Primary structure requirements for correct sorting of the yeast mitochondrial protein ADH III to the yeast mitochondrial matrix space.

Alcohol dehydrogenase isoenzyme III (ADH III) in Saccharomyces cerevisiae, the product of the ADH3 gene, is located in the mitochondrial matrix. The ADH III protein was synthesized as a larger precursor in vitro when the gene was transcribed with the SP6 promoter and translated with a reticulocyte lysate. A precursor of the same size was detected when radioactively pulse-labeled proteins were immunoprecipitated with anti-ADH antibody. This precursor was rapidly processed to the mature form in vivo with a half-time of less than 3 min. The processing was blocked if the mitochondria were uncoupled with carbonyl cyanide m-chlorophenylhydrazone. Mutant enzymes in which only the amino-terminal 14 or 16 amino acids of the presequence were retained were correctly targeted and imported into the matrix. A mutant enzyme that was missing the amino-terminal 17 amino acids of the presequence produced an active enzyme, but the majority of the enzyme activity remained in the cytoplasmic compartment on cellular fractionation. Random amino acid changes were produced in the wild-type presequence by bisulfite mutagenesis of the ADH3 gene. The resulting ADH III protein was targeted to the mitochondria and imported into the matrix in all of the mutants tested, as judged by enzyme activity. Mutants containing amino acid changes in the carboxyl-proximal half of the ADH3 presequence were imported and processed to the mature form at a slower rate than the wild type, as judged by pulse-chase studies in vivo. The unprocessed precursor appeared to be unstable in vivo. It was concluded that only a small portion of the presequence contains the necessary information for correct targeting and import. Furthermore, the information for correct proteolytic processing of the presequence appears to be distinct from the targeting information and may involve secondary structure information in the presequence.     

Sapogeninbildung in suspensionskulturen von Digitalis purpurea

The effect of various growth conditions on the saponin level in suspension cultures of Digitalis purpurea has been investigated. The effect of time cultivation, 2,4-D concentration and 2,4-D combined with NAA with/or without casein hydrolysate, on saponin concentrations is described.     

Quantification of angiogenesis in estrogen receptor-positive and negative breast carcinoma

Background

The objective of this study was to evaluate angiogenesis according to CD34 antigen expression in estrogen receptor (ER)-positive and negative breast carcinomas.

Methods

This study comprised 64 cases of infiltrating ductal carcinoma in postmenopausal women divided into two groups: Group A: ER-positive, n = 35; and Group B: ER-negative, n = 29. The anti-CD34 monoclonal antibody was used as a marker for endothelial cells. Microvessel count was carried out in 10 fields per slide using a 40× objective lens (magnification 400×). Statistical analysis of the data was performed using Student's t-test (p < 0.05).

Results

The mean number of vessels stained with the anti-CD34 antibody in the estrogen receptor-positive and negative tumors was 23.51 ± 1.15 and 40.24 ± 0.42, respectively. The number of microvessels was significantly greater in the estrogen receptor-negative tumors (p < 0.001).

Conclusion

ER-negative tumors have significantly greater CD34 antigen expression compared to ER-positive tumors.

     

Differential responsiveness to immunoablative therapy in refractory rheumatoid arthritis is associated with level and avidity of anti-cyclic citrullinated protein autoantibodies: a case study

