Comparison of batch and perfusion culture in combination with pilot-scale expanded bed purification for the production of soluble recombinant beta-secretase

beta-Secretase is one of the prime targets for therapeutic intervention of Alzheimer's disease. For the development of a secretase inhibitor a steady supply of large quantities of a homogeneous and active recombinant beta-secretase is a prerequisite. Therefore various culture modes were investigated using HEK-293 cells stably transfected with soluble recombinant beta-secretase. The coupling of the Fc part of human IgG1 to the ectodomain of beta-secretase (residues 1-460) allowed a fast purification of the protein with rProtA expanded bed chromatography. Batch cultures of 5 to 50 L working volume run for 7 days showed reproducible cell growth and product yields of 3 mg/L purified protein. A 20 L perfusion culture was operated for 21 days, reaching a cell density of 30 x 10(6) cells/mL at a dilution rate of 2/d. The total product yield of the perfusion culture was 1.4 g of purified protein. The effect of different perfusion rates on cell growth, protein yield, and quality was investigated and compared to the results obtained in batch cultures. Protein quality was consistent as analyzed on 1D SDS-PAGE, and the final product contained both the mature and the pro form of beta-secretase. Although the cell specific protein expression was slightly reduced in perfusion culture, a substantial increase in specific activity of over 75% was achieved. Some of the increase in activity can be explained by an increase in the percentage of the mature form of the recombinant protein.     

Site-Specific Derivation of the Acute Copper Criteria for the Clinch River,Virginia

Previous studies indicated that inputs from the Clinch River Plant (CRP), a coal-burning, power-generating facility in Carbo, Virginia, caused impairment to transplanted freshwater mussels. These findings necessitated the examination of instream biological responses to CRP effluents. Therefore, our objective was to derive a site-specific Criterion Maximum Concentration (CMC) for copper (Cu), the toxic constituent of the effluent. This was accomplished by conducting acute toxicity tests with Cu using 17 different genera native to or currently residing in the Clinch River watershed. Four native unionids and a mayfly (Isonychia bicolor) had genus mean acute values of 60?µJ.g/L or lower (ranging from 37 to 60?µJ,g/L). With a GMAV of 88?µJ,g/L, Ceriodaphnia dubia ranked sixth among the 17 genera tested, and the fathead minnow, Pimephales promelas, ranked fourteenth with a GMAV of 310?µJ.g/L. Despite the sensitivity of the indigenous mussels tested, the site-specific CMC of 18?µJg/L was not substantially lower than the national CMC for Cu (20?µJ,g/L, hardness = 150?mg/L as CaCO3). While we conducted acute exposure tests, future investigations should incorporate both biomarker and population or community-level studies to determine low-level contaminant impacts on freshwater mussels.     

Involvement of UBE1L in ISG15 conjugation during retinoid-induced differentiation of acute promyelocytic leukemia

Acute promyelocytic leukemia (APL) cases expressing the t(15,17) product, promyelocytic leukemia (PML)/retinoic acid receptor alpha (RARalpha), have clinical remissions through leukemic cell differentiation after all-trans-retinoic acid (RA) treatment. This differentiation therapy propelled interest in uncovering molecular mechanisms for RA-dependent APL differentiation. We previously identified the ubiquitin-activating enzyme-E1-like protein (UBE1L) as an RA-regulated target gene in APL that triggers PML/RARalpha degradation and apoptosis. This study reports that conjugation of the ubiquitin-like species, interferon-stimulated gene, 15-kDa protein (ISG15), also occurs during RA-induced APL differentiation. Knock-down of UBE1L expression inhibited this conjugation. RA treatment of APL and other RA-responsive leukemic cells induced expression of UBE1L and ISG15 as well as intracellular ISG15 conjugates. Notably, ISG15 conjugation did not occur in RA-resistant NB4-R1 APL cells. Induction of UBE1L and ISG15 along with ISG15 conjugation in RA-sensitive NB4-S1 APL cells were detected following treatment with specific retinoids and type I interferon (IFN). UBE1L and ISG15 mRNAs were co-expressed in normal human tissues that were examined. In contrast, UBE1L mRNA expression was markedly repressed in several cancer cell lines. A physical association was found between UBE1L and ISG15 in vivo. This required the conserved diglycine motif in the carboxyl terminus of ISG15. Targeting UBE1L expression with small inhibitory RNA or small hairpin RNA inhibited IFN and RA-induced ISG15 conjugation. Formation of ISG15 conjugates through induction of an activating enzyme represents a novel pharmacologic mechanism for regulation of this ubiquitin-related species. Taken together, the observed rela tionship between expression of UBE1L and ISG15, their physical association and coordinate regulation, and induced ISG15 conjugation during leukemic cell differentiation implicate an important role for these proteins in retinoid response.     

