Rapid adaptation to microgravity in mammalian macrophage cells.

Despite the observed severe effects of microgravity on mammalian cells, many astronauts have completed long term stays in space without suffering from severe health problems. This raises questions about the cellular capacity for adaptation to a new gravitational environment. The International Space Station (ISS) experiment TRIPLE LUX A, performed in the BIOLAB laboratory of the ISS COLUMBUS module, allowed for the first time the direct measurement of a cellular function in real time and on orbit. We measured the oxidative burst reaction in mammalian macrophages (NR8383 rat alveolar macrophages) exposed to a centrifuge regime of internal 0 g and 1 g controls and step-wise increase or decrease of the gravitational force in four independent experiments. Surprisingly, we found that these macrophages adapted to microgravity in an ultra-fast manner within seconds, after an immediate inhibitory effect on the oxidative burst reaction. For the first time, we provided direct evidence of cellular sensitivity to gravity, through real-time on orbit measurements and by using an experimental system, in which all factors except gravity were constant. The surprisingly ultra-fast adaptation to microgravity indicates that mammalian macrophages are equipped with a highly efficient adaptation potential to a low gravity environment. This opens new avenues for the exploration of adaptation of mammalian cells to gravitational changes.

The oxidative burst reaction in mammalian cells depends on gravity.

Gravity has been a constant force throughout the Earth's evolutionary history. Thus, one of the fundamental biological questions is if and how complex cellular and molecular functions of life on Earth require gravity. In this study, we investigated the influence of gravity on the oxidative burst reaction in macrophages, one of the key elements in innate immune response and cellular signaling. An important step is the production of superoxide by the NADPH oxidase, which is rapidly converted to H2O2 by spontaneous and enzymatic dismutation. The phagozytosis-mediated oxidative burst under altered gravity conditions was studied in NR8383 rat alveolar macrophages by means of a luminol assay. Ground-based experiments in "functional weightlessness" were performed using a 2 D clinostat combined with a photomultiplier (PMT clinostat). The same technical set-up was used during the 13th DLR and 51st ESA parabolic flight campaign. Furthermore, hypergravity conditions were provided by using the Multi-Sample Incubation Centrifuge (MuSIC) and the Short Arm Human Centrifuge (SAHC). The results demonstrate that release of reactive oxygen species (ROS) during the oxidative burst reaction depends greatly on gravity conditions. ROS release is 1.) reduced in microgravity, 2.) enhanced in hypergravity and 3.) responds rapidly and reversible to altered gravity within seconds. We substantiated the effect of altered gravity on oxidative burst reaction in two independent experimental systems, parabolic flights and 2D clinostat / centrifuge experiments. Furthermore, the results obtained in simulated microgravity (2D clinorotation experiments) were proven by experiments in real microgravity as in both cases a pronounced reduction in ROS was observed. Our experiments indicate that gravity-sensitive steps are located both in the initial activation pathways and in the final oxidative burst reaction itself, which could be explained by the role of cytoskeletal dynamics in the assembly and function of the NADPH oxidase complex.

A system for repertoire cloning and phage display of murine and leporid antibody fragments.

Even though rabbit antibodies (Abs) are known to exceed murine Abs with respect to specificity, affinity, and stability, cloned leporid immune repertoires have been rarely considered in recombinant Ab preparation for environmental analysis. We have developed a set of four tet(p/o)-based phasmid vectors that allow the efficient cloning of both murine and leporid Ab repertoires. These vectors differ in the design of the cloning sites, choice of signal peptides, and antibiotic selection markers. A set of 39 primer oligodeoxynucleotides has been developed for the PCR amplification of rabbit Ab genes, representing the most exhaustive coverage of the leporid immune repertoire described so far. The atrazine-specific murine Fab fragment K411B and a cloned V-gene repertoire from sulfonamide-immunized rabbits were used to compare these phasmids with respect to expression of Fab fragments, phagemid titers, and number of Fab displaying phagemid particles. Our results show that the ratio of recombinant phagemids could be increased up to 65% of total phage titer by utilizing the appropriate phasmid. Based on this system, the selection of two sulfonamide-specific rabbit Abs, SA2 23 and SA2 90, was accomplished after a single phagemid panning round.

