δN and δC analysis of a Posidonia sinuosa seagrass bed

Potential food sources and dominant invertebrates and fishes were collected for the examination of variability in ¹³C/¹²C and ¹⁵N/¹⁴N to determine the sources of carbon available to consumers within a Western Australian Posidonia sinuosa-dominated seagrass bed. Autotrophs showed a wide distribution of δ¹³C values, with P. sinuosa at −11.3 ± 0.8‰ and macroalgae ranging from −16.6 to −31.7‰. This variation allowed us to successfully identify macroalgae as the main contributor of carbon to the trophic structure, although no distinction could be made between epiphytic macroalgae on seagrass, or allochthonous macroalgal sources. The range in δ¹⁵N ratios among potential food items at the trophic base was too small to make it useful as tracer of nitrogen flow pathways, but it consistently increased from macrophytes and detritus (4.1–6.8‰), to invertebrates (5.7–7.4‰) located near the middle of the food web, to fishes (8.3–11.9‰), with piscivorous species such as Leviprora inops generally having a higher ¹⁵N. δ¹³C of seston (−12.8‰) and sedimentary organic matter (−8.7‰) indicate that seagrass material is the main contributor to these two carbon pools, and that very little of it contributes to animal biomass.     

Variation among diets in discrimination of δC and δN in the amphipod Allorchestes compressa

Discrimination of stable isotopes of carbon (δ¹³C) and nitrogen (δ¹⁵N) was examined for the amphipod Allorchestes compressa Dana using controlled laboratory experiments. Amphipods were fed exclusively on single diets (fresh or decomposed macroalgae or seagrass) for three weeks. Macrophyte type (i.e. seagrass, brown algae or red algae) had a greater influence on the stable isotope ratios of A. compressa than the state of decomposition of the macrophyte material. The experiments revealed that δ¹³C in A. compressa stabilised at values lower than those of the diets, which contrasts to the general assumption that consumer-diet discrimination of δ¹³C ranges from 0 to + 1‰. Amphipods fed on seagrass yielded the lowest δ¹³C values, which were 9 to 10‰ lower than their diet, while amphipods fed on macroalgae had values 2 to 4‰ lower than their diet. In addition, contrary to the general assumption that consumer-diet discrimination of δ¹⁵N ranges from + 3 to + 5‰, discrimination of δ¹⁵N was as low as − 1 and + 1 when A. compressa was fed on brown and red algae, respectively, but as high as + 3‰ when fed on seagrass. The results show that discrimination of stable isotopes of carbon and nitrogen can vary considerably depending on the food source, demonstrating that validation of assumptions about discrimination are critical for interpreting stable isotope data from field studies.     

Effects of body size and environment on diet-tissue δC fractionation in fishes

δ¹³C data are often used in trophodynamic research where diet-tissue fractionation (Δδ¹³C) is assumed to be 0-1‰ per trophic level and unaffected by the size of animals or their environment. Variation in Δδ¹³C will influence conclusions about food sources, energy pathways and trophic level. To assess the effects of body size, age and environmental conditions on Δδ¹³C, European sea bass (Dicentrarchus labrax) were reared on constant diets of dab (Limanda limanda) or (Ammodytes marinus) for 2years under natural environmental regimes. Bass were sampled approximately monthly to determine Δδ¹³C for muscle, heart and liver tissue and were 1.66‰, − 0.18‰, − 1.77‰ (sandeel diet) and 1.34‰, − 1.18‰, − 1.75‰ (dab diet) respectively. Arithmetic lipid correction increased Δδ¹³C to > 2‰ for muscle and liver. Δδ¹³C was dependent on body mass and experimental duration (age) and generally declined with weight or time even after correction for lipid content. For liver, increasing temperature increased Δδ¹³C. The Δδ¹³C estimates from this study were compared with all available published Δδ¹³C estimates for fish. Bass muscle Δδ¹³C was similar to previous estimates for fish white muscle Δδ¹³C (1.56 ± 1.10‰) and whole body Δδ¹³C (1.52 ± 1.13‰). Fractionations derived in this study, combined with those from the literature, support the use of diet-tissue fractionation values of between 1‰-2‰ for δ¹³C, rather than the commonly used 0‰ − 1‰. For muscle Δδ¹³C, 1.5‰ is appropriate.     

