Heterologous expression of mouse cytochrome P450 2e1 in V79 cells: construction and characterisation of the cell line and comparison with V79 cell lines stably expressing rat P450 2E1 and human P450 2E1

A V79 Chinese hamster cell line was constructed for stable expression of mouse cytochrome P450 2e1 (Cyp2e1), as an addition to the existing cell battery consisting of cell lines stably expressing rat CYP2E1 and human CYP2E1 (V79 Cell Battery). The aim was to establish a cell battery that offers the in vitro possibility of investigating species-specific differences in the toxicity and metabolism of chemicals representing substrates for CYP2E1. The newly established cell line (V79m2E1) effectively expressed Cyp2e1 in the catalytically active form. The expression of catalytically active CYP2E1 in V79m2E1 cells was maintained over several months in culture, as demonstrated by Western Blotting and chlorzoxazone (CLX) 6-hydroxylase activity. The cells exhibited CLX 6-hydroxylase activity with a Km of 27.8 microM/l and Vmax of 40 pmol/mg protein/minute, compared with a Km of 28.2/28.6 microM/l and a Vmax of 130/60 pmol/mg protein/minute from V79r2E1/V79h2E1 cells. Furthermore, the CYP2E1-dependent mutagenicity of N-nitrosodimethylamine could be demonstrated in the V79m2E1 cells. Therefore, the new cell battery permits the interspecies comparison of CYP2E1-dependent toxicity and of metabolism of chemicals between humans and the two major rodent species--the rat and the mouse--that are usually used in classical toxicity studies.     

TRiC/CCT cooperates with different upstream chaperones in the folding of distinct protein classes

The role in protein folding of the eukaryotic chaperonin TRiC/CCT is only partially understood. Here, we show that a group of WD40 beta-propeller proteins in the yeast cytosol interact transiently with TRiC upon synthesis and require the chaperonin to reach their native state. TRiC cooperates in the folding of these proteins with the ribosome-associated heat shock protein (Hsp)70 chaperones Ssb1/2p. In contrast, newly synthesized actin and tubulins, the major known client proteins of TRiC, are independent of Ssb1/2p and instead use the co-chaperone GimC/prefoldin for efficient transfer to the chaperonin. GimC can replace Ssb1/2p in the folding of WD40 substrates such as Cdc55p, but combined deletion of SSB and GIM genes results in loss of viability. These findings expand the substrate range of the eukaryotic chaperonin by a structurally defined class of proteins and demonstrate an essential role for upstream chaperones in TRiC-assisted folding.     

Zebrafish: a new model on the pharmaceutical catwalk

Zebrafish is recognized as one of the most important vertebrate model organisms; however, its value in pharmacological studies has not been extensively explored and exploited. In this review, I summarize significant findings about the effects of drugs and medicines on important physiological processes in zebrafish. Our experiments have shown that cardiovascular, anti-angiogenic and anti-cancer drugs elicit comparable responses in zebrafish embryos to those in mammalian systems. Similar observations have been reported by other laboratories, exposing zebrafish to a variety of pharmaceutical active compounds affecting a range of different processes. All the data summarized indicate that zebrafish represents a very valuable organism for different kinds of pharmacological studies, such as screenings of chemical libraries, lead validation and optimization, mode-of-action studies, analysis of gene function, predictive toxicology and teratogenicity, pharmacogenomics and toxicogenomics. Zebrafish pharmacological assays have specific advantages compared to in vitro cell culture studies and in vivo experiments using mice, complementing these assays to give valuable guides for future tests of new drugs for human therapy.     

Tautomerism of 4-hydroxy-2,5-dimethyl-3(2H)-furanone: evidence for its enantioselective biosynthesis

Chiral natural flavor compounds exhibit characteristic enantiomeric excesses due to stereoselective, enzymatically catalyzed reactions during biogenesis. Although the enzymatic formation of the strawberry key flavor compound 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF; Furaneol(R)) is anticipated, the naturally occurring compound is racemic. As racemization due to keto-enol-tautomerism of HDMF could account for this observation, HDMF was investigated by (1)H-NMR spectroscopy tracing the exchange of the proton bound to the furanone-ring at C2 with deuteron from the medium (D(2)O). In addition, the racemization rate of HDMF was directly determined by cyclodextrin-modified capillary electrophoresis of enantiomerically enriched HDMF stored at different pH values. Tautomerism and the racemization rate of HDMF was lowest at pH values between 4 and 5. However, tautomerism and thus racemization was catalyzed under stronger acidic conditions (pH 2) and especially at pH values greater than 7, the value published for plant cell cytosol. Approximately 50% of the protons at C2 were exchanged with deuteron within 1 h at pH 7.2. Therefore, in order to demonstrate the enzymatic formation of HDMF, incubation experiments with Zygosaccharomyces rouxii as well as strawberry protein extract were carried out under slightly acidic conditions (pH 5), the most suitable pH value for studies on the enantiomeric ratio of HDMF. In both experiments the formation of enantiomerically enriched HDMF could be demonstrated for the first time, whereas incubation experiments under neutral conditions resulted in the detection of racemic HDMF.     

