GPCR expression using baculovirus-infected Sf9 cells

Expression of proteins in insect cells using recombinant baculoviruses has gained wide use in the G protein-coupled receptor (GPCR) community. This expression system produces high yields of functional receptor, is able to perform post-translational modifications, and is readily adaptable to large-scale culture. Here, we describe the generic methods for expressing a GPCR using baculovirus-infected insect cells, including the maintenance of insect cell culture. Data are presented for polyhedrin promoter-driven expression of a C-terminal 6 x histidine-tagged mammalian M(2) muscarinic receptor in Sf9 cells. Results demonstrate that expressed receptor could be detected and quantified using radiolabeled ligand binding, that expression was maximal at approximately 72 h post-infection, and that expression levels could be altered by addition of various ligands to cultures of infected insect cells.Amanda L. Aloia, Richard V. Glatz, Edward J. McMurchie, and Wayne R. Leifer

Buccal cell cytokeratin 14 identifies mild cognitive Impairment and Alzheimer’ s Disease in the AIBL Study of Aging

Previous studies have suggested that mild cognitive impairment (MCI) may be reflective of the early stages of neurodegenerative disorders such as Alzheimer’s disease (AD). The hypothesis was that cytokeratin (CK) 14 expression can be used as a biomarker in isolated buccal mucosa to identify individuals with MCI or AD from the Australian Imaging, Biomarkers and Lifestyle (AIBL) flagship study of aging. Visual assessment of buccal cell CK14 expression was carried out using immunofluorescence techniques. The frequency of basal buccal cells expressing CK14 was significantly lower in the MCI (P=0.0002) and AD (P<0.05) groups compared with the control group. Receiver-operating characteristic (ROC) curves were carried out for CK14 expression and yielded an area under the curve (AUC) of 0.899 for the MCI (P<0.0001) group and 0.772 for the AD (P=0.004) group. When the CK14 expression data were combined with plasma homocysteine concentration, the AUC was further improved to 0.932 and 0.788 for the MCI (P=0.0001) and AD (P=0.004) groups, respectively. APOE ε4 carriers in the control group had 21% lower CK14 expression compared with control non APOE ε4 carriers, however this difference was not statistically significant. The changes in the buccal cell CK14 expression observed in this pilot study could prove useful as a potential biomarker in identifying individuals with an increased risk of developing MCI and eventually AD. These promising results need to be replicated in a larger subset of the AIBL cohort and in cohorts of other neurodegenerative disorders to determine changes specific to AD

Effect of iodine and selenium on proliferation, viability, and oxidative stress in HTR-8/SVneo placental cells

Adequate maternal micronutrition is vital for placental formation, fetal growth, and development. Oxidative stress adversely affects placental development and function and an association between deficient placental development, oxidative stress, and micronutrient deficiency has been observed. Selenium and iodine are two essential micronutrients with antioxidant properties. Epidemiological studies have shown that poor micronutrient status in pregnant women is associated with a higher incidence of pregnancy complications. The aim of this study was to determine how selenium, iodine, and their combination impact oxidative stress in placental trophoblast cells. HTR8/SVneo extravillous trophoblasts were supplemented with a concentration range of organic and inorganic selenium, potassium iodide, or their combination for 24 h. Oxidative stress was then induced by treating cells with menadione or H2O2 for 24 h. Cell viability and lipid peroxidation as the biomarker of oxidative stress were assessed at 48 h. Both menadione and H2O2 reduced cell viability and increased lipid peroxidation (P < 0.05). Greater cell viability was found in selenium-supplemented cells when compared with vehicle treated cells (P < 0.05). Selenium and iodine supplementation separately or together were associated with lower lipid peroxidation compared with vehicle control (P < 0.05). Supplementation with the combination of selenium and iodine resulted in a greater reduction in lipid peroxidation compared with selenium or iodine alone (P < 0.05). Oxidative stress negatively impacts trophoblast cell survival and cellular integrity. Selenium and iodine protect placental trophoblasts against oxidative stress. Further research is warranted to investigate the molecular mechanisms by which selenium and iodine act in the human placenta.Nahal Habibi, Tanja Jankovic-Karasoulos, Shalem Yiner-Lee Leemaqz, Maxime Francois, Shao Jia Zhou, Wayne R. Leifert ... et al

