The biochemical mechanisms of action of tigliane and phorbol esters

Initial studies adding TPA and Sap A to Human Mononuclear cell (HMNC) cultures found that the mitogenic action of Sap A was abolished if cells were incubated for 3 days prior to phorbol ester addition. Previously the phorbol ester receptor has been found to be a family of protein kinases (PKC's). The relationship between biochemical action and biological effect was investigated using rat brain as the enzyme source. Phorbol ester-stimulated kinase activities in rat brain hydroxylapatite FPLC fractions were investigated using a modified PS/Triton micellar assay. Proteins in the elution profile were separated by SDS-PAGE and immuno-blotted with antisera specific for PKC α, β1, γ, δ and ϵ to identify the PKC isotypes present.These investigations were coordinated with phorbol ester activation studies on PKC Isotypes (α, β1, γ, δ and ϵ) purified from bovine brain. This methodology was then applied to screen eluted proteins from subpopulations of HMNC as well as Daudi cells and mouse macrophages. These approaches revealed several phorbol ester-sensitive protein kinase activities, which could not be identified as PKC α, β1, γ, δ and ϵ Isotypes by elution or immunological definition, in crudely fractionated brain and cell extracts. Some of these activities demonstrated tissue specificity when elution positions characteristic of brain were compared to those seen for cultured cells. Preparations of HMNC's were also found to contain an entirely novel kinase activity which was stimulatable by Resiniferatoxin (Rx, a daphnane ester) only in the absence of added calcium (termed Rx-Kinase). A similar activity was also isolated, in greater quantity, from starch-elicited mouse peritoneal macrophages under identical conditions. Fractions of mouse Rx-Kinase activity were found to potently activate the reconstituted mouse NADPH oxidase system to generate superoxide in vitro In the presence of Rx and the absence of calcium (i.e. conditions corresponding to its histone kinase requirements in vitro).This suggests major differences between this putative phorbol ester receptor and the PKC family of isotypes

Cupid, a cell permeable peptide derived from amoeba, capable of delivering GFP into a diverse range of species

Cell permeating peptides (CPPs) are attracting great interest for use as molecular delivery vehicles for the transport of biologically active cargo across the cell membrane. The sequence of a novel CPP sequence, termed ‘Cupid’, was identified from the genome of Dictyostelium discoideum. A Cupid-Green Fluorescent Protein (Cupid-GFP) fusion protein was tested on mammalian, whole plant cells, plant leaf protoplast and fungal cell cultures and observed using confocal microscopy. GFP fluorescence builds up within the cell cytosol in 60 min, demonstrating Cupid-GFP has permeated them and folded correctly into its fluorescent form. Our combined data suggest Cupid can act as a molecular vehicle capable of delivering proteins, such as GFP, into the cytosol of a variety of cells

Mid- to late Holocene geomorphological and hydrological changes in the south Taihu area of the Yangtze delta plain, China

The Taihu Plain of the Lower Yangtze valley, China was a centre of rice agriculture during the Neolithic period. Reasons for the rapid development of rice cultivation during this period, however, have not been fully understood for this coastal lowland, which is highly sensitive to sea-level change. To improve understanding of the morphological and hydrological context for evolution of prehistoric rice agriculture, two sediment cores (DTX4 and DTX10) in the East Tiaoxi River Plain, south Taihu Plain, were collected, and analysed for radiocarbon dating, diatoms, organic carbon and nitrogen stable isotopes (δ13C and δ15N), grain size and lithology. These multiproxy analyses revealed that prior to ca. 7500 cal. yr BP, the East Tiaoxi River Plain was a rapidly aggrading high-salinity estuary (the Palaeo-Taihu Estuary). After ca. 7500 cal. yr BP, low salinity conditions prevailed as a result of strong Yangtze freshwater discharge. Subsequently, seawater penetration occurred and saltmarsh developed between ca. 7000 and 6500 cal. yr BP due to accelerated relative sea-level rise. This transgression event influenced a large area of the Taihu Plain during the Holocene, as shown by multiple sediment records from previous studies. Persistent freshwater marsh (or subaerial land) formed due to dramatic shrinkage/closure of the Palaeo-Taihu Estuary after ca. 5600 cal. yr BP when sea level was relatively stable. We speculate that morphological and hydrological changes of the East Tiaoxi River Plain played an important role in agricultural development across the Taihu Plain during the Neolithic period. The closure of the Palaeo-Taihu Estuary and the formation of stable freshwater marsh (or subaerial land) after ca. 5600 cal. yr BP were critical preconditions encouraging the rapid rise of rice productivity in the Liangzhu period (5500-4500 cal. yr BP). This development changed the landscape and river systems, and thus provided adequate freshwater supply to the Taihu Plain