The mode of bone morphogenetic protein (BMP) receptor oligomerization determines different BMP-2 signaling pathways.

Bone morphogenetic proteins (BMPs) are multifunctional proteins regulating cell growth, differentiation, and apoptosis. BMP-2 signals via two types of receptors (BRI and BRII) that are expressed at the cell surface as homomeric as well as heteromeric complexes. Prior to ligand binding, a low but measurable level of BMP-receptors is found in preformed hetero-oligomeric complexes. The major fraction of the receptors is recruited into hetero-oligomeric complexes only after ligand addition. For this, BMP-2 binds first to the high affinity receptor BRI and then recruits BRII into the signaling complex. However, ligand binding to the preformed complex composed of BRII and BRI is still required for signaling, suggesting that it may mediate activating conformational changes. Using several approaches we have addressed the following questions: (i) Are preformed complexes incompetent of signaling in the absence of BMP-2? (ii) Which domains of the BRII receptors are essential for this complex formation? (iii) Are there differences in signals sent from BMP-induced versus preformed receptor complexes? By measuring the activation of Smads, of p38 MAPK and of alkaline phosphatase, we show that the ability of kinase-deficient BRII receptor mutants to inhibit BMP signaling depends on their ability to form heteromeric complexes with BRI. Importantly, a BRII mutant that is incapable in forming preassembled receptor complexes but recruits into a BMP-induced receptor complex does not interfere with the Smad pathway but does inhibit the induction of alkaline phosphatase as well as p38 phosphorylation. These results indicate that signals induced by binding of BMP-2 to preformed receptor complexes activate the Smad pathway, whereas BMP-2-induced recruitment of receptors activates a different, Smad-independent pathway resulting in the induction of alkaline phosphatase activity via p38 MAPK.     

A dominant negative mutant of 2-5A-dependent RNase suppresses antiproliferative and antiviral effects of interferon.

2-5A-dependent RNase is the terminal factor in the interferon-regulated 2-5A system thought to function in both the molecular mechanism of interferon action and in the general control of RNA stability. However, direct evidence for specific functions of 2-5A-dependent RNase has been generally lacking. Therefore, we developed a strategy to block the 2-5A system using a truncated form of 2-5A-dependent RNase which retains 2-5A binding activity while lacking RNase activity. When the truncated RNase was stably expressed to high levels in murine cells, it prevented specific rRNA cleavage in response to 2-5A transfection and the cells were unresponsive to the antiviral activity of interferon alpha/beta for encephalomyocarditis virus. Remarkably, cells expressing the truncated RNase were also resistant to the antiproliferative activity of interferon. The truncated RNase is a dominant negative mutant that binds 2-5A and that may interfere with normal protein-protein interactions through nine ankyrin-like repeats.     

Hyper-oceanic liverwort species of conservation concern: evidence for dispersal limitation and identification of suitable uncolonised regions

In order to successfully manage and conserve species and plant communities, it is important to have a good understanding of their ecology and distributions. The three liverwort species Anastrophyllum donnianum, Scapania ornithopodioides and Scapania nimbosa, are restricted to the mixed northern hepatic mat community found in the most oceanic parts of north-western Europe. These species are of conservation concern because they are globally rare with strict environmental requirements and a limited dispersal potential, which makes them vulnerable to disturbance and climate change. In this study we used species distribution modelling to (1) predict their potential distribution in Norway (2) to assess whether they are limited by dispersal or suitable climate, (3) identify which climatic factors are most important in determining their distribution and (4) suggest regions for further field based surveys. Maximum entropy (MaxEnt) models were developed for each species using target-group background data, and five environmental coverage layers. Our results indicate that all three species are limited by dispersal rather than the availability of suitable areas in Norway. In particular, A. donnianum seems to be limited from reaching uncolonised highly suitable areas in northern Norway due to a barrier unsuitable region with insufficient summer rain. S. ornithopodioides is absent from northern Norway despite the presence of highly suitable regions scattered along the coast. The models locate highly suitable areas where conservation measures should be focused when they overlap with known populations. Areas of interest for targeting searches for potentially undiscovered populations are indicated.     

