Investigations on the mutagenicity of 1,4-dichlorobenzene and its main metabolite 2,5-dichlorophenol in vivo and in vitro

The genotoxic potential of 1,4-dichlorobenzene (1,4-DCB) has been extensively evaluated in vitro and in vivo. The majority of the studies demonstrated the absence of a genotoxic potential for 1,4-DCB. At variance are a bone marrow micronucleus test (MNT) after intraperitoneal (i.p.) treatment of NMRI mice [Mohtashamipur et al., Mutagenesis 2 (1987) 111–113] and a gene mutation assay on mouse lymphoma cells [McGregor et al., Environ. Mol. Mutagen. 12 (1988) 85–145]. Therefore, we investigated 1,4-DCB and its main metabolite 2,5-dichlorophenol (2,5-DCP) for both endpoints. In an MNT, male and female NMRI mice were treated orally with single doses of 2500 mg/kg 1,4-DCB and 1500 mg/kg 2,5-DCP, respectively. Smears were prepared 24, 48 and 72 h thereafter. No induction of micronuclei was detected for both compounds. Also under the conditions of Mohtashamipur et al. (1987), intraperitoneal treatments of male and female mice with 2 × 177.5 and 2 × 355 mg/kg 1,4-DCB failed to induce micronuclei. In addition, CHO/HPRT-gene mutation tests with 1,4-DCB and 2,5-DCP yielded negative results for both compounds with and without metabolic activation system. Therefore, 1,4-DCB and 2,5-DCP are considered to be non-mutagenic in these test systems.     

Approaching the secrets of N-glycosylation in Aspergillus fumigatus: characterization of the AfOch1 protein

The mannosyltransferase Och1 is the key enzyme for synthesis of elaborated protein N-glycans in yeast. In filamentous fungi genes implicated in outer chain formation are present, but their function is unclear. In this study we have analyzed the Och1 protein of Aspergillus fumigatus. We provide first evidence that poly-mannosylated N-glycans exist in A. fumigatus and that their synthesis requires AfOch1 activity. This implies that AfOch1 plays a similar role as S. cerevisiae ScOch1 in the initiation of an N-glycan outer chain. A Δafoch1 mutant showed normal growth under standard and various stress conditions including elevated temperature, cell wall and oxidative stress. However, sporulation of this mutant was dramatically reduced in the presence of high calcium concentrations, suggesting that certain proteins engaged in sporulation require N-glycan outer chains to be fully functional. A characteristic feature of AfOch1 and Och1 homologues from other filamentous fungi is a signal peptide that clearly distinguishes them from their yeast counterparts. However, this difference does not appear to have consequences for its localization in the Golgi. Replacing the signal peptide of AfOch1 by a membrane anchor had no impact on its ability to complement the sporulation defect of the Δafoch1 strain. The mutant triggered a normal cytokine response in infected murine macrophages, arguing against a role of outer chains as relevant Aspergillus pathogen associated molecular patterns. Infection experiments provided no evidence for attenuation in virulence; in fact, according to our data the Δafoch1 mutant may even be slightly more virulent than the control strains.     

Heterogeneity ofHLA defined in PLT: A cellular assay detects differences not seen using HLA-DRw serology

The primed lymphocyte typing test (PLT) is used to detect the gene products of theHLA-D region which are responsible for secondary restimulation of cells primed in MLC. Alternatively, products of theHLA-D region may be detected serologically using antisera directed against a subpopulation of lymphocytes; these are the so-called DRw determinants. The PLT was used to see if it were possible to detect heterogeneity within a given serologically defined group using a cellular test. As priming combinations, we used family members identical for one haplotype and differing in theHLA-A, B andC regions, but not theD region of the second haplotype. Our results indicated that it was possible to prime against this second haplotype and that the segregation of the difference followedHLA. Therefore, using a cellular test it was possible to detect differences among cells belonging to a given DRw group. This suggests that PLT can be a useful tool to identify those serological groups which are composed of heterogenous determinants. In addition, it points out the problem in using any one test to establish identity of theHLA-D region, especially for clinical purposes.     

