Modulation of Conotoxin Structure and Function Is Achieved through a Multienzyme Complex in the Venom Glands of Cone Snails

The oxidative folding of large polypeptides has been investigated in detail; however, comparatively little is known about the enzyme-assisted folding of small, disulfide-containing peptide substrates. To investigate the concerted effect of multiple enzymes on the folding of small disulfide-rich peptides, we sequenced and expressed protein-disulfide isomerase (PDI), peptidyl-prolyl cis-trans isomerase, and immunoglobulin-binding protein (BiP) from Conus venom glands. Conus PDI was shown to catalyze the oxidation and reduction of disulfide bonds in two conotoxins, α-GI and α-ImI. Oxidative folding rates were further increased in the presence of Conus PPI with the maximum effect observed in the presence of both enzymes. In contrast, Conus BiP was only observed to assist folding in the presence of microsomes, suggesting that additional co-factors were involved. The identification of a complex between BiP, PDI, and nascent conotoxins further suggests that the folding and assembly of conotoxins is a highly regulated multienzyme-assisted process. Unexpectedly, all three enzymes contributed to the folding of the ribbon isomer of α-ImI. Here, we identify this alternative disulfide-linked species in the venom of Conus imperialis, providing the first evidence for the existence of a “non-native” peptide isomer in the venom of cone snails. Thus, ER-resident enzymes act in concert to accelerate the oxidative folding of conotoxins and modulate their conformation and function by reconfiguring disulfide connectivities. This study has evaluated the role of a number of ER-resident enzymes in the folding of conotoxins, providing novel insights into the enzyme-guided assembly of these small, disulfide-rich peptides.     

17(E)-Picolinylidene androstane derivatives as potential inhibitors of prostate cancer cell growth: Antiproliferative activity and molecular docking studies

We report a rapid and efficient synthesis of A-ring modified 17α-picolyl and 17(E)-picolinylidene androstane derivatives from dehydroepiandrosterone. Compounds were validated spectroscopically and structurally characterized by X-ray crystallography. Virtual screening by molecular docking against clinical targets of steroidal anticancer drugs (ERα, AR, Aromatase and CYP17A1) suggests that 17(E)-picolinylidene, but not 17α-picolyl androstanes could specifically interact with CYP17A1 (17α-hydroxylase) with similar geometry and affinity as Abiraterone, a 17-pyridinyl androstane drug clinically used in the treatment of prostate cancer. In addition, several 17(E)-picolinylidene androstanes demonstrated selective antiproliferative activity against PC3 prostate cancer cells, which correlates with Abiraterone antiproliferative activity and predicted CYP17A1 binding affinities. Based on these preliminary results, 17(E)-picolinylidene androstane derivatives could be a promising starting point for the development of new compounds for the treatment of prostate cancer.     

Targeting of fluorescent Lactococcus lactis to colorectal cancer cells through surface display of tumour-antigen binding proteins

Development of targeted treatment for colorectal cancer is crucial to avoid side effects. To harness the possibilities offered by microbiome engineering, we prepared safe multifunctional cancer cell-targeting bacteria Lactococcus lactis. They displayed, on their surface, binding proteins for cancer-associated transmembrane receptors epithelial cell adhesion molecule (EpCAM) and human epidermal growth factor receptor 2 (HER2) and co-expressed an infrared fluorescent protein for imaging. Binding of engineered L. lactis to tumour antigens EpCAM and HER2 was confirmed and characterised in vitro using soluble receptors. The proof-of-principle of targeting was demonstrated on human cell lines HEK293, HT-29 and Caco-2 with fluorescent microscopy and flow cytometry. The highest L. lactis adhesion was seen for the HEK293 cells with the overexpressed tumour antigens, where colocalisation with their tumour antigens was seen for 39% and 67% of EpCAM-targeting and HER2-targeting bacteria, respectively. On the other hand, no binding was observed to HEK293 cells without tumour antigens, confirming the selectivity of the engineered L. lactis. Apart from cell targeting in static conditions, targeting ability of engineered L. lactis was also shown in conditions of constant flow of bacterial suspension over the HEK293 cells. Successful targeting by engineered L. lactis support the future use of these bacteria in biopharmaceutical delivery for the treatment of colorectal cancer.     