Community structure and food web based on stable isotopes (δN and δC) analysis of a North Eastern Atlantic maerl bed

Maerl beds are highly biodiverse biogenic substrata that have been receiving increasing attention in the last decade. Although maerl beds represent important nursery areas for commercial fishes and molluscs, little is known on the trophic web of their communities. Community structure parameters of maerl bed of the Bay of Brest (species richness, abundance, biomass and dominating species) were studied in parallel with the carbon and nitrogen isotopic composition of their main benthic species (macrofaunal, and megafaunal organisms) in order to assess the trophic levels and differences in the potential food sources of maerl inhabitants. The major potential sources of energy were identified to originate either from epiphytic macroalgae and microphytobenthos both growing on maerl thalli, together with sedimenting (sedimentary) particulate organic matter (POM) originating from the water column. The majority of the macro- and megafaunal organisms investigated were filter feeders, selective-deposit feeders and predators/scavengers. Filter feeders fall into three different groups representing different trophic pathways (i) sponges feeding directly on POM (water column filter feeders I), (ii) ascidians and holothurians feeding on POM and probably captured pelagic preys (water column filter feeders II), and (iii) filter feeding molluscs and crustaceans were hypothesised to feed on microphytobenthos or on decaying sedimented POM (Interface filter feeders). Selective deposit feeders were also divided into two subgroups. Carnivores were also distinguished between those with scavenging habits and true predators. Coupling of the trophic levels observed with the community biomass structure revealed that most of the benthic biomass derives its food from detritic sedimented POM and/or microphytobenthos, with interface filter feeders (23% of the biomass), selective deposit feeders (12%). Carnivores made up to 14% of the total biomass. Generally stable isotopes ratio mean values overlap and cover a large range within feeding types, indicating a strong overlap in food sources and a high degree of complexity of the food web presumably due to the diversity of the potential food sources.     

Variability of stable isotope signatures (δC and δN) in two spider crab populations (Maja brachydactyla) in Western Europe

The ontogenic variations of nitrogen and carbon stable isotopic signatures (δ¹⁵N and δ¹³C) were investigated in two spider crab (Maja brachydactyla) populations inhabiting in different biotopes of Western Europe. The Iroise Sea population is localized in Western Brittany and characterized by a seasonal migration occurring on a large bathymetric and habitat gradient while the Seine Bay population, in the Eastern English Channel, remains in a more homogeneous environment during its migration. In the Iroise Sea population, δ¹³C values increased significantly both with body size and age, revealing a shift towards “benthic-component” prey with spider crab growth. On the contrary, neither body size nor ageing gave rise to a significant trophic level change (derived from the δ¹⁵N). In this M. brachydactyla population, the seasonal migrations from coastal waters in summer to offshore habitats in winter involved significant but slight differences in both δ¹³C and δ¹⁵N. In the Seine Bay population, low variations for both carbon and nitrogen were recorded related to either sex or size or seasonal migration. Thus, the δ¹³C and δ¹⁵N variability in the spider crab depends on the availability and diversity of prey in its different living habitats, as well as on the morphological aptitudes of individuals to feed on prey (individual's size).     

Biosynthetic origin of the saw-toothed profile in δC and δΗ of n-alkanes and systematic isotopic differences between n-, iso- and anteiso-alkanes in leaf waxes of land plants