Lack of Bdnf and TrkB signalling in the postnatal cochlea leads to a spatial reshaping of innervation along the tonotopic axis and hearing loss

Members of the neurotrophin gene family and their high-affinity Trk receptors control innervation of the cochlea during embryonic development. Lack of neurotrophin signalling in the cochlea has been well documented for early postnatal animals, resulting in a loss of cochlear sensory neurones and a region-specific reduction of target innervation along the tonotopic axis. However, how reduced neurotrophin signalling affects the innervation of the mature cochlea is currently unknown. Here, we have analysed the consequences of a lack of the TrkB receptor and its ligand, the neurotrophin brain-derived neurotrophic factor (Bdnf), in the late postnatal or adult cochlea using mouse mutants. During early postnatal development, mutant animals show a lack of afferent innervation of outer hair cells in the apical part of the cochlea, whereas nerve fibres in the basal part are maintained. Strikingly, this phenotype is reversed during subsequent maturation of the cochlea, which results in a normal pattern of outer hair cell innervation in the apex and loss of nerve fibres at the base in adult mutants. Measurements of auditory brain stem responses of these mice revealed a significant hearing loss. The observed innervation patterns correlate with opposing gradients of Bdnf and Nt3 expression in cochlear neurones along the tonotopic axis. Thus, the reshaping of innervation may be controlled by autocrine signalling between neurotrophins and their receptors in cochlear neurones. Our results indicate a substantial potential for re-innervation processes in the mature cochlea, which may also be of relevance for treatment of hearing loss in humans.     

Automated whole mount localisation techniques for plant seedlings

Plant biology is currently experiencing a growing demand for easy and reliable mRNA and protein localisation techniques. Here, we present novel whole mount in situ hybridisation and immunolocalisation protocols, suitable to localise mRNAs and proteins in Arabidopsis seedlings. We demonstrate that these methods can be used in different organs of Arabidopsis seedlings as well as in other plant species. In order to achieve better reproducibility and higher throughput, we modified these protocols for automation to be performed by a liquid handling robot. In addition, we show that other procedures such as reporter enzyme assays and tissue clearing can be similarly automated. We present examples of application of our protocols including mRNA localisation and proteins and epitope tag (co)localisations which demonstrate that these methods provide reliable and versatile tools for expression, localisation and anatomical studies in plants.     

Mechanism of regulation of the Epac family of cAMP-dependent RapGEFs

Epac1 (cAMP-GEFI) and Epac2 (cAMP-GEFII) are closely related guanine nucleotide exchange factors (GEFs) for the small GTPase Rap1, which are directly regulated by cAMP. Here we show that both GEFs efficiently activate Rap2 as well. A third member of the family, Repac (GFR), which lacks the cAMP dependent regulatory sequences, is a constitutive activator of both Rap1 and Rap2. In contrast to Epac1, Epac2 contains a second cAMP binding domain at the N terminus, as does the Epac homologue from Caenorhabditis elegans. Affinity measurements show that this distal cAMP binding domain (the A-site) binds cAMP with much lower affinity than the cAMP binding domain proximal to the catalytic domain (the B-site), which is present in both Epac1 and Epac2. Deletion mutant analysis shows that the high affinity cAMP binding domains are sufficient to regulate the GEFs in vitro. Interestingly, isolated fragments containing the B-sites of either Epac1 or Epac2, but not the A-site from Epac2, inhibit the catalytic domains in trans. This inhibition is relieved by the addition of cAMP. In addition to the cAMP binding domains, both Epac1 and Epac2 have a DEP domain. Deletion of this domain does not affect regulation of Epac1 activity but affects membrane localization. From these results, we conclude that all three members of the Epac family regulate both Rap1 and Rap2. Furthermore, we conclude that the catalytic activity of Epac1 is constrained by a direct interaction between GEF and high affinity cAMP binding domains in the absence of cAMP. Epac1 becomes activated by a release of this inhibition when cAMP is bound.     

RalGEF2, a pleckstrin homology domain containing guanine nucleotide exchange factor for Ral

Ral is a ubiquitously expressed Ras-like small GTPase. Several guanine nucleotide exchange factors for Ral have been identified, including members of the RalGDS family, which exhibit a Ras binding domain and are regulated by binding to RasGTP. Here we describe a novel type of RalGEF, RalGEF2. This guanine nucleotide exchange factor has a characteristic Cdc25-like catalytic domain at the N terminus and a pleckstrin homology (PH) domain at the C terminus. RalGEF2 is able to activate Ral both in vivo and in vitro. Deletion of the PH domain results in an increased cytoplasmic localization of the protein and a corresponding reduction in activity in vivo, suggesting that the PH domain functions as a membrane anchor necessary for optimal activity in vivo.     

Retention of bacteria on a substratum surface with micro-patterned hydrophobicity

Bacteria adhere to almost any surface, despite continuing arguments about the importance of physico-chemical properties of substratum surfaces, such as hydrophobicity and charge in biofilm formation. Nevertheless, in vivo biofilm formation on teeth and also on voice prostheses in laryngectomized patients is less on hydrophobic than on hydrophilic surfaces. With the aid of micro-patterned surfaces consisting of 10-microm wide hydrophobic lines separated by 20-microm wide hydrophilic spacings, we demonstrate here, for the first time in one and the same experiment, that bacteria do not have a strong preference for adhesion to hydrophobic or hydrophilic surfaces. Upon challenging the adhering bacteria, after deposition in a parallel plate flow chamber, with a high detachment force, however, bacteria were easily wiped-off hydrophobic lines, most notably when these lines were oriented parallel to the direction of flow. Adhering bacteria detached slightly less from the hydrophilic spacings in between, but preferentially accumulated adhering on the hydrophilic regions close to the interface between the hydrophilic spacings and hydrophobic lines. It is concluded that substratum hydrophobicity is a major determinant of bacterial retention while it hardly influences bacterial adhesion.