Molecular and Pharmacological Characterization of Serotonin 5-HT2α and 5-HT7 Receptors in the Salivary Glands of the Blowfly Calliphora vicina

Secretion in blowfly (Calliphora vicina) salivary glands is stimulated by the biogenic amine serotonin (5-hydroxytryptamine, 5-HT), which activates both inositol 1,4,5-trisphosphate (InsP₃)/Ca²⁺ and cyclic adenosine 3′,5′-monophosphate (cAMP) signalling pathways in the secretory cells. In order to characterize the signal-inducing 5-HT receptors, we cloned two cDNAs (Cv5-ht2α, Cv5-ht7) that share high similarity with mammalian 5-HT₂ and 5-HT₇ receptor genes, respectively. RT-PCR demonstrated that both receptors are expressed in the salivary glands and brain. Stimulation of Cv5-ht2α-transfected mammalian cells with 5-HT elevates cytosolic [Ca²⁺] in a dose-dependent manner (EC₅₀ = 24 nM). In Cv5-ht7-transfected cells, 5-HT produces a dose-dependent increase in [cAMP]_i (EC₅₀ = 4 nM). We studied the pharmacological profile for both receptors. Substances that appear to act as specific ligands of either Cv5-HT_2α or Cv5-HT₇ in the heterologous expression system were also tested in intact blowfly salivary gland preparations. We observed that 5-methoxytryptamine (100 nM) activates only the Cv5-HT_2α receptor, 5-carboxamidotryptamine (300 nM) activates only the Cv5-HT₇ receptor, and clozapine (1 µM) antagonizes the effects of 5-HT via Cv5-HT₇ in blowfly salivary glands, providing means for the selective activation of each of the two 5-HT receptor subtypes. This study represents the first comprehensive molecular and pharmacological characterization of two 5-HT receptors in the blowfly and permits the analysis of the physiological role of these receptors, even when co-expressed in cells, and of the modes of interaction between the Ca²⁺- and cAMP-signalling cascades.     

Long-Term Persistence of Bi-functionality Contributes to the Robustness of Microbial Life through Exaptation

Modern enzymes are highly optimized biocatalysts that process their substrates with extreme efficiency. Many enzymes catalyze more than one reaction; however, the persistence of such ambiguities, their consequences and evolutionary causes are largely unknown. As a paradigmatic case, we study the history of bi-functionality for a time span of approximately two billion years for the sugar isomerase HisA from histidine biosynthesis. To look back in time, we computationally reconstructed and experimentally characterized three HisA predecessors. We show that these ancient enzymes catalyze not only the HisA reaction but also the isomerization of a similar substrate, which is commonly processed by the isomerase TrpF in tryptophan biosynthesis. Moreover, we found that three modern-day HisA enzymes from Proteobacteria and Thermotogae also possess low TrpF activity. We conclude that this bi-functionality was conserved for at least two billion years, most likely without any evolutionary pressure. Although not actively selected for, this trait can become advantageous in the case of a gene loss. Such exaptation is exemplified by the Actinobacteria that have lost the trpF gene but possess the bi-functional HisA homolog PriA, which adopts the roles of both HisA and TrpF. Our findings demonstrate that bi-functionality can perpetuate in the absence of selection for very long time-spans.     

The evolution of multicellularity in animals as a shift in biological autonomy

The hypothesis is advanced that major evolutionary innovations are characterized by an increase of organismal autonomy in the sense of an emancipation from the environment. After a brief overview of the literature on this concept, increasing autonomy is defined as the evolutionary shift in the individual system-environment relationship, so that the direct influences of the environment are gradually reduced and a stabilization of self-referential, intrinsic functions within the system is generated. This is described as relative autonomy because numerous interconnections with the environment and dependencies upon it are retained. Elements of an increasing autonomy are spatial separations, an increase in homeostatic functions, internalizations and an increase in physiological and behavioral flexibility. These elements are described by taking the transition from single cells to metazoans as a case study. The principle of increasing autonomy is of central relevance for understanding this transition. The hypothesis does not contradict the principle of adaptation, but rather contributes to a further understanding of its elements as it supplies aspects for a reconsideration of the relationship between the outside and the inside, between organism and environment.     