The effect of zinc on human trophoblast proliferation and oxidative stress

Adequate Zinc (Zn) intake is required to prevent multiple teratogenic effects however deviations from adequate Zn intake, including high maternal Zn status, have been linked to increased incidence of pregnancy complications, including those associated with inadequate placentation. Using placental trophoblast HTR8/SVneo cells and first trimester human placental explants ( n = 12), we assessed the effects of varying Zn concentrations on trophoblast proliferation, viability, apoptosis and oxidative stress. Compared to physiologically normal Zn levels (20 μM), HTR-8/SVneo cell proliferation index was significantly lower in the presence of physiologically elevated (40 μM; P = .020) and supra-physiological (80 μM; P = .007) Zn. The latter was also associated with reduced proliferation ( P = .004) and viability ( P < .0 0 01) in cultured placental explants, but not apoptosis. Reactive oxygen species production in HTR8/SVneo cultures was significantly higher in the presence of 80 μM Zn compared to all physiologically relevant levels. Oxidative stress, induced by an oxidizing agent menadione, was further exacerbated by high (80 μM) Zn. Zn did not affect lipid peroxidation in either HTR8/SVneo cells or placental explants or antioxidant defense mechanisms that included glutathione reductase and superoxide dismutase. Further study should focus on elucidating mechanisms behind impaired trophoblast proliferation and increased oxidative stress as a result of elevated Zn levels.Tanja Jankovic-Karasoulos, Dale McAninch, Clare Dixon, Shalem Y.-L. Leemaqz, Maxime François, Wayne R. Leifert, Dylan McCullough, Carmela Ricciardelli, Claire T. Roberts, Tina Bianco-Miott

Cell-free receptor-based biosensors

The ability to express and purify modified recombinant signalling proteins such that they retain their biological function in a cell-free context has provided a basis for production of molecular biosensors. Here the authors utilise G-protein coupled receptors (GPCRs) and their G-proteins to detect various binding partners in a cell-free environment. Molecular biology approaches were employed to express these proteins using baculovirus and bacteria, and to alter their characteristics to improve surface-attachment and fluorescent labelling capabilities. Ligand-mediated signalling of a GPCR could be measured (using [35S]GTPgammaS-binding assays) in a reconstituted system with recombinant proteins either free in solution or attached to Ni2+-coated beads. Affinity of histidine-tagged proteins for a Ni2+-coated surface was significantly enhanced by addition of extra histidine residues to the tag, as determined by surface plasmon resonance. This was due to the longer tag occupying, on average, a greater number of available histidine-binding sites. Further, a novel homogeneous fluorescence resonance energy transfer (FRET)-based assay has been developed to detect rearrangements in the G-protein heterotrimer. Investigation of small peptides that can be fused to G-protein subunits, allowing for site-specific fluorescent labelling, was undertaken in order to improve the resolution of the "first generation" FRET assay. By utilizing this improved G-protein heterotrimer "molecular switch", we are developing a generic technology such that a range of GPCRs could be assayed for ligand-mediated activation while attached to surfaces (e.g. on beads or as microarrays) or in solution (e.g. multi-well plates), with increased throughput.Richard V. Glatz, Wayne R. Leifert, Kelly Bailey, Tamara H. Cooper, Chris S. Barton, A. Scott Martin, Amanda Aloia, Olgatina Bucco, Lakshmi Waniganayake, Gang Wei, Burkhard Raguse, Lech Wieczorek, and Edward J. McMurchi

