Role of the reticulum in the stability and shape of the isolated human erythrocyte membrane

In order to examine the widely held hypothesis that the reticulum of proteins which covers the cytoplamsic surface of the human erythrocyte membrane controls cell stability and shape, we have assessed some of its properties. The reticulum, freed of the bilayer by extraction with Triton X-100, was found to be mechanically stable at physiological ionic strength but physically unstable at low ionic strength. The reticulum broke down after a characteristic lag period which decreased 500-fold between 0 degrees and 37 degrees C. The release of polypeptide band 4.1 from the reticulum preceded that of spectrin and actin, suggesting that band 4.1 might stabilize the ensemble but is not essential to its integrity. The time-course of breakdown was similar for ghosts, the reticulum inside of ghosts, and the isolated reticulum. However, at very low ionic strength, the reticulum was less stable within the ghost than when free; at higher ionic strength, the reverse was true. Over a wide range of conditions the membrane broke down to vesicles just as the reticulum disintegrated, presumably because the bilayer was mechanically stabilized by this network. The volume of both ghosts and naked reticula varied inversely and reversibly with ionic strength. The volume of the naked reticulum varied far more widely than the ghost, suggesting that its deformation was normally limited by the less extensible bilayer. The contour of the isolated reticulum was discoid and often dimpled or indented, as visualized in the fluorescence microscope after labeling of the ghosts with fluoroscein isothiocyanate. Reticula derived from ghosts which had lost the ability to crenate in isotonic saline were shriveled, even though the bilayer was smooth and expanded. Conversly, ghosts crenated by dinitrophenol yielded smooth, expanded reticula. We conclude that the reticulum is a durable, flexible, and elastic network which assumes and stabilizes the contour of the membrane but is not responsible for its crenation.     

Aggregation risk prediction for antibodies and its application to biotherapeutic development

Aggregation is a common problem affecting biopharmaceutical development that can have a significant effect on the quality of the product, as well as the safety to patients, particularly because of the increased risk of immune reactions. Here, we describe a new high-throughput screening algorithm developed to classify antibody molecules based on their propensity to aggregate. The tool, constructed and validated on experimental aggregation data for over 500 antibodies, is able to discern molecules with a high aggregation propensity as defined by experimental criteria relevant to bioprocessing and manufacturing of these molecules. Furthermore, we show how this tool can be combined with other computational approaches during early drug development to select molecules with reduced risk of aggregation and optimal developability properties.     

Analysis of the genomic response of human prostate cancer cells to histone deacetylase inhibitors

Histone deacetylases (HDACs) have emerged as important targets for cancer treatment. HDAC-inhibitors (HDACis) are well tolerated in patients and have been approved for the treatment of patients with cutaneous T-cell lymphoma (CTCL). To improve the clinical benefit of HDACis in solid tumors, combination strategies with HDACis could be employed. In this study, we applied Analysis of Functional Annotation (AFA) to provide a comprehensive list of genes and pathways affected upon HDACi-treatment in prostate cancer cells. This approach provides an unbiased and objective approach to high throughput data mining. By performing AFA on gene expression data from prostate cancer cell lines DU-145 (an HDACi-sensitive cell line) and PC3 (a relatively HDACi-resistant cell line) treated with HDACis valproic acid or vorinostat, we identified biological processes that are affected by HDACis and are therefore potential treatment targets for combination therapy. Our analysis revealed that HDAC-inhibition resulted among others in upregulation of major histocompatibility complex (MHC) genes and deregulation of the mitotic spindle checkpoint by downregulation of genes involved in mitosis. These findings were confirmed by AFA on publicly available data sets from HDACi-treated prostate cancer cells. In total, we analyzed 375 microarrays with HDACi treated and non-treated (control) prostate cancer cells. All results from this extensive analysis are provided as an online research source (available at the journal’s website and at http://luigimarchionni.org/HDACIs.html). By publishing this data, we aim to enhance our understanding of the cellular changes after HDAC-inhibition, and to identify novel potential combination strategies with HDACis for the treatment of prostate cancer patients.     

Nine new polymorphic microsatellite loci for the amplification of archived otolith DNA of Atlantic cod,Gadus morhua L.

Nine out of 22 microsatellite primers tested were successfully amplified on three samples of cod Gadus morhua L. (two contemporary and one archived otolith samples). All loci were polymorphic (5–23 alleles/locus). The average observed heterozygosity across loci and samples was 0.625, ranging from 0.294 to 0.895 at each locus. All loci were under Hardy–Weinberg equilibrium, except PGmo56 that showed significant excess of heterozygotes in all studied samples. The isolated loci were suitable for degraded DNA and therefore useful for conducting a long‐term temporal study with DNA obtained from archived otoliths of cod.     

Seed weevils living on the edge: pressures and conflicts over body size in the endoparasitic Curculio larvae

Abstract 1. Body size in parasitic insects can be subjected to contrasting selective pressures, especially if they complete their development within a single host. On the one hand, a larger body size is associated with a higher fitness. On the other hand, the host offers a discrete amount of resources, thus constraining the evolution of a disproportionate body size. 2. The present study used the weevil Curculio elephas as a study model. Larvae develop within a single acorn, feeding on its cotyledons, and larval body size is strongly related to individual fitness. 3. The relationship between larval and acorn size was negatively exponential. Larval growth was constrained in small acorns, which did not provide enough food for the weevils to attain their potential size. Larval size increased and levelled off in acorns over a certain size (inflexion point), in which cotyledons were rarely depleted. When there were more than one larva per acorn, a larger acorn was necessary to avoid food depletion. 4. The results show that C. elephas larvae are sometimes endoparasitic, living on the edge of host holding capacity. If they were smaller they could avoid food depletion more easily, but the fitness benefits linked to a larger size have probably promoted body size increase. The strong negative effects of conspecific competition may have possibly influenced female strategy of laying a single egg per seed. 5. Being larger and fitter, but always within the limits of the available host sizes, may be one main evolutionary dilemma in endoparasites.     

