A PEX6-defective peroxisomal biogenesis disorder with severe phenotype in an infant, versus mild phenotype resembling Usher syndrome in the affected parents

Sensorineural deafness and retinitis pigmentosa (RP) are the hallmarks of Usher syndrome (USH) but are also prominent features in peroxisomal biogenesis defects (PBDs); both are autosomal recessively inherited. The firstborn son of unrelated parents, who both had sensorineural deafness and RP diagnosed as USH, presented with sensorineural deafness, RP, dysmorphism, developmental delay, hepatomegaly, and hypsarrhythmia and died at age 17 mo. The infant was shown to have a PBD, on the basis of elevated plasma levels of very-long- and branched-chain fatty acids (VLCFAs and BCFAs), deficiency of multiple peroxisomal functions in fibroblasts, and complete absence of peroxisomes in fibroblasts and liver. Surprisingly, both parents had elevated plasma levels of VLCFAs and BCFAs. Fibroblast studies confirmed that both parents had a PBD. The parents' milder phenotypes correlated with relatively mild peroxisomal biochemical dysfunction and with catalase immunofluorescence microscopy demonstrating mosaicism and temperature sensitivity in fibroblasts. The infant and both of his parents belonged to complementation group C. PEX6 gene sequencing revealed mutations on both alleles, in the infant and in his parents. This unique family is the first report of a PBD with which the parents are themselves affected individuals rather than asymptomatic carriers. Because of considerable overlap between USH and milder PBD phenotypes, individuals suspected to have USH should be screened for peroxisomal dysfunction.     

Human stereovision without localized image features

Many theories of human stereovision are based on feature matching and the related correspondence problem. In this paper, we present psychophysical experiments indicating that localized image features such as Laplacian zerocrossings, intensity extrema, or centroids are not necessary for binocular depth perception. Smooth one-dimensional intensity profiles were combined into stereograms with mirror-symmetric half-images such that these localized image features were either absent or did not carry stereo information. In a discrimination task, subjects were asked to distinguish between stereograms differing only by an exchange of these half-images (ortho- vs. pseudoscopic stereograms). In a depth ordering task, subjects had to judge which of the two versions appeared in front. Subjects are able to solve both tasks even in the absence of the mentioned image features. The performance is compared to various possible stereo mechanisms. We conclude that localized image features and the correspondences between them are not necessary to perceive stereoscopic depth. One mechanism accounting for our data is correlation or mean square difference. Received: 8 February 1994 / Accepted in revised form: 15 September 1994     

Further indications of the multicomponent nature of the acrosome reaction-inducing substance of human follicular fluid

Human follicular fluid (hFF), which has been treated with either unspecific proteases or dextran-coated charcoal (DCC) to remove proteins and/or steroids, cannot successfully induce the acrosome reaction (AR). After the removal of steroids, AR-inducing activity can be restored to hFF by supplementation with exogenous progesterone, but only in the presence of intact protein. Gel filtration experiments with ³H-progesterone-labelled hFF showed elution of the radioactive signal in the high molecular weight range, corresponding to bound progesterone. AR-inducing activity was seen in exactly the same fraction. Based on these results, the acrosome reaction-inducing substance (ARIS) appears to be a complex of progesterone and a progesterone-binding protein, which was shown to be identical with the plasma protein corticosteroid-binding globulin (CBG) by immunological techniques. AR induction was only observed in the presence of both CBG and progesterone, suggesting a combined effect of the two components. © 1995 wiley-Liss, Inc.     