Nitrogênio e fósforo no crescimento de plantas de ginseng brasileiro [Pfaffia glomerata(Spreng.) Pedersen] cultivadas in vitro Nitrogen and phosphorus on growth of brazilian ginseng [Pfaffia glomerata (Spreng.) Pedersen] in vitro cultured plants

O ginseng brasileiro [Pfaffia glomerata (Spreng.) Pedersen] apresenta propriedades medicinais marcantes e, por isso, atualmente, é largamente explorado de forma extrativista, tanto por parte dos coletores de ervas como pela indústria farmacêutica. Este trabalho objetivou caracterizar o efeito da variação isolada da concentração de N e P do meio MS no crescimento de plantas de P. glomerata cultivadas in vitro. Segmentos nodais de 1,0cm de comprimento e sem folhas, de plantas já estabelecidas in vitro, foram cultivados em meio MS contendo cinco concentrações (0, 25, 50, 100 e 150% da concentração padrão do meio de cultura MS) de nitrogênio ou fósforo. Aos 15 dias após a inoculação (DAI), o número de raízes e o percentual de enraizamento são maiores na concentração de N e P equivalentes a 50% daquela do meio de cultura MS. Aos 40 DAI, o crescimento em altura das brotações, número de segmentos nodais, índice de área foliar, número de folhas, matéria seca de raízes, da parte aérea e total da planta é maior na concentração de N e P, em média, próxima a 80% daquela do meio de cultura MS.<br>Brazilian ginseng [Pfaffia glomerata (Spreng.) Pedersen] has expressionable medicinal properties, and therefore, nowadays it is extensively exploited either by the herb collectors as well as the pharmaceutical industry. The aim of this work was to characterize the effect of N and P concentrations from the MS medium on growth of P. glomerata in vitro cultured plants. 1-node segment without leaves, from in vitro cultivated plants, were grown on five levels (0, 25, 50, 100, and 150% of the strength of the MS medium) of N and P. After 15 days of inoculation, root number and rooting percentage are greater on N and P levels of the MS medium with 50% strength. After 40 days of inoculation, growth, based on sprout height, number of nodal segments per plant, leave area index per plant, number of leaves per plant, dry weight of aerial parts, roots and of the whole plant, is greater when the N and P levels of the MS medium is near to 80% strength

High content, multi-parameter analyses in buccal cells to identify Alzheimer's disease

Alzheimer’s disease (AD) is a degenerative brain disorder and is the most common form of dementia. Minimally invasive approaches are required that combine biomarkers to identify individuals who are at risk of developing mild cognitive impairment (MCI) and AD, to appropriately target clinical trials for therapeutic discovery as well as lifestyle strategies aimed at prevention. Buccal mucosa cells from the Australian Imaging, Biomarkers and Lifestyle Flagship Study of Ageing cohort (n=60) were investigated for cytological markers that could be used to identify both MCI and AD individuals. Visual scoring of the buccal cytome demonstrated a significantly lower frequency of basal and karyorrhectic cells in the MCI group compared with controls. A high content, automated assay was developed using laser scanning cytometry to simultaneously measure cell types, nuclear DNA content and aneuploidy, neutral lipid content, putative Tau and amyloid-β (Aβ) in buccal cells. DNA content, aneuploidy, neutral lipids and Tau were similar in all groups. However, there was significantly lower Tau protein in both basal and karyolytic buccal cell types compared with differentiated buccal cells. Aβ, as measured by frequency of cells containing Aβ signal, as well as area and integral of Aβ signal, was significantly higher in the AD group compared with the control group. Buccal cell Aβ was correlated with mini-mental state examination (MMSE) scores (r = -0.436, P=0.001) and several blood-based biomarkers. Combining newly identified biomarkers from buccal cells with those already established may offer a potential route for more specific biomarker panels which may substantially increase the likelihood of better predictive markers for earlier diagnosis of AD