2,4-dichlorophenoxyacetate-utilizing strain Ralstonia eutropha (Alcaligenes eutrophus) JMP134 (29, 33, 37, 44), tcbF on pP51 from the 1,2,4-trichlorobenzene-degrading strain Pseudomonas sp. strain P51 (45), and clcE from the 3-chlorobenzoate catabolizing strains Pseudomonas sp. strain B13 and Pseudomonas putida AC866(pAC27) (15, 20, 21). Catechol degradation, in contrast, does not require a maleylacetate reductase activity, and corresponding genes do not belong to the known catechol operons. Thus, while at least two of the chlorocatechol catabolic enzymes, i.e., the dioxygenases and cycloisomerases, appear to have been recruited from catechol catabolism, maleylacetate reductase genes must have had a different origin and original function (34).The postulated original function of the maleylacetate reductases is still under discussion. In bacteria, these enzymes have been shown to play a role, for example, in quinol, resorcinol, and 2,4-dihydroxybenzoate degradation (6, 25, 41). Other aromatic growth substrates involving the action of maleylacetate reductase are more exotic, since they carry a fluorine substituent (35), a sulfo group (14), a nitro group (18, 40), or several chlorine substituents (8, 26, 48). Maleylacetate reductase genes have been shown to be part of a specialized gene cluster for 2,4,5-trichlorophenoxyacetate degradation (8, 9) and of a gene cluster for hydroxyquinol conversion which contributes to 4-nitrophenol turnover (4).The chlorocatechol pathway of the chlorophenol-utilizing strain Rhodococcus opacus (erythropolis) 1CP obviously evolved functionally convergent to the corresponding pathway in the proteobacteria mentioned above (13, 39). Thus, it is not surprising that the chlorocatechol gene cluster of strain 1CP is organized differently from the corresponding proteobacterial operons; in fact, its characterization showed that it does not comprise a maleylacetate reductase gene (13). Thus, the nature of the gene cluster(s) encoding a maleylacetate reductase in R. opacus remained to be elucidated. Such gene clusters could complement otherwise incomplete pathways, and they might also have provided the source from which the maleylacetate reductase gene was recruited during evolution of dedicated pathways, such as the proteobacterial chlorocatechol catabolic route.(Some of the results presented here have previously been reported in a preliminary communication [38].)     

The synthesis of the tripodal phosphine CH3C{CH2P(m-CF3C6H4)2}3 and its rhodium coordination chemistry

The new tripodal phosphine CH₃C{CH₂P(m-CF₃C₆H₄)₂}₃, CF₃PPP, was prepared by reacting CH₃C(CH₂Br)₃ with Li⁺P(m-CF₃C₆H₄)₂⁻, the latter being best obtained by adding Li⁺NⁱPr₂⁻ to PH(m-CF₃C₆H₄)₂. The rhodium complexes [RhCl(CO)(CF₃PPP)], [Rh(LL)(CF₃PPP)](CF₃SO₃) (LL = 2 CO or NBD), [RhX₃(CF₃PPP)], [RhX(MeCN)₃(CF₃PPP)](CF₃SO₃)₂ (X = H and Cl), [RhCl₂(MeCN)(CF₃PPP)](CF₃SO₃) and [Rh(MeCN)₃(CF₃PPP)](CF₃SO₃)₃ were prepared and characterized. The X-ray crystal structure of [Rh(NBD)(CF₃PPP)](CF₃SO₃) is reported. The lower oxygen sensitivity of the CF₃PPP rhodium(I) complexes, relative to the corresponding species with the parent ligand CH₃C(CH₂PPh₂)₃, is attributed to the higher effective nuclear charge on the metal centers caused by the presence of the six CF₃ substituents on the terdentate phosphine. A similar effect may be responsible for the easier hydrolysis of the CF₃PPP-containing, cationic rhodium(III) complexes relative to the corresponding compounds of the parent ligand.     

On the Fine Structure of the Nervous System of Tubiluchus philippinensis (Tubiluchidae,Priapulida)

At the mouth tube/introvert border a circumenteric intraepithelial nerve ring occupies a circular ridge protruding into the body cavity. The ring has a centrally located neuropile nearly free of perikarya and two zones of different perikarya above and below the neuropile. Presumably non-neuronal perikarya have an oval nucleus, large heterochromatin clumps and marked filament bundles. Such elements resemble tanycytic glial cells. Two types of presumably neuronal perikarya contain small cytoplasmic granules, similar to those in nerve fibre profiles. One of these neurons has a pale nucleus with a prominent nucleolus, the other a rather inconspicuous nucleus similar to that of the tanycytic cells. The neuronal processes of the fibre ring differ in diameter and contain clear and dense core vesicles, small granules (high or medium electron density) or granules with a dense periphery and a light centre. Sometimes neighbouring processes seem interconnected by electrical synapses. Images suggesting chemical synapses are rare. A large intraepithelial nerve lies in the wall of the introvert and ventral body wall close to the musculature, possibly innervated by this nerve. Frontal of the anus lies an intraepithelial ganglion demonstrating again a central neuropile. two neuronal types and tanycytic elements with filament bundles. Comparative aspects of the characters of the Tubiluchus nervous system are also discussed.     