Monitoring the redox and protonation dependent contributions of cardiolipin in electrochemically induced FTIR difference spectra of the cytochrome bc1 complex from yeast

Biochemical studies have shown that cardiolipin is essential for the integrity and activity of the cytochrome bc₁ complex and many other membrane proteins. Recently the direct involvement of a bound cardiolipin molecule (CL) for proton uptake at center N, the site of quinone reduction, was suggested on the basis of a crystallographic study. In the study presented here, we probe the low frequency infrared spectroscopy region as a technique suitable to detect the involvement of the lipids in redox induced reactions of the protein. First the individual infrared spectroscopic features of lipids, typically present in the yeast membrane, have been monitored for different pH values in micelles and vesicles. The pK_a values for cardiolipin molecule have been observed at 4.7 ± 0.3 and 7.9 ± 1.3, respectively. Lipid contributions in the electrochemically induced FTIR spectra of the bc₁ complex from yeast have been identified by comparing the spectra of the as isolated form, with samples where the lipids were digested by lipase-A₂. Overall, a noteworthy perturbation in the spectral region typical for the protein backbone can be reported. Interestingly, signals at 1159, 1113, 1039 and 980 cm^− 1 have shifted, indicating the perturbation of the protonation state of cardiolipin coupled to the reduction of the hemes. Additional shifts are found and are proposed to reflect lipids reorganizing due to a change in their direct environment upon the redox reaction of the hemes. In addition a small shift in the alpha band from 559 to 556 nm can be seen after lipid depletion, reflecting the interaction with heme b_H and heme c. Thus, our work highlights the role of lipids in enzyme reactivity and structure.     

EXPOSE, an Astrobiological Exposure Facility on the International Space Station - from Proposal to Flight

Following an European Space Agency announcement of opportunity in 1996 for ”Externally mounted payloads for 1st utilization phase” on the International Space Station (ISS), scientists working in the fields of astrobiology proposed experiments aiming at long-term exposure of a variety of chemical compounds and extremely resistant microorganisms to the hostile space environment. The ESA exposure facility EXPOSE was built and an operations´ concept was prepared. The EXPOSE experiments were developed through an intensive pre-flight experiment verification test program. 12 years later, two sets of astrobiological experiments in two EXPOSE facilities have been successfully launched to the ISS for external exposure for up to 1.5 years. EXPOSE-E, now installed at the balcony of the European Columbus module, was launched in February 2008, while EXPOSE-R took off to the ISS in November 2008 and was installed on the external URM-D platform of the Russian Zvezda module in March 2009.     

Polychlorinated biphenyl (PCB)-degrading bacteria associated with trees in a PCB-contaminated site

The abundance, identities, and degradation abilities of indigenous polychlorinated biphenyl (PCB)-degrading bacteria associated with five species of mature trees growing naturally in a contaminated site were investigated to identify plants that enhance the microbial PCB degradation potential in soil. Culturable PCB degraders were associated with every plant species examined in both the rhizosphere and root zone, which was defined as the bulk soil in which the plant was rooted. Significantly higher numbers of PCB degraders (2.7- to 56.7-fold-higher means) were detected in the root zones of Austrian pine (Pinus nigra) and goat willow (Salix caprea) than in the root zones of other plants or non-root-containing soil in certain seasons and at certain soil depths. The majority of culturable PCB degraders throughout the site and the majority of culturable PCB degraders associated with plants were identified as members of the genus Rhodococcus by 16S rRNA gene sequence analysis. Other taxa of PCB-degrading bacteria included members of the genera Luteibacter and Williamsia, which have not previously been shown to include PCB degraders. PCB degradation assays revealed that some isolates from the site have broad congener specificities; these isolates included one Rhodococcus strain that exhibited degradation abilities similar to those of Burkholderia xenovorans LB400. Isolates with broad congener specificity were widespread at the site, including in the biostimulated root zone of willow. The apparent association of certain plant species with increased abundance of indigenous PCB degraders, including organisms with outstanding degradation abilities, throughout the root zone supports the notion that biostimulation through rhizoremediation is a promising strategy for enhancing PCB degradation in situ.     

