Rubredoxin-related Maturation Factor Guarantees Metal Cofactor Integrity during Aerobic Biosynthesis of Membrane-bound [NiFe] Hydrogenase

The membrane-bound [NiFe] hydrogenase (MBH) supports growth of Ralstonia eutropha H16 with H₂ as the sole energy source. The enzyme undergoes a complex biosynthesis process that proceeds during cell growth even at ambient O₂ levels and involves 14 specific maturation proteins. One of these is a rubredoxin-like protein, which is essential for biosynthesis of active MBH at high oxygen concentrations but dispensable under microaerobic growth conditions. To obtain insights into the function of HoxR, we investigated the MBH protein purified from the cytoplasmic membrane of hoxR mutant cells. Compared with wild-type MBH, the mutant enzyme displayed severely decreased hydrogenase activity. Electron paramagnetic resonance and infrared spectroscopic analyses revealed features resembling those of O₂-sensitive [NiFe] hydrogenases and/or oxidatively damaged protein. The catalytic center resided partially in an inactive Ni_u-A-like state, and the electron transfer chain consisting of three different Fe-S clusters showed marked alterations compared with wild-type enzyme. Purification of HoxR protein from its original host, R. eutropha, revealed only low protein amounts. Therefore, recombinant HoxR protein was isolated from Escherichia coli. Unlike common rubredoxins, the HoxR protein was colorless, rather unstable, and essentially metal-free. Conversion of the atypical iron-binding motif into a canonical one through genetic engineering led to a stable reddish rubredoxin. Remarkably, the modified HoxR protein did not support MBH-dependent growth at high O₂. Analysis of MBH-associated protein complexes points toward a specific interaction of HoxR with the Fe-S cluster-bearing small subunit. This supports the previously made notion that HoxR avoids oxidative damage of the metal centers of the MBH, in particular the unprecedented Cys₆[4Fe-3S] cluster.     

3D QSAR study of hypolipidemic asarones by comparative molecular surface analysis

Three-dimensional quantitative structure-activity relationship (3D QSAR) modeled for alpha-asarone derivatives using the comparative molecular surface analysis (CoMSA) allowed us to reveal a correlation between the activity of these compounds and the electrostatic potential at the molecular surface. The grid formalism (s-CoMSA) allowed us to indicate a pharmacophore that is of key importance for compound activity. The CoMSA formalism coupled with the iterative variable elimination method gives a highly predictive model.     

Sterol carrier protein-2 expression alters sphingolipid metabolism in transfected mouse L-cell fibroblasts

The influence of sterol carrier protein-2 (SCP-2) on the cellular metabolism of sphingolipids was examined in control mouse L-cells and stably transfected clones expressing the protein SCP-2. Three approaches were used to examine for differences; (1) compositional analysis of endogenous sphingolipid classes, (2) metabolism of NBD-ceramide, and (3) live cell labelling via endocytic uptake of BODIPY-sphingomyelin. SCP-2 over expression significantly altered the endogenous levels of both neutral and acidic sphingolipid classes. Among the neutral sphingolipids, expression of SCP-2 induced a 1.7-fold increase in the level of lactosylceramide (LacCer, p < 0.05) and a similar fold decrease in the level of the higher-order neutral glycosylceramides (p < 0.05). Among the acidic sphingolipids, SCP-2 resulted in a 5.2-fold decrease in the endogenous plasma membrane level of ganglioside GM1 (p < 0.03). Incubation of both control and transfected cell lines with NBD-ceramide resulted in the rapid establishment of a steady-state distribution of NBD-labelled sphingomyelin (NBD-SM) and glucosylceramide (NBD-GlcCer). In the SCP-2 expressing clones the conversion of NBD-Cer to NBD-GlcCer was 30% lower during incubation periods between 5 and 30 min (p < 0.025). Inspection of the cells by fluorescence microscopy after incubation with BODIPY labelled sphingomyelin (BODIPY-SM) revealed similar punctuated patterns with no distinguishable differences between the cell types. These results imply that SCP-2 plays a role in modulating enzymatic steps involved in metabolism of sphingolipid homeostasis. Portions of this work were presented in abstract at the 2004 annual meeting of the American Society of Biochemistry and Molecular Biology, Boston Mass FASEB J, 18(8): C271, 2004     