Combining the enantioselectivity of a cyclodextrin and a diamide selector in a mixed binary gas‐chromatographic chiral stationary phase

The present work reports on the investigation of a mixed binary chiral stationary phase (CSP) prepared by simultaneous attachment of permethylated‐β‐cyclodextrin ( D selector) and resorcinarene with pendant l ‐ or d ‐valine diamide groups ( L′ and D′ selectors, respectively) to a polysiloxane matrix via platinum‐catalyzed hydrosilylation. The gas‐chromatographic investigation of a number of racemates on the four different CSPs ( D, D′, DD′, and DL′ ) showed that the enantioselectivity of the individual chiral selectors was retained in the mixed binary CSPs. As a consequence, hydrocarbons, underivatized alcohols, ketones, and almost all proteinogenic amino acid derivatives could be separated simultaneously on each of the mixed CSPs. Matched and mismatched cases of enantioseparation on the mixed binary CSPs were observed but turned out to be of minor importance for enantiomeric separation. In general, more racemates were separated with α ≥ 1.02 on the mixed phases as compared to the single phases. In order to analyze the influence of the presence of the diamide selector on the enantioselectivity of the cyclodextrin selector, a mixed ternary CSP containing the selector D and a racemic mixture of the selectors D′ and L′ [ D ( D′L′ )] was prepared and investigated. Merits and limitations of the approach of mixed binary CSPs are discussed. © 2005 Wiley‐Liss, Inc. Chirality     

Optimized conditions for rapd analysis inPinus radiata

Summary Pinus radiata is the most important softwood plantation species in Australia and New Zealand. The improtance of this species in forestry has led to an increasing demand to improve the efficiency of selection time of the production population, which currently takes 13 yr by traditional methods. With the application of molecular biology techniques such as random amplified polymorphic DNA (RAPD) the selection period can be reduced to 6 yr. In this study, the conditions for RAPD were optimized and the feasibility of this marker system was investigated with different families ofPinus radiata from Tasmania and South Australia. Best concentrations of Taq-polymerase (1 U), magnesium chloride (2 mM), and template DNA (20 ng) were selected to test different polymerase chain reaction (PCR) thermocycler profiles. Devey's et al. (1996) program was the most effective for production of clear RAPD bands. Best conditions were investigated to screen 10–12 bp arbitrary Breasatec and Operon primers. Both types were found useful at detecting genetic variation between families. Seventy percent and thirty percent of the selected Bresatec and Operon primers, respectively, produced polymorphic bands.     

Community composition and sensitivity of periphyton to atrazine in flowing waters: the role of environmental factors

The relationship between environmental variables, community composition and the sensitivity of periphyton on artificial substrata to the herbicide atrazine (EC₅₀ values obtained by concentration-effect curves of photosynthesis to atrazine) was studied for 20 stream and river sites on a latitudinal across Europe (Sweden, The Netherlands, Spain). Sensitivity to atrazine was higher in Swedish than in the Spanish or Dutch sites. Direct gradient analyses were used to relate diatom taxa and algal groups with environmental variables. A first redundancy analysis (RDA) based on diatom taxa showed a pollution gradient (atrazine and nutrient concentration) associated to diatom taxa that are indicators of different degrees of pollution. A second RDA based on algal groups showed that diatom-dominated communities corresponded both to sites at higher altitudes and less industrialized areas and to sites with higher atrazine concentration; Cyanobacteria were the most common in industrial areas, whereas Chlorophyceae dominated in sites with high water temperature and alkalinity. Linear regression analyses were applied to find the relationship between the ordination axes obtained and the EC₅₀ values. First axes of both RDA showed significant or marginally significant relationship with atrazine sensitivity. Regression analyses for the Spanish sites indicated that the sensitivity to atrazine was related with light conditions (EC₅₀ was positively correlated with light) and the percentage of different algal groups (EC₅₀ was positively correlated with the percentage of diatoms and negatively correlated with the percentage of green algae). The results indicating that differences in sensitivity are related to environmental variables such as light, nutrients or atrazine concentration, permitted us to identify biological indicators of sensitivity to atrazine in lotic systems: Bacillariophyceae-dominated periphyton communities were more tolerant than Chlorophyceae and Chrysophyceae-dominated communities. In addition, diatom taxa found to be tolerant to atrazine in this study have been considered in the literature to be tolerant to organic pollution. This revised version was published online in June 2006 with corrections to the Cover Date.     

Differential expression of annexins I, II and IV in human tissues: an immunohistochemical study

 Annexins constitute a family of Ca²⁺- and phospholipid-binding proteins. Although their functions are still not clearly defined, several members of the annexin family have been implicated in membrane-related events along exocytotic and endocytotic pathways. To elucidate a possible correlation of those functional proposals with the tissue distribution of annexins, we analysed immunohistochemically the expression of annexins I, II and IV in a broad variety of human tissues. Annexins I and II were chosen for this study since their functionally relevant N-terminal domains are structurally closely related, whilst annexin IV is structurally less related to the former two proteins. The study revealed distinct expression patterns of annexins I, II and IV throughout the body. Annexin I was found in leucocytes of peripheral blood, tissue macrophages and T-lymphocytes and in certain epithelial cells (respiratory and urinary system, superficial cells of non-keratinised squamous epithelium), annexin II in endothelial cells, myoepithelial cells and certain epithelial cells (mainly respiratory and urinary system), whereas annexin IV was almost exclusively found in epithelial cells. Epithelia of the upper respiratory system, Bowman’s capsule, urothelial cells, mesothelial cells, peripheral nerves, the choroid plexus, ependymal cells and pia mater and arachnoid of meninges generally strongly expressed all three annexins investigated. The characteristic expression in different tissues and the intracellular distribution indicates that the three annexins investigated are involved in aspects of differentiation and/or physiological functions specific to these tissues. Accepted: 15 January 1998