Enhanced flavour formation by combination of selected lactococci from industrial and artisanal origin with focus on completion of a metabolic pathway

E.H.E. AYAD, A. VERHEUL, W.J.M. ENGELS, J.T.M. WOUTERS AND G. SMIT. 2001 . Combinations of lactococcal strains from various origins with divers properties were developed as new starters for new dairy products. Flavour formation by such tailor-made cultures was studied. In some cases, a strongly enhanced flavour was observed. For instance, the combination of B1157 and SK110 strains in milk resulted in a very strong chocolate-like flavour. B1157 produces only a moderate chocolate-like flavour, whereas SK110 alone fails to produce this flavour. Headspace gas chromatography results corroborate the organoleptic evaluations. High levels of branched-chain aldehydes were found when B1157 and SK110 were grown together. The enzyme activities involved in this pathway were studied; both strains contain transaminase activity. Although B1157 had a very high amino acid decarboxylating activity, its release of amino acids from milk protein was limited. SK110 was strongly limited in decarboxylating activity, although this strain is very active in proteolysis. By combining these strains, the substrates released by SK110 can directly be used by the other strain, resulting in the completion of the whole flavour-formation pathway. This opens new avenues for the preparation of tailor-made cultures.     

Biogeographical analysis of two Polypodium species complexes (Polypodiaceae) in Mexico and Central America

We analyzed the geographical and elevational distributions of two Polypodium complexes from Mexico and Central America. Distribution data of nine species of the Polypodium colpodes complex and the Polypodium plesiosorum complex were obtained from almost 1500 herbarium specimens, field collections in Mexico and Costa Rica, and literature studies. The presence of each species was recorded for each Mesoamerican country, in 1° × 1° grid‐cells and biogeographical provinces. The rarity of species was also evaluated. Although the two complexes show extensive overlap, the P. colpodes complex is distributed mainly along the Pacific versant of Mexico and Central America, whereas the P. plesiosorum complex occurs mainly along the Atlantic versant. Those biogeographical provinces with maximum species diversity are Chiapas (seven species), Sierra Madre del Sur (six species), and the Trans‐Mexican Volcanic belt (six species). Grid‐cells with more species are located mainly in the mountains of central‐southern Mexico and northern Central America. Richness does not decrease or increase with latitude. Elevation distributions showed that most Polypodium species are concentrated in the montane interval and three species groups were recognized based on elevational preferences. Polypodium colpodes and P. plesiosorum are the most widely distributed species, whereas Polypodium castaneum and Polypodium flagellare are the only two species that possess the three attributes of rarity (narrow geographical distribution, high habitat specificity, and scarce local populations). Polypodium species of both complexes are present mainly in the montane regions of the study area and show some degree of geographical sympatry, especially in southern Mexico and northern Central America. This overlapping is explained by the elevation tolerance within montane systems and because most species inhabit three or more vegetation types. The distributional patterns of these complexes coincided with the three regional highlands of Mesoamerica, which are separated from each other by the Isthmus of Tehuantepec and by the lowlands of Nicaragua. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, ?? , ??–??.     

Tibolone is not converted by human aromatase to 7alpha-methyl-17alpha-ethynylestradiol (7alpha-MEE): analyses with sensitive bioassays for estrogens and androgens and with LC-MSMS

To exclude that aromatization plays a role in the estrogenic activity of tibolone, we studied the effect tibolone and metabolites on the aromatization of androstenedione and the aromatization of tibolone and its metabolites to 7alpha-methyl-17alpha-ethynylestradiol (7alpha-MEE) by human recombinant aromatase. Testosterone (T), 17alpha-methyltestosterone (MT), 19-nortestosterone (Nan), 7alpha-methyl-19-nortestosterone (MENT) and norethisterone (NET) were used as reference compounds. Sensitive in vitro bioassays with steroid receptors were used to monitor the generation of product and the reduction of substrate. LC-MSMS without derivatization was used for structural confirmation. A 10 times excess of tibolone and its metabolites did not inhibit the conversion of androstenedione to estrone by human recombinant aromatase as determined by estradiol receptor assay whereas T, MT, Nan, and MENT inhibited the conversion for 75, 53, 85 and 67%, respectively. Tibolone, 3alpha- and 3beta-hydroxytibolone were not converted by human aromatase whereas the estrogenic activity formed with the Delta4-isomer suggests a conversion rate of 0.2% after 120 min incubation. In contrast T, MT, Nan, and MENT were completely converted to their A-ring aromates within 15 min while NET could not be aromatized. Aromatization of T, MT, Nan and MENT was confirmed with LC-MSMS. Structure/function analysis indicated that the 17alpha-ethynyl-group prevents aromatization of (19-nor)steroids while 7alpha-methyl substitution had no effect. Our results with the sensitive estradiol receptor assays show that in contrast to reference compounds tibolone and its metabolites are not aromatized.