An expression system for the E. coli fermentation of recombinant antibody Fab fragments from mice and rabbits.

A mutually compatible vector system was developed for the bench-top fermenter production of mouse and rabbit Fab fragments comprising pASK85-pro-K411B and pASK85Rab-pro-WR13. These vectors provide a mouse- and rabbit-specific cloning site, respectively, the tetA promoter/operator and the proBA operon that complements the Pro biosynthetic deficiency of the Escherichia coli strain JM83 and can serve as an additional selection marker. Fermentation at elevated cell density (OD600 = 20-40) of the atrazine-specific mouse Fab fragment K411B using JM83 harboring pASK85-pro-K411B in a 2 L bench-top vessel resulted in a yield of 240 microg/L x OD600 affinity-purified protein (13.8 mg). In contrast, expression of leporid Fab fragments using pASK85Rab-pro-WR13 was unsuccessful due to the aggregation of rabbit light chains, which probably relates to a general problem of this specific class of immunoglobulins with their additional Cys residues. Coexpression of rabbit Fab fragments together with four periplasmic folding-helper proteins encoded on pTUM4 led to a significantly improved folding efficiency, resulting in a yield of 50 microg/L x OD600 affinity-purified rabbit Fab fragment (3.3 mg) from the 2 L bench-top fermenter.

Placental transfer of DDT in mother-infant pairs from Northern Thailand.

The present study objective was to investigate ratios and correlation coefficients between dichlorodiphenyltrichloroethane (DDT) compounds in cord and maternal sera of mother-infant pairs from northern Thailand. The study site was located in Chiang Dao District of Chiang Mai Province which was an agricultural and former malaria endemic area. DDT compounds were analyzed in 88 cord and maternal serum samples using gas chromatography-electron capture detection (GC-ECD). p,p'-DDE (1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene) was the major component and detected in every cord and maternal serum samples with geometric means of 1,255 and 1,793 n g(-1) lipids, respectively. p,p'-DDT (1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane) was detected at 89.8 and 100% of cord and maternal serum samples, respectively. The second and third highest levels detected were p,p'-DDD (1,1-dichloro-2,2-bis(p-chlorophenyl)ethane) and p,p'-DDT, respectively. The ratios between cord and maternal sera for p,p'-DDE, p,p'-DDT, and p,p'-DDD that were less than 1 had high correlation coefficients (ratio = 0.70, r = 0.82 for p,p'-DDE, ratio = 0.62, r = 0.66 for p,p'-DDT, and ratio = 0.79, r = 0.78 for p,p'-DDD). The high correlation coefficients indicate that cord serum levels of DDT compounds could be accurately estimated from maternal serum levels. It can be concluded that cord serum levels of p,p'-DDE, p,p'-DDT, and p,p'-DDD were approximately 70%, 62%, and 79% of maternal serum levels, respectively. Furthermore, our findings can be applied in public health to monitor and evaluate risk among infants from high DDT exposure area.

Biomarkers for exposure to estrogenic compounds: gene expression analysis in zebrafish (Danio rerio).

Gene expression analyses in male zebrafish (Danio rerio) were carried out using microarray technique and quantitative polymerase chain reaction. Genes responding to the exposure to 17beta-estradiol, bisphenol A and genistein were identified, among them genes involved in metabolism, reproductional and developmental processes. Threshold levels of 17beta-estradiol (200 ng/L), bisphenol A (2000 microg/L), and genistein (5000 microg/L) for the upregulation of the vtg1 gene in short-time exposures (11 days) were determined by qPCR. 14k microarrays were used to generate complete lists of genes regulated by these estrogenic compounds. For this purpose, liver samples from 10 exposed zebrafish and 10 controls were processed. In this case the expressions of 211 genes were significantly regulated by 17beta-estradiol, 47 by bisphenol A and 231 by genistein. Furthermore, it is shown that fish exposed to 17beta-estradiol and genistein have similarities in their gene expression patterns, whereas bisphenol A apparently affected gene expression in a different way. Only genes coding for egg-yolk precursor protein vitellogenin were found to be regulated by all three compounds, which shows that these genes are the only suitable markers for exposure to different estrogenic compounds. The regulated genes were assigned to gene ontology classes. All three estrogenic compounds regulated genes mainly involved in primary and cellular metabolism, but genistein regulated several genes involved in cell cycle-regulation and bisphenol A several genes involved in protein biosynthesis. Genistein also upregulated the expression of four eggshell proteins, which can be used as biomarkers for exposure to this chemical.

