A novel Arabidopsis thaliana protein is a functional peripheral-type benzodiazepine receptor

A key element in the regulation of mammalian steroid biosynthesis is the 18 kDa peripheral-type benzodiazepine receptor (PBR), which mediates mitochondrial cholesterol import. PBR also possess an affinity to the tetrapyrrole metabolite protoporphyrin. The bacterial homolog to the mammalian PBR, the Rhodobacter TspO (CrtK) protein, was shown to be involved in the bacterial tetrapyrrole metabolism. Looking for a similar mitochondrial import mechanism in plants, protein sequences from Arabidopsis and several other plants were found with significant similarities to the mammalian PBR and to the Rhodobacter TspO protein. A PBR-homologous Arabidopsis sequence was cloned and expressed in E. coli. The recombinant gene product showed specific high affinity benzodiazepine ligand binding. Moreover, the protein applied to E. coli protoplasts caused an equal benzodiazepine-stimulated uptake of cholesterol and protoporphyrin IX. These results suggest that the PBR like protein is involved in steroid import and is directing protoporphyrinogen IX to the mitochondrial site of protoheme formation.     

Identification of cardiac malformations in mice lacking <Emphasis Type="Italic">Ptdsr</Emphasis>using a novel high-throughput magnetic resonance imaging technique

Background  

Congenital heart defects are the leading non-infectious cause of death in children. Genetic studies in the mouse have been crucial to uncover new genes and signaling pathways associated with heart development and congenital heart disease. The identification of murine models of congenital cardiac malformations in high-throughput mutagenesis screens and in gene-targeted models is hindered by the opacity of the mouse embryo.     

Alterations in the phenotype of CMV-specific and total CD8+ T-cell populations in Wegener's granulomatosis

Wegener's granulomatosis (WG) is an autoimmune disease of as yet unknown etiology. To date it has remained obscure what causes WG or determines disease progression. Case reports suggest that viral infections such as cytomegalovirus (CMV) reactivation may contribute to disease flares. In this study we found a skewing of the phenotype of CMV-specific CD8+tet(ramer)+ T-cells in WG. A marked proportion of these cells displayed a late differentiated "effector memory" T-cell phenotype with decreased expression of CD28 and CD62L, and heterogeneous CD27 expression, features which were also seen in CD8+tet- T-cells in WG, but not in controls. Our results might reflect profound generalized changes in the CD8+ T-cell compartment also affecting virus-specific T-cell responses in WG.     

ERG6 and PDR5 regulate small lipophilic drug accumulation in yeast cells via distinct mechanisms

Diagnosis and circumvention of multi-drug resistance requires an understanding of the underlying cellular mechanisms. In the model organism Saccharomyces cerevisiae, deletions of PDR5 or ERG6 increase sensitivity to many small lipophilic drugs. Pdr5p is a plasma membrane ATP-binding cassette transporter that actively exports drugs, thereby lowering their intracellular levels. The mechanism by which ERG6, an enzyme in sterol biosynthesis, affects drug accumulation is less clear. We show here that ERG6 limits the rate of passive drug diffusion across the membrane, without affecting Pdr5p-mediated drug export. Consistent with their action by distinct mechanisms, PDR5 and ERG6 effects on drug accumulation are additive.     

Comparison of violaxanthin de-epoxidation from the stroma and lumen sides of isolated thylakoid membranes from Arabidopsis: implications for the mechanism of de-epoxidation

The enzyme violaxanthin de-epoxidase (VxDE) is localized in the thylakoid lumen and catalyzes the de-epoxidation of membrane-bound violaxanthin (Vx) to zeaxanthin. De-epoxidation from the opposite, stroma side of the membrane has been investigated in the npq1 mutant from Arabidopsis thaliana (L.) Heynh. - which lacks VxDE - by adding partially purified VxDE from spinach thylakoids. The accessibility of Vx to the exogenously added enzyme (exoVxDE) and the kinetics of Vx conversion by the exoVxDE in thylakoids from npq1 plants were very similar to the characteristics of Vx conversion by the endogenous enzyme (endoVxDE) in thylakoids from wild-type plants. However, the conversion of Vx by exoVxDE was clearly retarded at lower temperatures when compared with the reaction catalyzed by endoVxDE. Since the exoVxDE - in contrast to the endoVxDE - has no access to the stacked regions of the membrane, where the xanthophylls bound to photosystem II are located, these results support the assumption of pronounced diffusion of xanthophylls within the thylakoid membrane.     

