Olfactory proteins mediating chemical communication in the navel orangeworm moth, Amyelois transitella.

BackgroundThe navel orangeworm, Amyelois transitella Walker (Lepidoptera: Pyralidae), is the most serious insect pest of almonds and pistachios in California for which environmentally friendly alternative methods of control--like pheromone-based approaches--are highly desirable. Some constituents of the sex pheromone are unstable and could be replaced with parapheromones, which may be designed on the basis of molecular interaction of pheromones and pheromone-detecting olfactory proteins.MethodologyBy analyzing extracts from olfactory and non-olfactory tissues, we identified putative olfactory proteins, obtained their N-terminal amino acid sequences by Edman degradation, and used degenerate primers to clone the corresponding cDNAs by SMART RACE. Additionally, we used degenerate primers based on conserved sequences of known proteins to fish out other candidate olfactory genes. We expressed the gene encoding a newly identified pheromone-binding protein, which was analyzed by circular dichroism, fluorescence, and nuclear magnetic resonance, and used in a binding assay to assess affinity to pheromone components.ConclusionWe have cloned nine cDNAs encoding olfactory proteins from the navel orangeworm, including two pheromone-binding proteins, two general odorant-binding proteins, one chemosensory protein, one glutathione S-transferase, one antennal binding protein X, one sensory neuron membrane protein, and one odorant receptor. Of these, AtraPBP1 is highly enriched in male antennae. Fluorescence, CD and NMR studies suggest a dramatic pH-dependent conformational change, with high affinity to pheromone constituents at neutral pH and no binding at low pH

The metabolomics of psoriatic disease.

Metabolomics is an emerging new "omics" field involving the systematic analysis of the metabolites in a biologic system. These metabolites provide a molecular snapshot of cellular activity and are thus important for understanding the functional changes in metabolic pathways that drive disease. Recently, metabolomics has been used to study the local and systemic metabolic changes in psoriasis and its cardiometabolic comorbidities. Such studies have revealed novel insights into disease pathogenesis and suggest new biochemical signatures that may be used as a marker of psoriatic disease. This review will discuss common strategies in metabolomics analysis, current findings in the metabolomics of psoriasis, and emerging trends in psoriatic metabolomics

Detection of early osteogenic commitment in primary cells using Raman spectroscopy

Major challenges in the development of novel implant surfaces for artificial joints include osteoblast heterogeneity and the lack of a simple and sensitive in vitro assay to measure early osteogenic responses. Raman spectroscopy is a label-free, non-invasive and non-destructive vibrational fingerprinting optical technique that is increasingly being applied to detect biochemical changes in cells. In this study Raman spectroscopy has been used to obtain bone cell-specific spectral signatures and to identify any changes therein during osteoblast commitment and differentiation of primary cells in culture. Murine calvarial osteoblasts (COBs) were extracted and cultured and studied by Raman spectroscopy over a 14 day culture period. Distinct osteogenic Raman spectra were identified after 3 days of culture with strong bands detected for mineral: phosphate ν3 (1030 cm−1) and B-type carbonate (1072 cm−1), DNA (782 cm−1) and collagen matrix (CH2 deformation at 1450 cm−1) and weaker phosphate bands (948 and 970 cm−1). Early changes were detected by Raman spectroscopy compared to a standard enzymatic alkaline phosphatase (ALP) assay and gene expression analyses over this period. Proliferation of COBs was confirmed by fluorescence intensity measurements using the Picogreen dsDNA reagent. Changes in ALP levels were evident only after 14 days of culture and mRNA expression levels for ALP, Col1a1 and Sclerostin remained constant during the culture period. Sirius red staining for collagen deposition also revealed little change until day 14. In contrast Raman spectroscopy revealed the presence of amorphous calcium phosphate (945–952 cm−1) and carbonated apatite (957–962 cm−1) after only 3 days in culture and octacalcium phosphate (970 cm−1) considered a transient mineral phase, was detected after 5 days of COBs culture. PCA analysis confirmed clear separation between time-points. This study highlights the potential of Raman spectroscopy to be utilised for the early and specific detection of proliferation and differentiation changes in primary cultures of bone cells

A keratin scaffold regulates epidermal barrier formation, mitochondrial lipid composition, and activity.

Keratin intermediate filaments (KIFs) protect the epidermis against mechanical force, support strong adhesion, help barrier formation, and regulate growth. The mechanisms by which type I and II keratins contribute to these functions remain incompletely understood. Here, we report that mice lacking all type I or type II keratins display severe barrier defects and fragile skin, leading to perinatal mortality with full penetrance. Comparative proteomics of cornified envelopes (CEs) from prenatal KtyI(-/-) and KtyII(-/-)(K8) mice demonstrates that absence of KIF causes dysregulation of many CE constituents, including downregulation of desmoglein 1. Despite persistence of loricrin expression and upregulation of many Nrf2 targets, including CE components Sprr2d and Sprr2h, extensive barrier defects persist, identifying keratins as essential CE scaffolds. Furthermore, we show that KIFs control mitochondrial lipid composition and activity in a cell-intrinsic manner. Therefore, our study explains the complexity of keratinopathies accompanied by barrier disorders by linking keratin scaffolds to mitochondria, adhesion, and CE formation

Does Osmotic Stress Affect Natural Product Expression in Fungi?