Enzyme-linked immunosorbent assay (ELISA) and sol-gel-based immunoaffinity purification (IAP) of the pyrethroid bioallethrin in food and environmental samples.

Enzyme-linked immunosorbent assay (ELISA) and sol-gel-based immunoaffinity purification (IAP) methods for the pyrethroid bioallethrin were developed and applied for monitoring bioallethrin in spiked food, soil, and dust samples. Attempts to determine bioallethrin content in fruit and vegetable extracts revealed high variability between sample preparations and marked interferences with the assay. Sol-gel IAP followed by solid-phase sample concentration was effective in removing the interfering components and resulted in high recovery of bioallethrin from spiked crude acetonic extracts of fruits and vegetables, even in the presence of high extract concentrations (28%). Solid-phase treatment alone failed to remove the interfering components from the spiked sample. Gas chromatography-mass spectrometry analysis of the IAP samples revealed bioallethrin as a doublet unsolved peak because of the cis and trans isomer present in the standard with confirmation of its mass. Unlike fruit and vegetable extracts, soil and dust samples did not interfere with the ELISA, and the bioallethrin content in those samples could be determined with high precision without the need of any further purification.

Development of a high-throughput screen for soluble epoxide hydrolase inhibition.

Mammalian soluble epoxide hydrolase (sEH) represents a highly promising new target for drug development. Chemical inhibition of this enzyme in animal models was shown to treat hypertension and vascular inflammation as well as related syndromes. Existing sEH inhibitors are relatively potent and specific. However, the low solubility and relatively fast metabolism of described sEH inhibitors make them less than therapeutically efficient, stating the need for novel inhibitor structures. Therefore, a series of alpha-cyanoester and alpha-cyanocarbonate epoxides were evaluated as potential human sEH (HsEH) substrates for the high-throughput screen (HTS) of compound libraries. (3-Phenyl-oxiranyl)-acetic acid cyano-(6-methoxy-naphthalen-2-yl)-methyl ester (PHOME), which displayed the highest aqueous stability and solubility, was selected for the development of an HTS assay with long incubation times at room temperature. Concentrations of HsEH and PHOME were optimized to ensure assay sensitivity, reliability, and reproducibility. Assay validation, which employed these optimized concentrations, resulted in good accuracy (60-100%) and high precision (<7% relative standard deviation). In addition, an overall Z' value of 0.7 proved the system's robustness and potential for HTS. The developed assay system will be a valuable tool to discover new structures for the therapeutic inhibition of sEH to treat various cardiovascular diseases.

Fluorescent substrates for soluble epoxide hydrolase and application to inhibition studies.

Inhibition of the mammalian soluble epoxide hydrolase (sEH) is a promising new therapy in the treatment of disorders resulting from hypertension and vascular inflammation. A spectrophotometric assay (4-nitrophenyl-trans-2,3-epoxy-3-phenylpropyl carbonate, NEPC) is currently used to screen libraries of chemicals; however this assay lacks the required sensitivity to differentiate the most potent inhibitors. A series of fluorescent alpha-cyanoester and alpha-cyanocarbonate epoxides that produce a strong fluorescent signal on epoxide hydrolysis by both human and murine sEH were designed as potential substrates for an in vitro inhibition assay. The murine enzyme showed a broad range of specificities, whereas the human enzyme showed the highest specificity for cyano(6-methoxy-naphthalen-2-yl)methyl trans-[(3-phenyloxiran-2-yl)methyl] carbonate. An in vitro inhibition assay was developed using this substrate and recombinant enzyme. The utility of the fluorescent assay was confirmed by determining the IC(50) values for a series of known inhibitors. The new IC(50) values were compared with those determined by spectrophotometric NEPC and radioactive tDPPO assays. The fluorescent assay ranked these inhibitors on the basis of IC(50) values, whereas the NEPC assay did not. The ranking of inhibitor potency generally agreed with that determined using the tDPPO assay. These results show that the fluorescence-based assay is a valuable tool in the development of sEH inhibitors by revealing structure-activity relationships that previously were seen only by using the costly and labor-intensive radioactive tDPPO assay.