A systematic investigation of the protein kinases involved in NMDA receptor-dependent LTD: evidence for a role of GSK-3 but not other serine/threonine kinases

Background: The signalling mechanisms involved in the induction of N-methyl-D-aspartate (NMDA) receptor-dependent long-term depression (LTD) in the hippocampus are poorly understood. Numerous studies have presented evidence both for and against a variety of second messengers systems being involved in LTD induction. Here we provide the first systematic investigation of the involvement of serine/threonine (ser/thr) protein kinases in NMDAR-LTD, using whole-cell recordings from CA1 pyramidal neurons. Results: Using a panel of 23 inhibitors individually loaded into the recorded neurons, we can discount the involvement of at least 57 kinases, including PKA, PKC, CaMKII, p38 MAPK and DYRK1A. However, we have been able to confirm a role for the ser/thr protein kinase, glycogen synthase kinase 3 (GSK-3). Conclusion: The present study is the first to investigate the role of 58 ser/thr protein kinases in LTD in the same study. Of these 58 protein kinases, we have found evidence for the involvement of only one, GSK-3, in LTD

Understanding the transfer of contemporary temperature signals into lake sediments via paired oxygen isotope ratios in carbonates and diatom silica: problems and potential

Although the oxygen isotope composition (δ18O) of calcite (δ18Ocalcite) and, to a lesser extent, diatom silica (δ18Odiatom) are widely used tracers of past hydroclimates (especially temperature and surface water hydrology), the degree to which these two hosts simultaneously acquire their isotope signals in modern lacustrine environments, or how these are altered during initial sedimentation, is poorly understood. Here, we present a unique dataset from a natural limnological laboratory to explore these issues. This study compares oxygen and hydrogen isotope data (δ18O, δ2H) of contemporary lake water samples at ~2-weekly intervals over a 2-year period (2010–12) with matching collections of diatoms (δ18Odiatom) and calcite (δ18Ocalcite) from sediment traps (at 10 m and 25 m) at Rostherne Mere (maximum depth 30 m), a well-monitored, eutrophic, seasonally stratified monomictic lake in the UK. The epilimnion shows a seasonal pattern of rising temperature and summer evaporative enrichment in 18O, and while there is a temperature imprint in both δ18Odiatom and δ18Ocalcite, there is significant inter-annual variability in both of these signals. The interpretation of δ18Odiatom and δ18Ocalcite values is complicated due to in-lake processes (e.g. non-equilibrium calcite precipitation, especially in spring, leading to significant 18Ocalcite depletion), and for δ18Odiatom, by post-mortem, depositional and possibly dissolution or diagenetic effects. For 2010 and 2011 respectively, there is a strong temperature dependence of δ18Ocalcite and δ18Odiatom in fresh trap material, with the fractionation slope for δ18Odiatom of ca. −0.2‰/°C, in agreement with several other studies. The δ18Odiatom data indicate the initiation of rapid post-mortem secondary alteration of fresh diatom silica (within ~6 months), with some trap material undergoing partial maturation in situ. Diatom δ18O of the trap material is also influenced by resuspension of diatom frustules from surface sediments (notably in summer 2011), with the net effect seen as an enrichment of deep-trap 18Odiatom by about +0.7‰ relative to shallow-trap values. Contact with anoxic water and anaerobic bacteria are potentially key to initiating this silica maturation process, as deep-trap samples that were removed prior to anoxia developing do not show enrichment. Dissolution (perhaps enhanced by anaerobic bacterial communities) may also be responsible for changes to δ18Odiatom that lead to increasing, but potentially predictable, error in inferred temperatures using this proxy. High resolution, multi-year monitoring can shed light on the complex dynamics affecting δ18Odiatom and δ18Ocalcite and supports the careful use of sedimentary δ18Odiatom and δ18Ocalcite as containing valuable hydroclimatic signals especially at a multi-annual resolution, although there remain substantial challenges to developing a reliable geothermometer on paired δ18Odiatom and δ18Ocalcite. In particular, δ18Odiatom needs cautious interpretation where silica post-mortem secondary alteration is incomplete and diatom preservation is not perfect, and we recommend dissolution be routinely assessed on diatom samples used for isotopic analyses

δ18O-inferred salinity from Littorina littorea (L.) gastropods in a Danish shell midden at the Mesolithic–Neolithic transition