Acknowledgments: Russell Kerr acknowledges the assistance of Nadia Prigoda-Lee, Marius Grote, Kate McQuillan and Stephanie Duffy, and generous financial support from NSERC, the Canada Research Chair program, the Jeanne and Jean-Louis Lévesque Foundation and the Atlantic Canada Opportunities Agency. Ka-Lai Pang thanks the president of National Taiwan Ocean University, Ching-Fong Chang, for a special fund to attend the workshop held in Charlottetown, Canada in 2014 where this work was discussed. Rob Capon and Zhuo Shang acknowledge support from the University of Queensland, and the UQ Institute for Molecular Bioscience. Zhuo Shang acknowledges the provision of an International Postgraduate Research Scholarship (IPRS) and a Centennial Scholarship by the University of Queensland. Catherine Roullier acknowledges the assistance of Marie-Claude Boumard and Thibaut Robiou du Pont, and support from Region Pays de la Loire, FrancePeer reviewedPublisher PD

Recombinant expression and functional characterisation of regiospecific flavonoid glucosyltransferases from Hieracium pilosella L.

Five glucosyltransferases were cloned by RT-PCR amplification using total RNA from Hieracium pilosella L. (Asteraceae) inflorescences as template. Expression was accomplished in Escherichia coli, and three of the HIS-tagged enzymes, UGT90A7, UGT95A1, and UGT72B11 were partially purified and functionally characterised as UDP-glucose:flavonoid O-glucosyltransferases. Both UGT90A7 and UGT95A1 preferred luteolin as substrate, but possessed different regiospecificity profiles. UGT95A1 established a new subgroup within the UGT family showing high regiospecificity towards the C-3' hydroxyl group of luteolin, while UGT90A7 primarily yielded the 4'-O-glucoside, but concomitantly catalysed also the formation of the 7-O-glucoside, which could account for this flavones glucoside in H. pilosella flower heads. Semi quantitative expression profiles revealed that UGT95A1 was expressed at all stages of inflorescence development as well as in leaf and stem tissue, whereas UGT90A7 transcript abundance was nearly limited to flower tissue and started to develop with the pigmentation of closed buds. Other than these enzymes, UGT72B11 showed rather broad substrate acceptance, with highest activity towards flavones and flavonols which have not been reported from H. pilosella. As umbelliferone was also readily accepted, this enzyme could be involved in the glucosylation of coumarins and other metabolite

Peptide metabolism in the lactococci and its regulation : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University

Aspects of peptide metabolism in the lactococci have been investigated to increase the understanding of how these nutritionally fastidious bacteria, which have a central role in the dairy manufacturing industry, are able to grow in a complex medium such as milk. Peptide metabolism by lactococci in milk encompasses the processes by which large oligopeptides, produced from milk-caseins by the extracellular activity of the cell wall-associated proteinase, are converted into an intracellular pool of metabolisable amino acids. This involves the activities of both membrane-bound transport systems and peptidases. Early research into lactococcal peptide utilisation has proposed significant differences between Lactococcus lactis strains with respect to the mechanisms by which these bacteria utilise peptides in their environment. More recent studies of the lactococcal peptide carrier systems, based on intensive studies of only a single strain, have proposed a major role for a carrier system capable of transporting di- and tripeptides, and a subsidiary role for another system transporting oligopeptides containing four or more residues. Yet to date, peptidases with an extracellular location capable of degrading the large casein-peptides into smaller peptides have not been isolated. This current study has attempted to investigate more fully the in vivo activity of the oligopeptide transport system, and to assess whether it may have a more fundamental role in peptide utilisation than previous work has suggested. For this study a model series of homologous peptides of increasing size from the dipeptide Val-Gly to the octapeptide Val-Gly7 , all based on the essential amino acid valine, was used. The larger peptides in this series, Val-Gly3 , Val-Gly4 and Val-Gly7 , were synthesised for this work. The ability of Lactococcus lactis subsp. cremoris E8 8 to transport these peptides, and to grow in a chemically defined medium where they constitute the sole source of the essential amino acid valine, was studied . Preliminary peptide uptake studies were also performed using oligopeptides derived from a proteolytic cleavage of β-casein. The collective results of these studies suggest that the upper size limit, and the relative activity of this transport system, may be sufficient to permit this strain to utilise relatively large casein-derived oligopeptides without the need for hydrolysis into smaller peptides and free amino acids. A comparative study of peptide transport by a number of Lactococcus lactis strains was undertaken to investigate previously published observations indicating significant differences in the mechanisms of peptide uptake between lactococcal strains. While the results of this comparative study are consistent with the general model proposing two separate peptide carrier systems, they have revealed that significant differences can exist between strains in the relative activities and possible substrate specificities of these transport systems consistent with previous work that the lactococci have only two peptide carrier systems. These observations imply the need for caution in extrapolating the results obtained from the study of a single strain to lactococci as a whole. In contrast to the finding of significant strain differences with respect to the relative rates of peptide transport, a comparative study of the relative activity of six different intracellular peptidases showed relatively few differences in peptidase activity between strains. An investigation was also carried out to assess whether the peptidases and transport systems involved in the utilisation of peptides were nutritionally regulated. No clear evidence was obtained for the significant induction of either the intracellular peptidase complement or the di-/tripeptide transport system. An attempt was also made to isolate a mutant of Lactococcus lactis subsp. cremoris E8 unable to utilise dipeptides, to assess whether the di-/tripeptide transport system or the intracellular dipeptidase of this strain were essential to casein utilisation. This attempt was not successful