The n-fatty acids containing an even number of carbons (ECN-n-FAs) in higher plants are biosynthesised by repetitive addition of a two carbon unit from malonyl-ACP. The n-alkanes containing an odd number of carbon atoms (OCN-n-alkanes) are generally formed by the decarboxylation of ECN-n-FAs, but it is unknown how the less abundant even-carbon-numbered alkanes (ECN-n-alkanes) are biosynthesised in higher plants.There is a distinctive compositional pattern of incorporation of stable carbon (¹³C) and hydrogen (²H) isotopes in co-existing ECN- and OCN-n-alkanes in leaves of higher plants, such that the OCN n-alkanes are relatively enriched in ¹³C but relatively depleted in ²H against the ECN-n-alkanes. This is consistent with the OCN-n-fatty acids having a propionate precursor which is derived from reduction of pyruvate. A tentative pathway is presented with propionate produced by enzymatic reduction of pyruvate which is then thio-esterified with CoSH (coenzyme A thiol) in the chloroplast to form the terminal precursor molecule propionyl-CoA. This is then repetitively extended/elongated with the 2-carbon unit from malonyl-ACP to form the long chain OCN-n-fatty acids.The anteiso- and iso-alkanes in Nicotiana tabacum leaf waxes have previously been found to be systematically enriched in ¹³C compared with the n-alkanes by Grice et al. (2008). This is consistent with the isotopic composition of their putative respective precursors (pyruvate as precursor for n-alkanes, valine for iso-alkanes and isoleucine for anteiso-alkanes). The current study complements that of Grice et al. (2008) and looks at the distribution of hydrogen isotopes. The n-alkanes were found to be more enriched in deuterium (²H) than the iso-alkanes which in turn were more enriched than the anteiso-alkanes. We propose therefore that the depletion of ²H in the iso-alkanes, relative to the n-alkanes is the consequence of accepting highly ²H-depleted hydrogen atoms from NADPH during their biosynthesis. The anteiso-alkanes are further depleted again because there are three NADPH-derived hydrogen atoms in their precursor isoleucine, as compared with only one NADPH-derived hydrogen in valine, the precursor of the iso-alkanes.     

Seasonal variations in bulk tissue, fatty acid and monosaccharide δC values of leaves from mesotrophic grassland plant communities under different grazing managements

Leaves of 26 grass, herb, shrub and tree species were collected from mesotrophic grasslands to assess natural variability in bulk, fatty acid and monosaccharide δ¹³C values under different grazing management (cattle- or deer-grazed) on three sample dates (May, July and October) such that interspecific and spatiotemporal variations in whole leaf tissues and compound-specific δ¹³C values could be determined. The total mean leaf bulk δ¹³C value for plants was −28.9‰ with a range of values spanning 7.5‰. Significant interspecific variation between bulk leaf δ¹³C values was only determined in October (P = <0.001) when δ¹³C values of the leaf tissues from both sites was on average 1.5‰ depleted compared to during July and May. Samples from May were significantly different between fields (P = 0.03) indicating an effect from deer- or cattle-grazing in young leaves. The average individual monosaccharide δ¹³C value was 0.8‰ higher compared with whole leaf tissues. Monosaccharides were the most abundant components of leaf biomass, i.e. arabinose, xylose, mannose, galactose and glucose, and therefore, fluctuations in their individual δ¹³C values had a major influence on bulk δ¹³C values. An average depletion of ca. 1‰ in the bulk δ¹³C values of leaves from the deer-grazed field compared to the cattle-grazed field could be explained by a general depletion of 1.1‰ in glucose δ¹³C values, as glucose constituted >50% total leaf monosaccharides. In October, δ¹³C values of all monosaccharides varied between species, with significant variation in δ¹³C values of mannose and glucose in July, and mannose in May. This provided an explanation for the noted variability in the tissue bulk δ¹³C values observed in October 1999. The fatty acids C_16:0, C_18:2 and C_18:3 were highly abundant in all plant species. Fatty acid δ¹³C values were lower than those of bulk leaf tissues; average values of −37.4‰ (C_16:0), −37.0‰ (C_18:2) and −36.5‰ (C_18:3) were determined. There was significant interspecific variation in the δ¹³C values of all individual fatty acids during October and July, but only for C_18:2 in May (P = <0.05). This indicated that seasonal trends observed in the δ¹³C values of individual fatty acids were inherited from the isotopic composition of primary photosynthate. However, although wide diversity in δ¹³C values of grassland plants ascribed to grazing management, interspecific and spatiotemporal influences was revealed, significant trends (P = <0.0001) for fatty acid and monosaccharide δ¹³C values: δ¹³C_16:0 < δ¹³C_18:2 < δ¹³C_18:3 and δ¹³C_arabinose > δ¹³C_xylose > δ¹³C_glucose > δ¹³C_galactose, respectively, previously described, appear consistent across a wide range of species at different times of the year in fields under different grazing regimes.     

