The periplasmic folding of a cysteineless autotransporter passenger domain interferes with its outer membrane translocation

Autotransporters are single polypeptides consisting of an outer membrane translocation domain mediating the translocation of a passenger domain. The periplasmic folding state of the passenger domain is controversial. By comparisons of passenger domains differing in their folding properties, our results suggest that periplasmic folding of passenger domains interferes with translocation.     

Characterization by Raman microspectroscopy of the strain-induced conformational transition in fibroin fibers from the silkworm Samia cynthia ricini

Raman microspectroscopy has been used to quantitatively study the effect of a mechanical deformation on the conformation and orientation of Samia cynthia ricini (S. c. ricini) silk fibroin. Samples were obtained from the aqueous solution stored in the silk gland and stretched at draw ratios (lambda) ranging from 0 to 11. Using an appropriate band decomposition procedure, polarized and orientation-insensitive spectra have been analyzed to determine order parameters and the content of secondary structures, respectively. The data unambiguously show that, in response to mechanical deformation, S. c. ricini fibroin undergoes a cooperative alpha-helix to beta-sheet conformational transition above a critical draw ratio of 4. The alpha-helix content decreases from 33 to 13% when lambda increases from 0 to 11, while the amount of beta-sheets increases from 15 to 37%. In comparison, cocoon silk is devoid of alpha-helical structure and always contains a larger amount of beta-sheets. Although the presence of isosbestic points in different spectral regions reveals that the conformational change induced by mechanical deformation is a two-state process, our results suggest that part of the glycine residues might be incorporated into beta-poly(alanine) structures. The beta-sheets are initially isotropically distributed and orient along the fiber axis as lambda increases, but do not reach the high level of orientation found in the cocoon fiber. The increase in the orientation level of the beta-sheets is found to be concomitant with the alpha --> beta conformational conversion, whereas alpha-helices do not orient under the applied strain but are rather readily converted into beta-sheets. The components assigned to turns exhibit a small orientation perpendicular to the fiber axis in stretched samples, showing that, overall, the polypeptide chains are aligned along the stretching direction. Our results suggest that, in nature, factors other than stretching contribute to the optimization of the amount of beta-sheets and the high degree of orientation found in natural cocoon silk.     

Pattern of segmental recombination in the distal inversion of mouse t haplotypes

To examine genetic exchange between t haplotypes and their wild-type homologs, four previously identified mouse Chromosome (Chr) 17 variants termed mosaic haplotypes were analyzed in detail. Three of these haplotypes-one from a Mus musculus population in Bulgaria, one from a Mus domesticus population in Chile, and one from a M. domesticus population in Germany-display properties indicative of the t complex. All four haplotypes are exceptional because they are characterized by the presence of a few wild-type DNA markers in the distal inversion [In(17)4] of a t haplotype chromosome: thus, they are classified as mosaic t haplotypes. The mosaic pattern for each haplotype is distinct, however. We compared the mosaic haplotypes with each other, and with several well-characterized laboratory t haplotypes, by analyzing several DNA markers in the In(17)4 region of the t complex, where all of the mosaicism occurs. We used a combination of high-resolution restriction mapping, DNA sequencing, and analysis of new DNA markers to classify the haplotypes. This analysis shows that segmental exchange, either by gene conversion or double crossing-over, has occurred at molecular markers in the vicinity of a gene, Dnahc8, that is a candidate for the t complex distorter locus Tcd2. While it is unclear whether segmental exchanges have included the Tcd2 gene, it is apparent that several independent recombination events have occurred in In(17)4 during the recent evolution of t haplotypes.     

Restoration of Petroleum-Contaminated Soil Using Phased Bioremediation

A conceptual approach is presented for the restoration of petroleum-contaminated sites by combining bioremediation with revegetation using native plants. Phased bioremediation includes active and passive treatment options for soil containing greater than 1% total petroleum hydrocarbons (TPHs). Phase I is used when initial soil TPH exceeds 1%. Phase I utilizes either active land treatment, with regular soil tillage, or passive bioremediation to attain a treatment endpoint of 1% soil TPH. Passive treatment utilizes static soil and TPH-tolerant plants. Phase II is utilized when soil contains 1% TPH or less. It combines passive bioremediation with revegetation using native plants to complete the site restoration process. The phased approach to bioremediation was developed from results of full-scale field bioremediation and laboratory treatability studies. This approach assumes that the kinetics of TPH biodegradation are initially rapid, followed by a much slower second stage. It provides active initial treatment, followed by lower-cost passive treatment. The selection of either active or passive treatment in Phase I depends on whether total cost or time of treatment is more important. Passive treatment, although less costly than active treatment, generally requires more time. Phased bioremediation may provide a flexible, cost-effective, and technically sound approach for restoration of petroleum-contaminated sites.

Vegetation used with passive bioremediation has several benefits. Plants stabilize soil, preventing erosion and thereby minimizing exposure to soil contaminants. Phytoremediation may also occur within the rhizosphere. The use of native plants has a strong ecological basis. They provide ecological diversity, are aesthetically pleasing and beneficial to wildlife, while requiring little maintenance. Phased bioremediation can provide a flexible, cost-effective, and technically sound approach for the restoration of petroleum-contaminated sites.     

Peroxiredoxin-4 interacts with and regulates the thromboxane A(2) receptor

We identified peroxiredoxin-4 (Prx-4) as a protein interacting with the beta isoform of the thromboxane A(2) receptor (TPbeta) by yeast two-hybrid analysis. Prx-4 co-immunoprecipitated constitutively with TPbeta in HEK293 cells. The second and third intracellular loops as well as the C-terminus of TPbeta interacted directly with Prx-4. Co-expression of Prx-4 caused a 60% decrease in cell surface expression of TPbeta. Prx-4 and TPbeta predominantly co-localized in the endoplasmic reticulum. Co-expression of Prx-4 in cells treated with H(2)O(2) targeted TPbeta for degradation. We show for the first time an interaction between a receptor involved in oxidative stress and Prx-4, an anti-oxidative enzyme.     

Disorganization is a completely dominant gain-of-function mouse mutation causing sporadic developmental defects.

Disorganization (Ds) is an exceptional mutation because of its diverse and profound developmental effects. Although other mouse mutations produce similar congenital defects, extreme pleiotropism, random occurrence, developmental independence of multiple defects, and type of anomaly make Ds unique. Examples of developmental defects include cranioschisis, rachischisis, thoracoschisis, exencephaly, hamartomas, and anomalies of appendages, digestive, genital and urinary tracts, sense organs, limbs and girdles, tail and pharynx. No other mutation in the mouse has such broad effects. Ds is therefore an important model for studying not only the genetic control of lineage determination and pattern formation, but also the occurrence of sporadic congenital defects. To characterize the effects of gene dosage, we examined the viability and phenotype of Ds homozygotes and the phenotype of +/+/Ds trisomic fetuses. Occurrence of homozygotes was tested by intercrossing Ds/+ heterozygotes, typing genetic markers that flank Ds, and examining homozygotes for morphological abnormalities. Not only were Ds homozygotes found in their expected frequency, homozygotes were not more severely affected than heterozygotes. Trisomies provide a direct test for determining whether Ds is a gain-of-function mutation. Trisomic fetuses were derived by crossing Ds/Ds homozygous mice to hybrid mice that were heterozygous for two related Robertsonian translocations. Two trisomic fetuses had developmental defects characteristic of Ds mice. Together these results demonstrate that Ds is a completely dominant, gain-of-function mutation.