A single-crystal ENDOR and density functional theory study of the oxidized states of the [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F

The catalytic center of the [NiFe] hydrogenase of Desulfovibrio vulgaris Miyazaki F in the oxidized states was investigated by electron paramagnetic resonance and electron–nuclear double resonance spectroscopy applied to single crystals of the enzyme. The experimental results were compared with density functional theory (DFT) calculations. For the Ni-B state, three hyperfine tensors could be determined. Two tensors have large isotropic hyperfine coupling constants and are assigned to the β-CH₂ protons of the Cys-549 that provides one of the bridging sulfur ligands between Ni and Fe in the active center. From a comparison of the orientation of the third hyperfine tensor with the tensor obtained from DFT calculations an OH⁻ bridging ligand has been identified in the Ni-B state. For the Ni-A state broader signals were observed. The signals of the third proton, as observed for the “ready” state Ni-B, were not observed at the same spectral position for Ni-A, confirming a structural difference involving the bridging ligand in the “unready” state of the enzyme. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users. Maurice van Gastel and Matthias Stein contributed equally to this work.     

Benzo[b]thiophene-2-carboxamides and benzo[b]furan-2-carboxamides are potent antagonists of the human H3-receptor

Benzo[b]thiophene-2-carboxamides and benzo[b]furan-2-carboxamides have been found to be antagonists on the human histamine-3-receptor, showing a Ki value of as low as 4 nM.     

Prenatal exposure of ethanol induces increased glutamatergic neuronal differentiation of neural progenitor cells

Background  

Prenatal ethanol exposure during pregnancy induces a spectrum of mental and physical disorders called fetal alcohol spectrum disorder (FASD). The central nervous system is the main organ influenced by FASD, and neurological symptoms include mental retardation, learning abnormalities, hyperactivity and seizure susceptibility in childhood along with the microcephaly. In this study, we examined whether ethanol exposure adversely affects the proliferation of NPC and de-regulates the normal ratio between glutamatergic and GABAergic neuronal differentiation using primary neural progenitor culture (NPC) and in vivo FASD models.     

Thymus development in Macaca fascicularis (Cynomolgus monkey): an approach for toxicology and embryology

Thymus development was studied in the cynomolgus monkey from day 35 of gestation (gd 35) to the stage of advanced involution in a 21-year-old monkey. Special emphasis was placed on thymus cell generation and cellular pattern formation. At gd 35, the epithelial bud of the thymus was visible in a sagittal position at the level of the thoracic aperture. At gd 50, first lymphocyte-like cells and few Human Leukocyte Antigen-D Region (HLA-DR) immunoreactive cells appeared. The cortico-medullary differentiation, Hassall’s body precursors and faint immunoreactivity for T-lymphocytes (CD 3-positive) were detected from gd 60 onwards. First macrophages (CD 68 positive) were apparent at day 70, first CD 20 immunoreactive cells (B-lymphocyte-like cells) at gd 85, and natural killer cells (M1014 immunoreactive) at gd 100. At gd 100 all evaluated cell populations present in the adult cynomolgus monkey thymus were in place, whereas no B- and T-cell precursors or (CD 34 and CD 117, respectively) dendritic cells (CD 35 positive cells) were present. All these immunopositive cells persisted, partly with changing distribution patterns, until the advanced age of 21 years with the exception of natural killer cells, which were present only until adult ages (evaluation at 4–7 years). The rationale of this study was to analyse thymic development in the cynomolgus monkey and to evaluate the relevance of the development of thymus in non-human primate as a model for corresponding human targeted toxicological research.