Characterization of conantokin Rl‐A: molecular phylogeny as structure/function study

A multidisciplinary strategy for discovery of new Conus venom peptides combines molecular genetics and phylogenetics with peptide chemistry and neuropharmacology. Here we describe application of this approach to the conantokin family of conopeptides targeting NMDA receptors. A new conantokin from Conus rolani, ConRl‐A, was identified using molecular phylogeny and subsequently synthesized and functionally characterized. ConRl‐A is a 24‐residue peptide containing three γ‐carboxyglutamic acid residues with a number of unique sequence features compared to conantokins previously characterized. The HPLC elution of ConRl‐A suggested that this peptide exists as two distinct, slowly exchanging conformers. ConRl‐A is predominantly helical (estimated helicity of 50%), both in the presence and absence of Ca⁺⁺. The order of potency for blocking the four NMDA receptor subtypes by ConRl‐A was NR2B > NR2D > NR2A > NR2C. This peptide has a greater discrimination between NR2B and NR2C than any other ligand reported so far. In summary, ConRl‐A is a new member of the conantokin family that expands our understanding of structure/function of this group of peptidic ligands targeted to NMDA receptors. Thus, incorporating phylogeny in the discovery of novel ligands for the given family of ion channels or receptors is an efficient means of exploring the megadiverse group of peptides from the genus Conus. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Altered Distribution of Peripheral Blood Memory B Cells in Humans Chronically Infected with Trypanosoma cruzi

Numerous abnormalities of the peripheral blood T cell compartment have been reported in human chronic Trypanosoma cruzi infection and related to prolonged antigenic stimulation by persisting parasites. Herein, we measured circulating lymphocytes of various phenotypes based on the differential expression of CD19, CD4, CD27, CD10, IgD, IgM, IgG and CD138 in a total of 48 T. cruzi-infected individuals and 24 healthy controls. Infected individuals had decreased frequencies of CD19+CD27+ cells, which positively correlated with the frequencies of CD4+CD27+ cells. The contraction of CD19+CD27+ cells was comprised of IgG+IgD-, IgM+IgD- and isotype switched IgM-IgD- memory B cells, CD19+CD10+CD27+ B cell precursors and terminally differentiated CD19+CD27+CD138+ plasma cells. Conversely, infected individuals had increased proportions of CD19+IgG+CD27-IgD- memory and CD19+IgM+CD27-IgD+ transitional/naïve B cells. These observations prompted us to assess soluble CD27, a molecule generated by the cleavage of membrane-bound CD27 and used to monitor systemic immune activation. Elevated levels of serum soluble CD27 were observed in infected individuals with Chagas cardiomyopathy, indicating its potentiality as an immunological marker for disease progression in endemic areas. In conclusion, our results demonstrate that chronic T. cruzi infection alters the distribution of various peripheral blood B cell subsets, probably related to the CD4+ T cell deregulation process provoked by the parasite in humans.     

Detecting psychogeriatric problems in primary care: factors related to psychiatric symptoms in older community patients

