The relationship between aromatase in primary breast tumors and response to treatment with aromatase inhibitors in advanced disease

Aromatase inhibitors are proving to be more effective than tamoxifen for postmenopausal patients with breast cancer. Estrogen concentrations in the breast are similar in both premenopausal and postmenopausal women, and several fold higher than circulating levels in postmenopausal women. In order to investigate the importance of intratumoral aromatase in stimulating the proliferation of the tumor, we used immunocytochemistry to determine the extent of aromatase expression in relationship to the response of the patient to aromatase inhibitor treatment. The relationship between positive staining for aromatase in the primary tumor and response to treatment with an aromatase inhibitor was investigated in a retrospective study of 102 patients with advanced breast cancer. Immunohistochemical staining using a monoclonal antibody against aromatase was performed on paraffin embedded tumor tissue. Response was evaluated using UICC criteria. Nine out of 13 patients with objective response to treatment stained positive and 49 of 89 patients with stable or progressive disease stained positive. No significant relationship between positive staining and objective response to treatment could be found. When patients with 'clinical benefit' (i.e. objective response plus prolonged stable disease of at least 6 months) were considered, also no relationship could be found. Further analysis of subgroups with positive hormone receptors, treatment with newer generation aromatase inhibitors, single metastatic site, non-visceral metastases and previous treatment only with tamoxifen did not show any relationship. Tumor aromatase expression did not correlate with response of patients with advanced breast cancer to aromatase inhibitor treatment. Most patients had relapsed from other treatments before receiving an aromatase inhibitor. It seems likely that many of these patients had tumors that may have progressed to hormone independence at this stage of the disease. Research in patients who have received treatment with aromatase inhibitors in earlier stages of disease (first line and adjuvant treatment) may provide further information on the relationship between tumor aromatase, steroid receptors and response to inhibitor treatment.     

Aberrant profiles of native and oxidized glycoproteins in Alzheimer plasma

A proteomic approach was employed to elucidate possible differential expression of native and oxidized glycoproteins using pooled plasma samples derived from ten patients with sporadic Alzheimer's disease (AD) and pooled plasma samples from nine normal elderly control (NEC) subjects. The plasma samples were fractionated by sequential affinity chromatography on heparin-agarose (HepA) and concanavalin A-agarose (ConA) columns followed by separation on one-dimensional and two-dimensional polyacrylamide gels. Carbonylation (oxidation) of proteins was monitored by in-strip derivatization with 2,4-dinitrophenylhydrazine (DNP) and anti-DNP immunoblotting. Nine spots representing glycoproteins which showed enrichment or high specific oxidation indices in AD HepA-ConA 2-D gels relative to NEC samples were analyzed by matrix-assisted laser desorption-time of flight-mass spectrometry and identified with high probability (p < 0.001) as isoforms of human transferrin (Tf), hemopexin (Hpx) and alpha-1-antitrypsin (alpha-1-AT). These glycoproteins were concentrated, respectively, 5-, 6.5- and 107-fold in HepA-ConA eluates derived from AD plasma relative to the NEC samples. Specific oxidation indices of the identified Tf and Hpx isoforms in AD plasma were respectively, 7.4 and 2.8 relative to NEC. Our findings provide further evidence for systemic derangements in heme/iron/redox homeostasis and activation of the acute phase response in sporadic AD. Moreover, the data implicate isoforms of Tf, Hpx and alpha-1-AT as potential biological markers of this condition.     

