Temperature-induced shifts in associations of longevity with body size in Drosophila melanogaster

One of the hypotheses of growing interest in studies of responses to thermal environments suggests that trade-offs and other trait associations may be altered by temperature. Here, the commonly observed positive association between body size and longevity was examined at two adult test temperatures, 14 degrees C and 25 degrees C, in cold-stress-selected lines (S) and their controls (C) in 25 degrees C-reared Drosophila melanogaster. Thorax length (TL) and developmental time (DT) were also scored in 25 degrees C-reared individuals before and after one generation of truncation selection on longevity. The topography of the selection surface that relates longevity to thorax and wing size was temperature dependent and differed both between lines and between sexes. Longevity increased monotonically with body size (TL) in C and S females at 25 degrees C but, surprisingly, longevity decreased with body size in S individuals at 14 degees C. Body size did not diverge between S and C lines and showed no response to longevity selection. However, DT increased by 25 degrees C-longevity selection in C individuals and decreased by 14 degrees C-longevity selection in S individuals. These results suggest that trait associations (including the commonly observed trade-off between body size and DT) can greatly depend on temperature, as a shift in the sign of the correlation is possible at low temperature. Genotype x temperature interaction is an important source of variation in the relationship between soma size and longevity.     

Scale-up and optimization of an acoustic filter for 200 L/day perfusion of a CHO cell culture

Acoustic cell retention devices have provided a practical alternative for up to 50 L/day perfusion cultures but further scale-up has been limited. A novel temperature-controlled and larger-scale acoustic separator was evaluated at up to 400 L/day for a 10(7) CHO cell/mL perfusion culture using a 100-L bioreactor that produced up to 34 g/day recombinant protein. The increased active volume of this scaled-up separator was divided into four parallel compartments for improved fluid dynamics. Operational settings of the acoustic separator were optimized and the limits of robust operations explored. The performance was not influenced over wide ranges of duty cycle stop and run times. The maximum performance of 96% separation efficiency at 200 L/day was obtained by setting the separator temperature to 35.1 degrees C, the recirculation rate to three times the harvest rate, and the power to 90 W. While there was no detectable effect on culture viability, viable cells were selectively retained, especially at 50 L/day, where there was a 5-fold higher nonviable washout efficiency. Overall, the new temperature-controlled and scaled-up separator design performed reliably in a way similar to smaller-scale acoustic separators. These results provide strong support for the feasibility of much greater scale-up of acoustic separations.     

In vitro hemocompatibility testing of biomaterials according to the ISO 10993-4

The development of synthetic materials, textured polymers and metals and their increasing use in medicine make research of biomaterials' hemocompatibility very relevant. Problems arise from the polymorphism and diversity of the different materials, the static and dynamic test models and the patients' individual biologic factors. First, methods, models, tests as well as preanalytical factors have to be standardized according to the current knowledge in medicine laid down in the ISO 10993 part 4. The routine controls used in clinical chemistry and hematology have to be performed. Information about normal ranges (mean value, standard deviation, 95% confidence interval) should be provided. Tests have to be performed within a minimal delay of usually 2 h since some properties of blood change rapidly following collection. Various conditions (depending on the wall shear rate) were simulated within the centrifugation system and a Chandler system. Qualities and aspects of hemocompatibility such as platelet activation, oxidative burst, hemolysis, fibrinolysis, fibrin formation, generation of thrombin, contact activation, and complement activation were analysed and the results were entered non-dimensionally into a non-dimensional score system, where 0 points stand for the best and 65 points for the worst evaluation. We found a good correlation between the total score and contact activation, thrombin generation and leukocyte activation in a low shear stress system and a good correlation between the total score and thrombin generation, hemolysis and platelet activation in the high shear stress system. Further on the effect of additives and sterilization procedures can be measured. The concepts presented underline the relevance/importance of an efficient diagnostic approach to hemocompatibility that takes account of clinical and socio-economic concerns.     

Phylogenetic relationships and taxonomic position of Chlorella -like isolates from low pH environments (pH < 3.0)

Background  

Little is known about phytoplankton communities inhabiting low pH environments such as volcanic and geothermal sites or acidic waters. Only specialised organisms are able to tolerate such extreme conditions. There is, thus, low species diversity. We have characterised the previously isolated acid tolerant Chlorella -like microalgae Viridiella fridericiana and Chlorella protothecoides var. acidicola by microscopical and biomolecular methods in order to assess their phylogenetic relationships.     

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.     

Response of beech (Fagus sylvatica) to elevated CO2 and N: Influence on larval performance of the gypsy moth Lymantria dispar (Lep., Lymantriidae)

Two-year-old beech seedlings were kept from germination to bioassays with Lymantria dispar under the following conditions: ambient CO₂/low N, elevated CO₂/low N, ambient CO₂/elevated N, and elevated CO₂/elevated N. The effect of these growing conditions of the trees on the performance of the defoliator L. dispar was studied 2 years after initiating the tree cultivation. The developmental success of third-instar larvae of L. dispar was characterized by the weight gained, percentage of weight gain, relative growth rate (RGR), relative consumption rate (RCR), and efficiency of conversion of ingested food into body substance (ECI). Contrary to our expectations, additional N-fertilization did not increase and elevated CO₂ did not delay larval growth rate. However, the environmental treatments of the beech seedlings were found to affect the larval performance. Larvae consumed significantly higher amounts of foliage (RCR) on beech trees under controlled conditions (ambient CO₂ and low N) compared to those under elevated CO₂ and enhanced N. The opposite was true for ECI. The lowest efficacy to convert consumed food to body substance was observed under control conditions and the highest when the larvae were kept on beech trees grown under elevated CO₂ and additional N-fertilization. These opposite effects resulted in the weight gain-based parameters (absolute growth, percentage of growth, and RGR) of the gypsy moth larvae remaining unaffected. The results indicate that the gypsy moth larvae are able to change their ECI and RCR to obtain a specific growth rate. This is discussed as an adaptation to specific food qualities.