Norsminde Fjord has received extensive geoarchaeological investigation, hosting one of the classic Stone Age shell midden sites in Denmark, and one of the best examples of the widespread oyster decline at the Mesolithic–Neolithic transition. Here, intra-shell δ18O (and δ13C) analyses from the common periwinkle Littorina littorea (L.) are used to infer inter-annual environmental changes at the Mesolithic–Neolithic transition (four from each period). This study utilises a modern δ18O L. littorea-salinity training set previously developed for the Limfjord, Denmark to quantify winter salinity. δ18O values range between +1.6% and +4.0% in the late Mesolithic and ‒6.3% to +2.0% in the early Neolithic. Using maximum δ18O values, winter salinity at the known temperature of growth cessation in L. littorea (i.e. +3.7 ± 1°C) for the first annual cycle of each shell ranges between 25.5 and 26.8 psu (standard deviation (SD): 0.56) for the late Mesolithic, with an average salinity of 26.1 psu. Early-Neolithic shells range between 19.4 and 28.2 psu (SD: 4.59) with an average salinity of 23.7 psu. No statistically significant change in salinity occurs between the late Mesolithic and early Neolithic. This result supports recent diatom/mollusc-based inferences that salinity was not the sole cause of the oyster decline, although some evidence is presented here for more variable seasonal salinity conditions in the early Neolithic, which (along sedimentary change and temperature deterioration) might have increased stress on oyster populations in some years. It is recommended here that for robust palaeoenvironmental inferences, where possible, multiple specimens should be used from the same time period in conjunction with multiproxy data

The shellfish enigma across the Mesolithic-Neolithic transition in southern Scandinavia

The well-known and widespread replacement of oysters (abundant during the Mesolithic period) by cockles and mussels in many Danish Stone Age shell middens ca. 5900 cal yrs BP coincides with the transition to agriculture in southern Scandinavia. This human resource shift is commonly believed to reflect changing resource availability, driven by environmental and/or climatic change at the Mesolithic-Neolithic transition rather than cultural choice. While several hypotheses have been proposed to explain the “Mesolithic-Neolithic oyster decline”, an explanation based on a sudden freshening of the inner Danish waters has received most attention. Here, for the first time, we test and refute this long-standing hypothesis that declining salinity explains the marked reduction in oysters identified within numerous shell middens across coastal Denmark at the Mesolithic-Neolithic transition using quantitative and qualitative salinity inference from several, independent proxies (diatoms, molluscs and foraminifera) from multiple Danish fjord sites. Alternatively, we attribute the oyster decline to other environmental causes (particularly changing sedimentation), ultimately driven by external climatic forcing. Critical application of such high-quality environmental archives can reinvigorate archaeological debates and can aid in understanding and managing environmental change in increasingly impacted coastal regions

Lithium Inhibits Glycogen Synthase Kinase-3 by Competition for Magnesium

The mechanism by which lithium (Li+) inhibits the protein kinase glycogen synthase kinase-3 (GSK-3) is unknown. Here, we demonstrate that Li+ is a competitive inhibitor of GSK-3 with respect to magnesium (Mg2+), but not to substrate or ATP. This mode of inhibition is conserved between mammalian and Dictyostelium GSK-3 isoforms, and is not experienced with other group I metal ions. As a consequence, the potency of Li+ inhibition is dependent on Mg2+ concentration. We also found that GSK-3 is sensitive to chelation of free Mg2+ by ATP and is progressively inhibited when ATP concentrations exceed that of Mg2+. Given the cellular concentrations of ATP and Mg2+, our results indicate that Li+ will have a greater effect on GSK-3 activity in vivo than expected from in vitro studies and this may be a factor relevant to its use in the treatment of depression

The function of secondary metabolites in plant carnivory

Abstract Background Carnivorous plants are an ideal model system for evaluating the role of secondary metabolites in plant ecology and evolution. Carnivory is a striking example of convergent evolution to attract, capture and digest prey for nutrients to enhance growth and reproduction and has evolved independently at least ten times. Though the roles of many traits in plant carnivory have been well studied, the role of secondary metabolites in the carnivorous habit is considerably less understood. Scope This review provides the first synthesis of research in which secondary plant metabolites have been demonstrated to have a functional role in plant carnivory. From these studies we identify key metabolites for plant carnivory and their functional role, and highlight biochemical similarities across taxa. From this synthesis we provide new research directions for integrating secondary metabolites into understanding of the ecology and evolution of plant carnivory. Conclusions Carnivorous plants use secondary metabolites to facilitate prey attraction, capture, digestion and assimilation. We found ~170 metabolites for which a functional role in carnivory has been demonstrated. Of these, 26 compounds are present across genera that independently evolved a carnivorous habit, suggesting convergent evolution. Some secondary metabolites have been co-opted from other processes, such as defence or pollinator attraction. Secondary metabolites in carnivorous plants provide a potentially powerful model system for exploring the role of metabolites in plant evolution. They also show promise for elucidating how the generation of novel compounds, as well as the co-option of pre-existing metabolites, provides a strategy for plants to occupy different environments