Effects of body size and environment on diet-tissue δN fractionation in fishes

Nitrogen stable isotope natural abundance data are often used in trophodynamic research. The assumed nitrogen diet-tissue fractionation (Δδ¹⁵N) determines conclusions about trophic level, potential food sources and ontogenetic diet shifts. Δδ¹⁵N is usually assumed to be 3.0-3.4‰ per trophic level and unaffected by the size or age of animals or their environment. To assess the effects of body size, experimental duration and environmental conditions on fish tissue Δδ¹⁵N, two populations of European sea bass (Dicentrarchus labrax) were reared on constant diets of dab (Limanda limanda) muscle or sandeel (Ammodytes marinus) for 2 years under natural light and temperature regimes. Bass were sampled at approximately monthly intervals to determine Δδ¹⁵N for muscle, heart and liver tissue. Mean values of Δδ¹⁵N were 3.83‰, 3.54‰, 2.05‰ (sandeel diet) and 3.98‰, 3.32‰, 1.95‰ (dab diet) for muscle, heart and liver tissue respectively. The assumption that fractionation was independent of body mass was upheld for muscle and heart tissue, but not for liver. Time effects on muscle Δδ¹⁵N were explainable by a sinusoidal function with a period of 1 year and wave height ∼ 0.3‰. Time resulted in increases in heart δ¹⁵N and decreases in liver δ¹⁵N which were small compared to background variation, equating to 1/6 of a trophic level over 2 years, and unlikely to have great significance in ecological studies. Heart and liver δ¹⁵N were also affected by temperature probably reflecting the metabolic functions of these tissues and their associated rates of turnover. However in heart the explanatory power of temperature appeared tied to that of time. Although the Δδ¹⁵N for bass muscle on both diets approached 4‰, the Δδ¹⁵N values from this study, when combined with those from the literature, suggest that where fish species specific data are not available, a mean Δδ¹⁵N for fish muscle of 3.2‰ should be applied (mean white muscle Δδ¹⁵N = 3.15). The literature based mean Δδ¹⁵N for whole fish was lower than that of white muscle suggesting that a separate Δδ¹⁵N (2.9‰) should be applied when sampling whole fish.     

A 3D Structure Model of Integrin α4β1 Complex: I. Construction of a Homology Model of β1 and Ligand Binding Analysis

It is well established that integrin α4β1 binds to the vascular cell adhesion molecule (VCAM) and fibronectin and plays an important role in signal transduction. Blocking the binding of VCAM to α4β1 is thought to be a way of controlling a number of disease processes. To better understand how various inhibitors might block the interaction of VCAM and fibronectin with α4β1, we began constructing a structure model for the integrin α4β1 complex. As the first step, we have built a homology model of the β1 subunit based on the I domain of the integrin CD11B subunit. The model, including a bound Mg²⁺ ion, was optimized through a specially designed relaxation scheme involving restrained minimization and dynamics steps. The native ligand VCAM and two highly active small molecules (TBC772 and TBC3486) shown to inhibit binding of CS-1 and VCAM to α4β1 were docked into the active site of the refined model. Results from the binding analysis fit well with a pharmacophore model that was independently derived from active analog studies. A critical examination of residues in the binding site and analysis of docked ligands that are both potent and selective led to the proposal of a mechanism for β1/β7 ligand binding selectivity.     

Regio- and stereospecific synthesis of rac-carbasugar-based cyclohexane pentols; Investigations of their α- and β-glucosidase inhibitions

In the present study, (3aR,7aS)-1,3,3a,4,7,7a-hexahydroisobenzofuran was submitted to photooxygenation and two isomeric hydroperoxides were successfully obtained. Without any further purification, reduction of the hydroperoxides with titanium tetraisopropoxide catalyzed by dimethyl sulfide gave two alcohol isomers in high yields. After acetylation of alcohol with Ac₂O in pyridine, epoxidation reaction of formed monoacetates with m-CPBA, then chromatographed and followed by hydrolysis of the acetate groups with NH₃ in CH₃OH resulted in the formation of epoxy alcohol isomers respectively. These epoxy alcohol isomers were subjected to trans-dihydroxylation reaction with acid (H₂SO₄) in the presence of water to afford triols. Acetylation of the free hydroxyl groups produced benzofuran triacetates in high yields. Ring-opening reaction of furan triacetates with sulfamic acid catalyzed in the presence of acetic acid/acetic anhydrate and subsequently hydrolysis of the acetate groups with ammonia gave the targeted cyclohexane carbasugar-based pentols. All products were separated and purified by chromatographic and crystallographic methods. Structural analyses of all compounds were conducted by spectral techniques including NMR and X-ray analyses. The biological inhibition activity of the target compounds was tested against glycosidase enzymes, α- and β-glucosidase.     