Anaerobic degradation of n-hexane in a denitrifying bacterium: Further degradation of the initial intermediate (1-methylpentyl)succinate via C-skeleton rearrangement

The anaerobic degradation pathway of the saturated hydrocarbon n-hexane in a denitrifying strain (HxN1) was examined by gas chromatography-mass spectrometry of derivatized extracts from cultures grown with unlabeled and deuterated substrate; several authentic standard compounds were included for comparison. The study was focused on possible reaction steps that follow the initial formation of (1-methylpentyl)succinate from n-hexane and fumarate. 4-Methyloctanoic, 4-methyloct-2-enoic, 2-methylhexanoic, 2-methylhex-2-enoic and 3-hydroxy-2-methylhexanoic acids (in addition to a few other methyl-branched acids) were detected in n-hexane-grown but not in n-hexanoate-grown cultures. Labeling indicated preservation of the original carbon chain of n-hexane in these acids. Tracing of the deuterium label of 3- d1-(1-methylpentyl)succinate in tentative subsequent products indicated a deuterium/carboxyl carbon exchange in the succinate moiety. This suggests that the metabolism of (1-methylpentyl)succinate employs reactions analogous to those in the established conversion of succinyl-CoA via methylmalonyl-CoA to propionyl-CoA. Accordingly, a pathway is proposed in which (1-methylpentyl)succinate is converted to the CoA-thioester, rearranged to (2-methylhexyl)malonyl-CoA and decarboxylated (perhaps by a transcarboxylase) to 4-methyloctanoyl-CoA. The other identified fatty acids match with a further degradation of 4-methyloctanoyl-CoA via rounds of conventional beta-oxidation. Such a pathway would also allow regeneration of fumarate (for n-hexane activation) from propionyl-CoA formed as intermediate and hence present a cyclic process.     

Epizootiologic and ecologic investigations of European brown hares (Lepus europaeus) in selected populations from Schleswig-Holstein, Germany

From 1997-99 European brown hare (Lepus europaeus) population densities were estimated by spotlight surveys within different areas in Schleswig-Holstein, Germany. These areas showed a wide variation in local hare population densities. In addition, red fox (Vulpes vulpes) densities were estimated in 1997 by surveys of fox dens and litters. Sera of 321 hares (shot between 1998-2000) from four study areas were examined for antibodies against European brown hare syndrome virus (EBHSV) by enzyme linked immunosorbent assay (ELISA), Yersinia spp. (n = 299) and Francisella tularensis (n = 299) by western blotting, Brucella spp. by Rose Bengal test, and Toxoplasma gondii by Sabin-Feldman test (n = 318). Tissue samples comprising lung, liver, spleen, kidney, heart, and adrenal glands were collected for histopathology. Liver (n = 201) and spleen (n = 201) samples were processed for the detection of T. gondii-antigen in tissue sections and 321 liver and spleen samples were investigated for EBHSV-antigen by ELISA. Furthermore, 116 hares were examined macro- and microscopically for lungworms. Significant negative correlations between hare and fox densities were found in spring and autumn 1997. Antibodies against EBHSV were detected in 92 of 321 (29%), against Yersinia spp. in 163 of 299 (55%), and against T. gondii in 147 of 318 (46%) hares. We evaluated the potential influence of origin and hunting season on exposure rates of hares using logistic regression analysis. A strong association between hare densities and exposure rates was observed for various agents. One hundred and eight of 201 (57%) hares were positive for T. gondii-antigen. All sera were negative for antibodies against Brucella spp. and F. tularensis and all lung samples were negative for lungworms. In conclusion, variation in red fox densities may have an impact on the hare populations examined and the infectious diseases we studied seem to play a subordinate role in the dynamics of European brown hare populations from Schleswig-Holstein.     

A novel method for the determination of carbonyl groups in cellulosics by fluorescence labeling. 3. Monitoring oxidative processes

The fluorescence-based CCOA method for determination of carbonyl group profiles in cellulosic substrates was employed to study the mechanisms of various oxidative and degradation processes involving celluloses in greater detail. The approach comprises labeling with the marker carbazole-9-carboxylic acid [2-(2-aminooxyethoxy)ethoxy]amide (CCOA), followed by gel permeation chromatography in DMAc/LiCl with fluorescence, multiangle laser light scattering, and refractive index detection. At first, the CCOA method was applied to study solutions of pulp in N-methylmorpholine-N-oxide monohydrate (NMMO), as occurring in the production of Lyocell-type fibers. NMMO is a rather strong oxidant that on one hand converts reducing end groups to carboxyl structures, thus lowering the overall carbonyl content, but generates new keto structures along the chain by nonselective oxidation on the other hand. The CCOA method allowed for the first time to distinguish the carbonyl course in different molecular weight ranges. Second, alkalization and aging of pulp, which are used in the industrial preparation of cellulose derivatives, e.g., as an element of the preripening process in viscose rayon production, were investigated. The CCOA method shows a clear reduction of the molecular weight, accompanied by a fast loss of carbonyls in the first phase, which is due to removal of low-molecular weight material by dissolution, and a slow decrease in the second phase, which is caused by further oxidation of carbonyl groups. Also here, differences in the carbonyl course in different molecular weight regions were monitored. Third, electron beaming, proposed as a means of pulp activation, was shown to decrease and narrow the molecular weight distribution, under generation of comparatively low amounts of carbonyls, which, however, are also introduced into high molecular weight, crystalline domains, as shown by a comparison of homogeneous and heterogeneous CCOA labeling approach. Finally, as the fourth application, thermal treatment of cellulose at temperatures between 105 and 165 degrees C was shown to bring about a small reduction of the molecular weight, which only at higher drying temperatures is accompanied by an introduction of carbonyls over the whole molecular weight range.