Formation of the immunogenic α1,3-fucose epitope: elucidation of substrate specificity and of enzyme mechanism of core fucosyltransferase A

Glycans of glycoproteins are often associated with IgE mediated allergic immune responses. Hymenoptera venoms, e.g., carry α1,3-fucosyl residues linked to the proximal GlcNAc of glycoproteins. This epitope, formed selectively by α1,3-fucosyltransferase (FucTA), is xenobiotic and as such highly immunogenic and it also shows cross-reactivity if present on different proteins. Production of post-translationally modified proteins in insect cells is however commonly used and, thus, resulting glycoproteins can carry this highly immunogenic epitope with potentially significant side effects on mammals. To analyze mechanism, specificity and reaction kinetics of the key enzyme, we chose FucTA from Apis mellifera (honeybee) and characterized it by saturation transfer difference (STD) NMR and surface plasmon resonance (SPR) experiments. Specifically, we show here that the donor substrate, GDP-Fucose, binds mostly via its guanine and less so via pyrophosphate and fucosyl fragments and has a K_D = 37 μM. Affinity and kinetic studies with both the core α1,6-fucosylated and the unfucosylated octa- or heptasaccharides, respectively, as acceptor substrate revealed that honeybee FucTA prefers the latter structure with affinities of K_D ～ 10 mM. Establishment of progress curve analysis using an explicit solution of the integrated Michaelis–Menten equation allowed for determination of key constants of the transfer reaction of the glycosyl residue. The dominant minimum acceptor substrate is an unfucosylated heptasaccharide with K_m = 420 μM and k_cat = 6 min^?1. Time-resolved NMR spectra as well as STD NMR allow molecular insights into specificity, activity and interaction of the enzyme with substrates and acceptors.     

The human EMAP-like protein-70 (ELP70) is a microtubule destabilizer that localizes to the mitotic apparatus.

In this report, we show that the echinoderm microtubule (MT)-associated protein (EMAP) and related EMAP-like proteins (ELPs) share a similar domain organization with a highly conserved hydrophobic ELP (HELP) domain and a large tryptophan-aspartic acid (WD) repeat domain. To determine the function of mammalian ELPs, we generated antibodies against a 70-kDa human ELP and showed that ELP70 coassembled with MTs in HeLa cell extracts and colocalized with MTs in the mitotic apparatus. To determine whether ELP70 bound to MTs directly, human ELP70 was expressed and purified to homogeneity from baculovirus-infected Sf9 cells. Purified ELP70 bound to purified MTs with a stoichiometry of 0.40 +/- 0.04 mol of ELP70/mol of tubulin dimer and with an intrinsic dissociation constant of 0.44 +/- 0.13 microm. Using a nucleated assembly assay and video-enhanced differential interference contrast microscopy, we demonstrated that ELP70 reduced seeded nucleation, reduced the growth rate, and promoted MT catastrophes in a concentration-dependent manner. As a result, ELP70-containing MTs were significantly shorter than MTs assembled from tubulin alone. These data indicate that ELP70 is a novel MT destabilizer. A lateral destabilization model is presented to describe ELP70's effects on microtubules.     

Interferometric biosensor based on planar optical waveguide sensor chips for label-free detection of surface bound bioreactions

A label free optical biosensor based on a free-space Young interferometer configuration is presented. Commercial planar Ta(2) O(5) waveguides are used as sensing elements and allow the investigation of surface bound bioreactions like immunoreactions or biological affinity systems. Design criteria are discussed and a detailed characterization of the sensor performance is presented. The developed interferometer yields an effective refractive index resolution of 9 x 10(-9), corresponding to a surface coverage of approximately 13 fg/mm(2). The performance of the system is characterized by two different affinity systems: the antibody-antigen complex protein G-immunoglobulin G is used as a model system for monitoring reaction kinetics. Further measurements on a silanized surface show the formation of a streptavidin monolayer on a biotinylated surface.