Development of a new approach for screening combinatorial libraries using MALDI-TOF-MS and HPLC-ESI-MS/MS

Mass spectrometric techniques play a prominent role in the rapidly expanding field of high-throughput screening (HTS). In this paper, the authors present a novel qualitative approach for the screening of a small library of compounds using MALDI-TOF-MS and HPLC-ESI-MS/MS. Chymotrypsin (CT), a serine protease, was selected as the target protein. A well-known inhibitor of CT is chymostatin (CS), a naturally occurring peptide aldehyde, which is reported to be a mixture of 3 components-A, B, and C-differing only in one of the amino acids. The authors report that native CS mixture consists of 3 additional ring hydroxylated components and that each compound exists in 2 epimeric forms. In case of protein-binding compounds, only 1 of the epimers was found to be active. A unique feature of this study is the generation of a combinatorial library of CS derivatives applying a one-pot strategy followed by identification and structural elucidation of the library components. Analytical investigation of the library resulted in the identification of 22 compounds. After incubation with CT and centrifugal ultrafiltration, 10 compounds were detected as protein-binding ligands. Finally, the complementary potentials of MALDI-TOF-MS and HPLC-MS/MS in the screening of complex ligand mixtures have been discussed.     

Generation and differentiation of microtissues from multipotent precursor cells for use in tissue engineering

This protocol describes an effective method for the production of spherical microtissues (microspheres), which can be used for a variety of tissue-engineering purposes. The obtained microtissues are well suited for the study of osteogenesis in vitro when multipotent stem cells are used. The dimensions of the microspheres can easily be adjusted according to the cell numbers applied in an individual experiment. Thus, microspheres allow for the precise administration of defined cell numbers at well-defined sites. Here we describe a detailed workflow for the production of microspheres using unrestricted somatic stem cells from human umbilical cord blood and adapted protocols for the use of these microspheres in histological analysis. RNA extraction methods for mineralized microtissues are specifically modified for optimum yields. The duration of running the complete protocol without preparatory cell culture but including 2 weeks of microsphere incubation, histological staining and RNA isolation is about 3 weeks.     

Marine sulfate-reducing bacteria cause serious corrosion of iron under electroconductive biogenic mineral crust

Iron (Fe(0) ) corrosion in anoxic environments (e.g. inside pipelines), a process entailing considerable economic costs, is largely influenced by microorganisms, in particular sulfate-reducing bacteria (SRB). The process is characterized by formation of black crusts and metal pitting. The mechanism is usually explained by the corrosiveness of formed H(2) S, and scavenge of 'cathodic' H(2) from chemical reaction of Fe(0) with H(2) O. Here we studied peculiar marine SRB that grew lithotrophically with metallic iron as the only electron donor. They degraded up to 72% of iron coupons (10?mm?×?10?mm?×?1?mm) within five months, which is a technologically highly relevant corrosion rate (0.7?mm?Fe(0) year(-1) ), while conventional H(2) -scavenging control strains were not corrosive. The black, hard mineral crust (FeS, FeCO(3) , Mg/CaCO(3) ) deposited on the corroding metal exhibited electrical conductivity (50?S?m(-1) ). This was sufficient to explain the corrosion rate by electron flow from the metal (4Fe(0) →?4Fe(2+) +?8e(-) ) through semiconductive sulfides to the crust-colonizing cells reducing sulfate (8e(-) +?SO(4) (2-) +?9H(+) →?HS(-) +?4H(2) O). Hence, anaerobic microbial iron corrosion obviously bypasses H(2) rather than depends on it. SRB with such corrosive potential were revealed at naturally high numbers at a coastal marine sediment site. Iron coupons buried there were corroded and covered by the characteristic mineral crust. It is speculated that anaerobic biocorrosion is due to the promiscuous use of an ecophysiologically relevant catabolic trait for uptake of external electrons from abiotic or biotic sources in sediments.     

Production of viable pigs from fetal somatic stem cells

Fetal somatic stem cells (FSSCs) are a novel type of somatic stem cells that have recently been discovered in primary fibroblast cultures from pigs and other species. The goal of the present study was to produce viable piglets from FSSCs. NT complexes were prepared from both FSSCs and porcine fetal fibroblasts (pFF) to permit comparison of these two donor cell types. FSSCs from isolated attached colonies were compared with pFF in their ability to form blastocysts upon use in NT. Fusion and cleavage rates were similar between the two groups, while blastocyst rates were significantly higher when using pFF as donor cells. FSSCs of three different size categories derived from dissociation of spheroids yielded similar results. The use of FSSCs of 15-20 microm in size yielded similar cleavage and blastocyst rates as fetal fibroblasts. In the final experiment NT complexes produced from FSSCs were transferred to foster mothers. After transfer to prepubertal gilts, three of seven recipients established pregnancies and delivered seven piglets, of which three piglets were viable and showed normal development. Results for the first time demonstrate that FSSCs are able to produce cloned embryos, and that pregnancies can be established and viable piglets can be produced.