In order to identify pathogenic correlates of refractory rheumatoid arthritis (RA), antibodies against anti-cyclic citrullinated protein (ACPAs) were investigated in RA patients in whom the dysregulated immune system had been ablated by high-dose chemotherapy (HDC) and autologous haematopoietic stem cell transplantation (HSCT). Six patients with refractory RA were extensively characterized in terms of levels of total immunoglobulins, RA-specific autoantibodies (ACPAs and rheumatoid factor) and antibodies against rubella, tetanus toxoid (TT) and phosphorylcholine before and after HDC plus HSCT. Additionally, the avidity of ACPAs was measured before and after treatment and compared with the avidity of TT antibodies following repeated immunizations. Synovial biopsies were obtained by arthroscopy before HDC plus HSCT, and analyzed by immunohistochemistry. In the three patients with clinically long-lasting responses to HDC plus HSCT (median 423 days), significant reductions in ACPA-IgG levels after therapy were observed (median level dropped from 215 to 34 arbitrary units/ml; P = 0.05). In contrast, stable ACPA-IgG levels were observed in three patients who relapsed shortly after HDC plus HSCT (median of 67 days). Clinical responders had ACPA-IgG of lower avidity (r = 0.75; P = 0.08) and higher degree of inflammation histologically (r = 0.73; P = 0.09). Relapse (after 38 to 530 days) in all patients was preceded by rising levels of low avidity ACPA-IgG (after 30 to 388 days), in contrast to the stable titres of high avidity TT antibodies. In conclusion, humoral autoimmune responses were differentially modulated by immunoablative therapy in patients with synovial inflammation and low avidity ACPA-IgG autoantibodies as compared with patients with high levels of high avidity ACPA-IgG. The distinct clinical disease course after immunoablative therapy based on levels and avidity of ACPA-IgG indicates that refractory RA is not a single disease entity.     

Gdf6a is required for the initiation of dorsal–ventral retinal patterning and lens development

Dorsal–ventral patterning of the vertebrate retina is essential for accurate topographic mapping of retinal ganglion cell (RGC) axons to visual processing centers. Bone morphogenetic protein (Bmp) growth factors regulate dorsal retinal identity in vertebrate models, but the developmental timing of this signaling and the relative roles of individual Bmps remain unclear. In this study, we investigate the functions of two zebrafish Bmps, Gdf6a and Bmp4, during initiation of dorsal retinal identity, and subsequently during lens differentiation. Knockdown of zebrafish Gdf6a blocks initiation of retinal Smad phosphorylation and dorsal marker expression, while knockdown of Bmp4 produces no discernable retinal phenotype. These data, combined with analyses of embryos ectopically expressing Bmps, demonstrate that Gdf6a is necessary and sufficient for initiation of dorsal retinal identity. We note a profound expansion of ventral retinal identity in gdf6a morphants, demonstrating that dorsal BMP signaling antagonizes ventral marker expression. Finally, we demonstrate a role for Gdf6a in non-neural ocular tissues. Knockdown of Gdf6a leads to defects in lens-specific gene expression, and when combined with Bmp signaling inhibitors, disrupts lens fiber cell differentiation. Taken together, these data indicate that Gdf6a initiates dorsal retinal patterning independent of Bmp4, and regulates lens differentiation.     

Seasonal variation in terrestrial resource subsidies influences trophic niche width and overlap in two aquatic snake species: a stable isotope approach

Quantifying diet is essential for understanding the functional role of species with regard to energy processing, transfer, and storage within ecosystems. Recently, variance structure in the stable isotope composition of consumer tissues has been touted as a robust tool for quantifying trophic niche width, a task that has previously proven difficult due to bias in direct dietary analyses and difficulties in integrating diet composition over time. We used carbon and nitrogen stable isotope analyses to examine trophic niche width of two sympatric aquatic snakes, banded watersnakes Nerodia fasciata and black swamp snakes Seminatrix pygaea inhabiting an isolated wetland where seasonal migrations of amphibian prey cause dramatic shifts in resource availability. Specifically, we characterized snake and prey isotope compositions through time, space, and ontogeny and examined isotope values in relation to prey availability and snake diets assessed by gut content analysis. We determined that prey cluster into functional groups based on similarity of isotopic composition and seasonal availability. Isotope variance structure indicated that the trophic niche width of the banded watersnake was broader (more generalist) than that of the black swamp snake. Banded watersnakes also exhibited seasonal variation in isotope composition, suggesting seasonal diet shifts that track amphibian prey availability. Conversely, black swamp snakes exhibited little seasonal variation but displayed strong ontogenetic shifts in carbon and nitrogen isotope composition that closely paralleled ontogenetic shifts in their primary prey, paedomorphic mole salamanders Ambystoma talpoideum. Although niche dimensions are often treated as static, our results demonstrate that seasonal shifts in niche dimensions can lead to changes in niche overlap between sympatric species. Such short‐term fluctuations in niche overlap can influence competitive interactions and consequently the composition and dynamics of communities and ecosystems.