Analysis of disease-linked rhodopsin mutations based on structure, function, and protein stability calculations

Retinitis pigmentosa (RP) refers to a heterogeneous group of inherited diseases that result in progressive retinal degeneration, characterized by visual field constriction and night blindness. A total of 103 mutations in rhodopsin are linked to RP to date, and the phenotypes range from severe to asymptomatic. To study the relation between phenotype and rhodopsin stability in disease mutants, we used a structure-based approach. For 12 of the mutants located at the protein-lipid interphase, we used the von Heijne water-membrane transfer scale, and we find that 9 of the mutations could affect membrane insertion. For 91 mutants, we used the protein design algorithm FoldX. The 3 asymptomatic mutations had no significant reduced stability, 2 were unsuitable for FoldX analysis since the structure was incorrect in this region, 63 mutations had a significant change in protein stability (> 1.6 kcal/mol), and 23 mutations had energy change values under the prediction error threshold (< 1.6 kcal/mol). Out of these 23, the disease-causing effect could be explained by the involvement in other functions (e.g., glycosylation motifs, the interface with arrestin and transducin, and the cilia-binding motif) for 19 mutants. The remaining 4 mutants were probably incorrectly associated with RP or have functionalities not discovered yet. For destabilizing mutations where clinical data were available, we found a highly significant correlation between FoldX energy changes and the average age of night blindness and between FoldX energy changes and daytime vision loss onset. Our detailed structural, functional, and energetic analysis provides a complete picture of the rhodopsin mutations and can guide mutation-specific therapies.     

Glucose-induced and nitrogen-starvation-induced peroxisome degradation are distinct processes in Hansenula polymorpha that involve both common and unique genes

In the methylotrophic yeast Hansenula polymorpha non-selective autophagy, induced by nitrogen starvation, results in the turnover of cytoplasmic components, including peroxisomes. We show that the uptake of these components occurs by invagination of the vacuolar membrane without their prior sequestration and thus differs from the mechanism described for bakers yeast. A selective mode of autophagy in H. polymorpha, namely glucose-induced peroxisome degradation, involves sequestration of individual peroxisomes tagged for degradation by membrane layers that subsequently fuse with the vacuole where the organelle is digested. H. polymorpha pdd mutants are blocked in selective peroxisome degradation. We observed that pdd1-201 is also impaired in non-selective autophagy, whereas this process still normally functions in pdd2-4. These findings suggest that mechanistically distinct processes as selective and non-selective autophagy involve common but also unique genes.     

Microwave effects on immobilized peroxidase chemiluminescence

Protein gels formed by crosslinking bovine serum albumin and horseradish peroxidase with glutaraldehyde were used to measure effects on peroxidase activity of 400-MHz (CW) radiofrequency radiation (RFR) at an average specific absorption rate (SAR) of 1.45 W/kg. The enzyme activity was measured by luminol chemiluminescence recorded on photographic film after hydrogen peroxide activation. Activity was measured during RFR exposure of gels or after exposure of gels polymerized in the RFR field. During exposure, a significant (P less than .05) reversible increase occurred in overall mean peroxidase activity of gels activated with 0.88 M H2O2 but not in those activated with 8.8 M H2O2. Gels containing solubilized luminol and formed in the field showed no overall mean increase in peroxidase activity, but did display a highly significant (P less than .001) alteration in the distribution of local activities when compared to unexposed gels. These results are apparently due to changes in the rate of diffusion (concentration equilibration) of hydrogen peroxide in the gel.     

Initial effects of experimental warming on carbon exchange rates, plant growth and microbial dynamics of a lichen-rich dwarf shrub tundra in Siberia

Biological soil crusts can affect seed germination and seedling establishment. We have investigated the effect of biological soil crusts on seed water status as a potential mechanism affecting seed germination. The seed water potential of two annual grasses, one exotic Bromus tectorum L. and another native Vulpia microstachys Nutt., were analyzed after placing the seeds on bare soil, on a crust that contains various lichens and mosses (mixed crust), or on a crust dominated by the crustose lichen Diploschistes muscorum (Scop.) R. Sant. (Diploschistes crust). Seed water potential and germination were similar on the bare soil and the mixed crust, except for the initial germination of V. microstachys, which was higher on the mixed crust than on the bare soil. For the two grasses studied, seed water potential was significantly higher on the bare soil and mixed crust than on the Diploschistes crust. These differences in water potential correlated with differences in germination, which was much lower on the lichen crust. Experiments were conducted under two watering regimens. Increasing the frequency of watering amplified the differences in seed water potential and germination between the Diploschistes crust and the other two surfaces. For a particular watering regimen, the bare soil, mixed crust, and Diploschistes crust received the same amount of water, but they reached significantly different water potentials. Throughout the experiments, the water potential of the soil and mixed crust remained above −0.6 MPa, while there was a marked decline in the water potential of the Diploschistes surface to about −4 MPa. To ascertain that water was the major factor limiting germination on the Diploschistes crust, we conducted germination tests in an environment with 100% relative humidity. Under these conditions, germination on the Diploschistes crust was similar to that on the bare soil. However, the seeds that germinated on the Diploschistes crust did not penetrate this surface and approximately 60% of their root tips became necrotic. Our results indicate that the presence of D. muscorum can inhibit seedling establishment by two mechanisms: a reduction in seed water absorption and an increase in root tip mortality.     