Evaluating the ecological realism of plant species distribution models with ecological indicator values

Species distribution models (SDMs) are routinely applied to assess current as well as future species distributions, for example to assess impacts of future environmental change on biodiversity or to underpin conservation planning. It has been repeatedly emphasized that SDMs should be evaluated based not only on their goodness of fit to the data, but also on the realism of the modeled ecological responses. However, possibilities for the latter are hampered by limited knowledge on the true responses as well as a lack of quantitative evaluation methods. Here we compared modeled niche optima obtained from European-scale SDMs of 1476 terrestrial vascular plant species with empirical ecological indicator values indicating the preferences of plant species for key environmental conditions. For each plant species we first fitted an ensemble SDM including three modeling techniques (GLM, GAM and BRT) and extracted niche optima for climate, soil, land use and nitrogen deposition variables with a large explanatory power for the occurrence of that species. We then compared these SDM-derived niche optima with the ecological indicator values by means of bivariate correlation analysis. We found weak to moderate correlations in the expected direction between the SDM-derived niche optima and ecological indicator values. The strongest correlation occurred between the modeled optima for growing degree days and the ecological indicator values for temperature. Correlations were weaker for SDM-derived niche optima with a more distal relationship to ecological indicator values (notably precipitation and soil moisture). Further, correlations were consistently highest for BRT, followed by GLM and GAM. Our method gives insight into the ecological realism of modeled niche optima and projected core habitats and can be used to improve SDMs by making a more informed selection of environmental variables and modeling techniques.     

Allosteric modulation of caspase 3 through mutagenesis

A mutation in the allosteric site of the caspase 3 dimer interface of Val266 to histidine abolishes activity of the enzyme, and models predict that the mutation mimics the action of small molecule allosteric inhibitors by preventing formation of the active site. Mutations were coupled to His266 at two sites in the interface, E124A and Y197C. We present results from X-ray crystallography, enzymatic activity and molecular dynamics simulations for seven proteins, consisting of single, double and triple mutants. The results demonstrate that considering allosteric inhibition of caspase 3 as a shift between discrete 'off-state' or 'on-state' conformations is insufficient. Although His266 is accommodated in the interface, the structural defects are propagated to the active site through a helix on the protein surface. A more comprehensive view of allosteric regulation of caspase 3 requires the representation of an ensemble of inactive states and shows that subtle structural changes lead to the population of the inactive ensemble.     

Metagenomic Survey for Viruses in Western Arctic Caribou,Alaska, through Iterative Assembly of Taxonomic Units

Pathogen surveillance in animals does not provide a sufficient level of vigilance because it is generally confined to surveillance of pathogens with known economic impact in domestic animals and practically nonexistent in wildlife species. As most (re-)emerging viral infections originate from animal sources, it is important to obtain insight into viral pathogens present in the wildlife reservoir from a public health perspective. When monitoring living, free-ranging wildlife for viruses, sample collection can be challenging and availability of nucleic acids isolated from samples is often limited. The development of viral metagenomics platforms allows a more comprehensive inventory of viruses present in wildlife. We report a metagenomic viral survey of the Western Arctic herd of barren ground caribou (Rangifer tarandus granti) in Alaska, USA. The presence of mammalian viruses in eye and nose swabs of 39 free-ranging caribou was investigated by random amplification combined with a metagenomic analysis approach that applied exhaustive iterative assembly of sequencing results to define taxonomic units of each metagenome. Through homology search methods we identified the presence of several mammalian viruses, including different papillomaviruses, a novel parvovirus, polyomavirus, and a virus that potentially represents a member of a novel genus in the family Coronaviridae.     

The complex of the insect LDL receptor homolog, lipophorin receptor, LpR, and its lipoprotein ligand does not dissociate under endosomal conditions