Determination of Acaulospora longula and Glomus subgroup Aa in plant roots from grassland using new primers against the large subunit ribosomal DNA.

Molecular techniques have become increasingly important for the identification of arbuscular mycorrhizal fungi (AMF). In this work Acaulospora longula and Glomus mosseae have been detected in plant roots from pastures using specific nucleotide primers for the two species. Part of the 5' end of the large subunit of the ribosomal RNA gene was amplified by nested PCR and sequenced. The distribution of the fungi within three different plant species, Plantago lanceolata, Trifolium repens, and Holcus lanatus, and two different types of grassland, have been studied. Neither the fungi nor the plants showed specific preference for their symbiotic partnership.

Comparative study of estrogenic potencies of estradiol, tamoxifen, bisphenol-A and resveratrol with two in vitro bioassays.

This study was undertaken to compare the sensitivity of two in vitro screening test methods and to determine the accuracy of predicted response to spiked laboratory water samples. A newly developed enzyme-linked receptor assay (ELRA) and a widely used yeast estrogen screen (YES) assay were selected to evaluate the estrogenic responses. Four natural, pharmaceutical, xenobiotic or phytobiotic chemicals: 17beta-estradiol (E2), tamoxifen, bisphenol-A and resveratrol were examined, and 17beta-E2 was used as a positive control. 17beta-E2 can strongly induce estrogenic response in both test systems, however, ELRA was found to be more sensitive to 17beta-E2 with a detection limit of 0.07 microg/l compared to 0.88 microg/l in YES assay. Similar results were obtained for bisphenol-A and resveratrol, and their estrogen potencies relative to E2 (100%) determined by ELRA were at least 5.6 times greater than produced by YES assay. ELRA was unable to distinguish the anti-estrogen tamoxifen and YES assay is also poor at distinguishing. Comparison of response to spiked laboratory water samples show that ELRA can give accurate determination to all four chemicals with recoveries among 70-120%, while YES can only give accurate determination to 17beta-E2 and bisphenol-A with recoveries among 69-112%. The comparative results provide evidence that ELRA is more suitable for rapid screening estrogenic potency of the environmental samples. Combination of ELRA and mammalian cellular assay will constitute an advantageous test to specify agonistic or antagonistic effects.

Juvenile hormone (JH) esterase: why are you so JH specific?

Juvenile hormone esterases (JHEs) from six insects belonging to three orders (Lepidoptera, Coleoptera, and Diptera) were compared in terms of their deduced amino acid sequence and biochemical properties. The four lepidopteran JHEs showed from 52% to 59% identity to each other and about 30% identity to the coleopteran and dipteran JHEs. The JHE of Manduca sexta was remarkably resistant to the addition of organic co-solvents and detergent; in some cases, it demonstrated significant activation of activity. Trifluoromethylketone (TFK) inhibitors with chain lengths of 8, 10 or 12 carbons were highly effective against both lepidopteran and coleopteran JHEs. The coleopteran JHE remained sensitive to TFK inhibitors with a chain length of 6 carbons, whereas the lepidopteran JHEs were significantly less sensitive. When the chain was altered to a phenethyl moiety, the coleopteran JHE remained moderately sensitive, while the lepidopteran JHEs were much less sensitive. The lepidopteran and coleopteran JHEs did not show dramatic differences in specificity to alpha-naphthyl and rho-nitrophenyl substrates. However, as the chain length of the alpha-naphthyl substrates increased from propionate to caprylate, there was a trend towards reduced activity. The JHE of M. sexta was crystallized and the properties of the crystal suggest a high-resolution structure will follow.

Single-chain Fv antibody-alkaline phosphatase fusion proteins produced by one-step cloning as rapid detection tools for ELISA.