Characterization of AAV vector particle stability at the single-capsid level

Virus families have evolved different strategies for genome uncoating, which are also followed by recombinant vectors. Vectors derived from adeno-associated viruses (AAV) are considered as leading delivery tools for in vivo gene transfer, and in particular gene therapy. Using a combination of atomic force microscopy (AFM), biochemical experiments, and physical modeling, we investigated here the physical properties and stability of AAV vector particles. We first compared the morphological properties of AAV vectors derived from two different serotypes (AAV8 and AAV9). Furthermore, we triggered ssDNA uncoating by incubating vector particles to increasing controlled temperatures. Our analyses, performed at the single-particle level, indicate that genome release can occur in vitro via two alternative pathways: either the capsid remains intact and ejects linearly the ssDNA molecule, or the capsid is ruptured, leaving ssDNA in a compact entangled conformation. The analysis of the length distributions of ejected genomes further revealed a two-step ejection behavior. We propose a kinetic model aimed at quantitatively describing the evolution of capsids and genomes along the different pathways, as a function of time and temperature. This model allows quantifying the relative stability of AAV8 and AAV9 particles.     

Sample preservation for determination of organic compounds: microwave versus freeze-drying

In search of a reliable drying method, which might be used evenunder field conditions, microwave drying was compared to freeze-dryingof plant material. Leaves of Ananas comosus and Avicennia germinansas well as buds and phloem of Acer pseudoplatanus were usedand checked for one or more of the following substances: sugars,sugar alcohols, organic and amino acids, total nitrogen, andglycinebetaine. With most samples good agreement was achieved between the twodrying methods. Only in the case of the Ananas comosus leaves,which exhibited low pH and high water content, did appreciabledifferences occur in organic and amino acids. Besides that,sucrose was the compound most susceptible to alterations, whichwas especially evident when leaves of Sambucus nigra were driedin the two different compartments (condenser compartment, dryingbell jar) of the freeze-dryer in use. For Ananas comosus leaf samples it was shown that microwavingcan also be used prior to extraction of tissue sap. Key words: Microwave, freeze-drying, drying method, tissue sap, organic solutes     

Newly found selenium-containing proteins in the tissues of the rat

The Se-containing proteins in 27 tissues of the rat were investigated by in vivo labeling with⁷⁵Se-selenite, separation of the tissue homogenate proteins by SDS-polyacrylamide gel electrophoresis, and determination of the labeled proteins by autoradiography. By using Se-depleted rats and a⁷⁵Se-tracer with a high specific activity, Se compounds present at only very low concentrations could be detected. Besides the 13 Se-containing proteins previously described, for which apparent molecular masses of 12, 15, 18, 20, 22, 25, 28, 34, 56, 60, 65, 70, and 75 kD have been found here, a further 15⁷⁵Se-labeled bands, with apparent molecular masses of 8, 10, 15.5, 16.5, 24, 32, 34.5, 38, 40, 41, 44, 45, 46.5, 53 and 116 kD could be distinguished. Two-dimensional separation of the kidney homogenate proteins showed that some of the Se-containing bands could be resolved into several labeled spots. Most of the newly found compounds were present in various tissues, but with some the enrichment in certain tissues suggested specific sites of action.     

The Hansenula polymorpha PEX14 gene encodes a novel peroxisomal membrane protein essential for peroxisome biogenesis.

We have cloned the Hansenula polymorpha PEX14 gene by functional complementation of the chemically induced pex14-1 mutant, which lacked normal peroxisomes. The sequence of the PEX14 gene predicts a novel protein product (Pex14p) of 39 kDa which showed no similarity to any known protein and lacked either of the two known peroxisomal targeting signals. Biochemical and electron microscopical analysis indicated that Pex14p is a component of the peroxisomal membrane. The synthesis of Pex14p is induced by peroxisome-inducing growth conditions. In cells of both pex14-1 and a PEX14 disruption mutant, peroxisomal membrane remnants were evident; these contained the H.polymorpha peroxisomal membrane protein Pex3p together with a small amount of the major peroxisomal matrix proteins alcohol oxidase, catalase and dihydroxyacetone synthase, the bulk of which resided in the cytosol. Unexpectedly, overproduction of Pex14p in wild-type H. polymorpha cells resulted in a peroxisome-deficient phenotype typified by the presence of numerous small vesicles which lacked matrix proteins; these were localized in the cytosol. Apparently, the stoichiometry of Pex14p relative to one or more other components of the peroxisome biogenesis machinery appears to be critical for protein import.     

Host-cell-specific glycosylation of HIV-2 envelope glycoprotein

Neutral complex-type N-glycans of the envelope glycoprotein 120 of HIV-2, propagated in different host cells, display cell-type specific variations. In order to identify typical structural elements, glycans were analysed by gel filtration, by enzymic sequencing and, in part, by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The characteristic substituents of di- tri- and tetraantennary carbohydrate units thus observed include N-acetyllactosamine repeats, bisecting N-acetylglucosamine and fucose linked to the chitobiose core as well as to N-acetyllactosamine antennae. Each glycoprotein preparation displayed a characteristic set of glycoforms. This revised version was published online in November 2006 with corrections to the Cover Date.