The insect low-density lipoprotein (LDL) receptor (LDLR) homolog, lipophorin receptor (LpR), mediates endocytic uptake of the single insect lipoprotein, high-density lipophorin (HDLp), which is structurally related to LDL. However, in contrast to the fate of LDL, which is endocytosed by LDLR, we previously demonstrated that after endocytosis, HDLp is sorted to the endocytic recycling compartment and recycled for re-secretion in a transferrin-like manner. This means that the integrity of the complex between HDLp and LpR is retained under endosomal conditions. Therefore, in this study, the ligand-binding and ligand-dissociation capacities of LpR were investigated by employing a new flow cytometric assay, using LDLR as a control. At pH 5.4, the LpR-HDLp complex remained stable, whereas that of LDLR and LDL dissociated. Hybrid HDLp-binding receptors, containing either the beta-propeller or both the beta-propeller and the hinge region of LDLR, appeared to be unable to release ligand at endosomal pH, revealing that the stability of the complex is imparted by the ligand-binding domain of LpR. The LpR-HDLp complex additionally appeared to be EDTA-resistant, excluding a low Ca(2+) concentration in the endosome as an alternative trigger for complex dissociation. From binding of HDLp to the above hybrid receptors, it was inferred that the stability upon EDTA treatment is confined to LDLR type A (LA) ligand-binding repeats 1-7. Additional (competition) binding experiments indicated that the binding site of LpR for HDLp most likely involves LA-2-7. It is therefore proposed that the remarkable stability of the LpR-HDLp complex is attributable to this binding site. Together, these data indicate that LpR and HDLp travel in complex to the endocytic recycling compartment, which constitutes a key determinant for ligand recycling by LpR.     

The Low Biomass Yields of the Acetic Acid Bacterium Acetobacter pasteurianus Are Due to a Low Stoichiometry of Respiration-Coupled Proton Translocation

Growth energetics of the acetic acid bacterium Acetobacter pasteurianus were studied with aerobic, ethanol-limited chemostat cultures. In these cultures, production of acetate was negligible. Carbon limitation and energy limitation were also evident from the observation that biomass concentrations in the cultures were proportional to the concentration of ethanol in the reservoir media. Nevertheless, low concentrations of a few organic metabolites (glycolate, citrate, and mannitol) were detected in culture supernatants. From a series of chemostat cultures grown at different dilution rates, the maintenance energy requirements for ethanol and oxygen were estimated at 4.1 mmol of ethanol (middot) g of biomass(sup-1) (middot) h(sup-1) and 11.7 mmol of O(inf2) (middot) g of biomass(sup-1) (middot) h(sup-1), respectively. When biomass yields were corrected for these maintenance requirements, the Y(infmax) values on ethanol and oxygen were 13.1 g of biomass (middot) mol of ethanol(sup-1) and 5.6 g of biomass (middot) mol of O(inf2)(sup-1), respectively. These biomass yields are very low in comparison with those of other microorganisms grown under comparable conditions. To investigate whether the low growth efficiency of A. pasteurianus might be due to a low gain of metabolic energy from respiratory dissimilation, (symbl)H(sup+)/O stoichiometries were estimated during acetate oxidation by cell suspensions. These experiments indicated an (symbl)H(sup+)/O stoichiometry for acetate oxidation of 1.9 (plusmn) 0.1 mol of H(sup+)/mol of O. Theoretical calculations of growth energetics showed that this low (symbl)H(sup+)/O ratio adequately explained the low biomass yield of A. pasteurianus in ethanol-limited cultures.     

Characterization of potato proteinase inhibitor II reactive site mutants.

Potato proteinase inhibitor II (PI-2) is composed of two sequence repeats. It contains two reactive site domains. We developed an improved protocol for the production of PI-2 using the yeast Pichia pastoris as the expression host. We then assessed the role of its two reactive sites in the inhibition of trypsin and chymotrypsin by mutating each of the two reactive sites in various ways. From these studies it appears that the second reactive site strongly inhibits both trypsin (Ki = 0.4 nM) and chymotrypsin (Ki = 0.9 nM), and is quite robust towards mutations at positions P2 or P1'. In contrast, the first reactive site inhibits only chymotrypsin (Ki = 2 nM), and this activity is very sensitive to mutations. Remarkably, replacing the reactive site amino acids of domain I with those of domain II did not result in inhibitory activities similar to domain II. The fitness for protein engineering of each domain is discussed.