Objective The aim was to determine the relationship and influence of different variables on the psychiatric symptomatology of older people who reside in the community, as detected by family practitioners.Design A cross-sectional and multi-centre study.Setting Twenty-eight general practices and two psychiatric practices in Huesca, Spain, from 19 primary care health centres.Subjects A sample of 324 patients aged over 65 years, representative of the older people who reside in the community in the province of Huesca.Main outcome measures Symptoms of depression (Yesavage GDS), cognitive impairment (MMSE), anxiety (GADS), psychotic symptoms, obsessive symptoms and hypochondriacal ideas (GMS) were measured by family practitioner and were detected following specific questions from the Geriatric Mental State (GMS-B) examination, following DSM-IV criteria, being defined as 'concern and fear of suffering, or the idea of having a serious disease based on the interpretation of somatic symptoms'. Sociodemographic, physical and somatic, functional and social data were evaluated. Analysis was carried out in three phases: univariate, bivariate and multivariate with logistic regression.Results At the time of the study, 46.1% of the older people studied suffered from some psychiatric symptom; 16.4% had cognitive impairment, 15.7% anxiety, 14.3% depression, 6.1% hallucinations and delusions, 7.2% hypochondriacal ideas and 4.4% obsessive symptoms. Female gender was significantly associated with depression (prevalence ration (PR) 3.3) and anxiety (PR 3.9). Age was a factor associated with cognitive impairment (PR 4.4). Depression was significantly related to severity of the physical illness (PR 61.7 in extremely severe impairment). Isolation (PR 16.3) and being single (PR 13.4) were factors which were strongly associated with anxiety; living in a nursing home was associated with psychotic symptoms (PR 7.6).Conclusions Severity of physical illness, isolation, living in a nursing home and female gender, among others, are related to psychiatric symptoms in community-residing older people identified in primary healthcare centres.     

Combined bottom-up and top-down mass spectrometry analyses of the pattern of post-translational modifications of Drosophila melanogaster linker histone H1

Linker histone H1 is a major chromatin component that binds internucleosomal DNA and mediates the folding of nucleosomes into a higher-order structure, namely the 30-nm chromatin fiber. Multiple post-translational modifications (PTMs) of core histones H2A, H2B, H3 and H4 have been identified and their important contribution to the regulation of chromatin structure and function is firmly established. In contrast, little is known about histone H1 modifications and their function. Here we address this question in Drosophila melanogaster, which, in contrast to most eukaryotic species, contains a single histone H1 variant, dH1. For this purpose, we combined bottom-up and top-down mass-spectrometry strategies. Our results indicated that dH1 is extensively modified by phosphorylation, methylation, acetylation and ubiquitination, with most PTMs falling in the N-terminal domain. Interestingly, several dH1 N-terminal modifications have also been reported in specific human and/or mouse H1 variants, suggesting that they have conserved functions. In this regard, we also provide evidence for the contribution of one of such conserved PTMs, dimethylation of K27, to heterochromatin organization during mitosis. Furthermore, our results also identified multiple dH1 isoforms carrying several phosphorylations and/or methylations, illustrating the high structural heterogeneity of dH1. In particular, we identified several non-CDK sites at the N-terminal domain that appear to be hierarchically phosphorylated. This study provides the most comprehensive PTM characterization of any histone H1 variant to date.     

Seasonal dynamics of allelopathically significant phenolic compounds in globally successful invader Conyza canadensis L. plants and associated sandy soil

The seasonal dynamics of total phenolics and phenolic acids in the stems of the global invader Conyza canadensis, from March (young plants in the form of rosettes) to September (fruit abscission and the beginning of plant decline), and in sandy soil were monitored monthly in non-native areas. The highest amount of total free phenolics was found in its tissues (31,000 μg g⁻¹) during the flowering and fruiting time (August). Bound phenolics peaked (up to 8443 μg g⁻¹) during shoot elongation and intensive plant growth (May–June) and in September. In the stems, bound phenolic acids (p-coumaric, ferulic, p-hydroxybenzoic, vanillic and syringic) have a maximum twice, in May and in August, with ferulic acid predominating (up to 951.6 μg g⁻¹). Free phenolic acids in the plant's tissue peaked in May (plant elongation). In the soil under C. canadensis, the amount of bound phenolics decreased between March and June, before increasing up to the full bloom phase of the plants (August). The amount of bound phenolic acids was several times greater than that of free ones, with maximum values in August. C. canadensis is a highly important source of phenolics in the ruderal phytocoenosis in new areas. In order to better explain the mechanisms of the spread and domination of invasive plants in non-native areas, in which allelopathy plays a decisive role, it is necessary to measure the production of allelochemicals in tissue and their accumulation in soil at the shortest possible intervals and link this with the phases of plant development.