A system was constructed for the production of alkaline phosphatase (aP)-labeled antibody single-chain Fv (scFv) fragments in Escherichia coli. The expression vector pASK75 was modified by sequentially inserting the E. coli aP coding region and the scFv cloning cassette. Engineering the cloning sites SfiI and NotI located at the 5' and 3' end of the scFv gene provides an easy means to insert scFv fragments. These cloning sites are widely used in recombinant antibody technology and, thus, enable the one-step cloning of scFv fragments derived from corresponding antibody phage libraries into the expression vector. An expressed herbicide-specific scFv aP fusion protein retained both, analyte binding and enzymatic activity, as determined by ELISA. Therefore, this system permits the production of scFv-aP conjugates in E. coli, which can replace conventionally prepared aP-labeled antibodies in immunoassays. These fusion proteins are designed to accelerate the immunochemical detection of analytes, since the assay duration is essentially reduced by omitting the use of enzyme labeled secondary antibodies.

Cloning, functional expression and kinetic characterization of pesticide-selective Fab fragment variants derived by molecular evolution of variable antibody genes.

Fab antibody fragments were constructed by subcloning single chain Fv variable regions from the phagemid vector pCANTAB 5E into the expression vector pASK99. The vector was designed for bacterial secretion of Fab fragments and bears coding sequences for murine constant domains including the Strep-tag II at the carboxyl-terminal end of the constant heavy chain domain. The cloning procedure was carried out with the scFv antibodies IPR-7, IPR-53 and IPR-23. The second and third clone originated from the molecular evolution of the s-triazine selective antibody IPR-7. The Fab fragments were expressed under the transcriptional control of the tetA promoter system. Large-scale production benefits from the anhydrotetracycline-inducible system because of the lower costs for the inducer compared to IPTG and the tightly regulated expression of the recombinant antibody fragments. The Strep-tag purification technology facilitates the isolation of Fab fragments from the E. coli periplasm. The characteristics of functionally expressed Fab fragments were determined by employing a BIAcore 2000 system. The KD of the Fab variant IPR-23 (K(D)= 1.1 2 x 10(-9) M) optimized by molecular evolution was improved by a factor of 24 compared to the Fab IPR-7 (K(D) = 2.73 x 10(-8) M), which was derived from the template scFv antibody IPR-7. The affinity alteration was also reflected in the 22-fold reduction of the IC50 values of the variants Fab IPR-7 (IC50 = 60.5 microg/L) and Fab IPR-23 (IC50=2.7 microg/L) in the corresponding atrazine ELISA.

A generic strategy for subcloning antibody variable regions from the scFv phage display vector pCANTAB 5 E into pASK85 permits the economical production of F(ab) fragments and leads to improved recombinant immunoglobulin stability.

Apart from the decisive sensitivity and specificity of immunosensors, the employed antibodies essentially contribute to additional key factors like fabrication costs for sensor chips and sensor stability. A production scheme for recombinant antibody fragments has been optimised with respect to these particular issues of biosensor development. The phagemid vector pCANTAB 5 E is widely used for the selection of antibody fragments from corresponding libraries. However, large-scale production of the selected single-chain F(v) (scFv) fragments is substantially restricted by the high cost for the inducer IPTG and the anti-E-tag antibody. The latter is needed in significant amounts for the purification of the recombinant protein. A generic strategy was established for subcloning scFv variable regions from pCANTAB 5 E into the plasmid pASK85 for the expression of F(ab) fragments. pASK85 bears coding sequences for murine constant domains including a His(6) tag at the carboxyl-terminal end of the constant heavy chain domain. The anti-s-triazine antibody K47H served as a model system in this study. Biosynthesis of the F(ab) fragment in a high cell density fermenter was induced by addition of anhydrotetracycline. The F(ab) fragment was subsequently purified from the periplasmic extract in a single step by immobilized metal affinity chromatography (IMAC). A yield of 100 microg/lxOD(550) purified F(ab) fragment was obtained employing a standard fermentation scheme. The sensitivity and cross-reactivity of the F(ab) was comparable to the parent scFv when assayed by enzyme immunoassay. However, the F(ab) fragment exhibited significantly improved long-term stability.