Biocatalytic conversion of avermectin to 4'-oxo-avermectin: improvement of cytochrome p450 monooxygenase specificity by directed evolution

Discovery of the CYP107Z subfamily of cytochrome P450 oxidases (CYPs) led to an alternative biocatalytic synthesis of 4'-oxo-avermectin, a key intermediate for the commercial production of the semisynthetic insecticide emamectin. However, under industrial process conditions, these wild-type CYPs showed lower yields due to side product formation. Molecular evolution employing GeneReassembly was used to improve the regiospecificity of these enzymes by a combination of random mutagenesis, protein structure-guided site-directed mutagenesis, and recombination of multiple natural and synthetic CYP107Z gene fragments. To assess the specificity of CYP mutants, a miniaturized, whole-cell biocatalytic reaction system that allowed high-throughput screening of large numbers of variants was developed. In an iterative process consisting of four successive rounds of GeneReassembly evolution, enzyme variants with significantly improved specificity for the production of 4'-oxo-avermectin were identified; these variants could be employed for a more economical industrial biocatalytic process to manufacture emamectin.     

Antibody microarray-based profiling of complex specimens: systematic evaluation of labeling strategies

Antibody microarrays have often had limited success in detection of low abundant proteins in complex specimens. Signal amplification systems improve this situation, but still are quite laborious and expensive. However, the issue of sensitivity is more likely a matter of kinetically appropriate microarray design as demonstrated previously. Hence, we re-examined in this study the suitability of simple and inexpensive detection approaches for highly sensitive antibody microarray analysis. N-hydroxysuccinimidyl ester (NHS)- and Universal Linkage System (ULS)-based fluorescein and biotin labels used as tags for subsequent detection with anti-fluorescein and extravidin, respectively, as well as fluorescent dyes were applied for analysis of blood plasma. Parameters modifying strongly the performance of microarray detection such as labeling conditions, incubation time, concentrations of anti-fluorescein and extravidin and extent of protein labeling were analyzed and optimized in this study. Indirect detection strategies whether based on NHS- or ULS-chemistries strongly outperformed direct fluorescent labeling and enabled detection of low abundant cytokines with many dozen-fold signal-to-noise ratios. Finally, particularly sensitive detection chemistry was applied to monitoring cytokine production of stimulated peripheral T cells. Microarray data were in accord with quantitative cytokine levels measured by ELISA and Luminex, demonstrating comparable reliability and femtomolar range sensitivity of the established microarray approach.     

Thermodynamic characterization of allosteric glycogen phosphorylase inhibitors

Glycogen phosphorylase (GP) is a validated target for the treatment of type 2 diabetes. Here we describe highly potent GP inhibitors, AVE5688, AVE2865, and AVE9423. The first two compounds are optimized members of the acyl urea series. The latter represents a novel quinolone class of GP inhibitors, which is introduced in this study. In the enzyme assay, both inhibitor types compete with the physiological activator AMP and act synergistically with glucose. Isothermal titration calorimetry (ITC) shows that the compounds strongly bind to nonphosphorylated, inactive GP (GPb). Binding to phosphorylated, active GP (GPa) is substantially weaker, and the thermodynamic profile reflects a coupled transition to the inactive (tense) conformation. Crystal structures confirm that the three inhibitors bind to the AMP site of tense state GP. These data provide the first direct evidence that acyl urea and quinolone compounds are allosteric inhibitors that selectively bind to and stabilize the inactive conformation of the enzyme. Furthermore, ITC reveals markedly different thermodynamic contributions to inhibitor potency that can be related to the binding modes observed in the cocrystal structures. For AVE5688, which occupies only the lower part of the bifurcated AMP site, binding to GPb (Kd = 170 nM) is exclusively enthalpic (Delta H = -9.0 kcal/mol, TDelta S = 0.3 kcal/mol). The inhibitors AVE2865 (Kd = 9 nM, Delta H = -6.8 kcal/mol, TDelta S = 4.2 kcal/mol) and AVE9423 (Kd = 24 nM, Delta H = -5.9 kcal/mol, TDelta S = 4.6 kcal/mol) fully exploit the volume of the binding pocket. Their pronounced binding entropy can be attributed to the extensive displacement of solvent molecules as well as to ionic interactions with the phosphate recognition site.