Winter-spring precipitation as the principal control on predominance of C3 plants in Central Asia over the past 1.77 Myr: Evidence from δC of loess organic matter in Tajikistan

Although changes in atmospheric CO₂ levels are thought to be the major factor driving long-term C₃/C₄ vegetation evolution, recent studies tend to emphasize the effect of regional climate conditions on C₃/C₄ variations. The middle latitudes (30-45°), in which C₃/C₄ plants are highly sensitive to environmental changes, provide an optimal basis for the investigation of the relative impacts of climate and pCO₂ on shifts in C₃/C₄ cover. In order to assess the factors controlling these shifts as well as the complex interactions between environmental factors, the carbon isotopic composition of bulk organic matter from the Chashmanigar loess section (southern Tajikistan) was measured for the past 1.77 Myr. In general, the δ¹³C record shows mostly negative values throughout the sequence, almost all δ¹³C values falling between − 23‰ and − 26‰, indicating a predominance of C₃ plants in Central Asia over this time period, despite the presence of numerous glacial-interglacial cycles. From 0.85 Myr to the present, the δ¹³C values become increasingly positive, reflecting a growing C₄ signal. However, this C₄ component is not detectable prior to 0.25 Myr, after which minor peaks are evident at ∼ 228, ∼ 171 and ∼ 18 kyr. The δ¹³C record from Chashmanigar indicates that winter-spring precipitation, i.e., Mediterranean climatic conditions, have characterized Central Asia throughout the past 1.77 Myr, leading to the predominance of C₃ vegetation. In the context of glacial-interglacial-scale changes in atmospheric CO₂, therefore, it is climate rather than pCO₂ that controls C₃/C₄ variations in Asia’s middle latitudes. The gradual increase in the C₄ component since 0.85 Myr, especially the notable peaks after 0.25 Myr, may have been caused by an increase in summer precipitation due to an enhanced southward shift of the climate zones.     

A Model of Action Potentials and Fast Ca Dynamics in Pancreatic β-Cells

We examined the ionic mechanisms mediating depolarization-induced spike activity in pancreatic β-cells. We formulated a Hodgkin-Huxley-type ionic model for the action potential (AP) in these cells based on voltage- and current-clamp results together with measurements of Ca²⁺ dynamics in wild-type and Kv2.1 null mouse islets. The model contains an L-type Ca²⁺ current, a “rapid” delayed-rectifier K⁺ current, a small slowly-activated K⁺ current, a Ca²⁺-activated K⁺ current, an ATP-sensitive K⁺ current, a plasma membrane calcium-pump current and a Na⁺ background current. This model, coupled with an equation describing intracellular Ca²⁺ homeostasis, replicates β-cell AP and Ca²⁺ changes during one glucose-induced spontaneous spike, the effects of blocking K⁺ currents with different inhibitors, and specific complex spike in mouse islets lacking Kv2.1 channels. The currents with voltage-independent gating variables can also be responsible for burst behavior. Original features of this model include new equations for L-type Ca²⁺ current, assessment of the role of rapid delayed-rectifier K⁺ current, and Ca²⁺-activated K⁺ currents, demonstrating the important roles of the Ca²⁺-pump and background currents in the APs and bursts. This model provides acceptable fits to voltage-clamp, AP, and Ca²⁺ concentration data based on in silico analysis.     

Effect of 6α,7β-dihydroxyvouacapan-17β-oic acid and its lactone derivatives on the growth of human cancer cells

The furanditerpene 6α,7β-dihydroxyvouacapan-17β-oic acid (1) is a natural product biosynthesized by some species from the genus Pterodon (Leguminosae). This secondary metabolite has multiple biological activities that include anti-inflammatory, analgesic, plant growth regulatory, anti-edematogenic, photosystem II inhibitory and photosynthesis uncoupler, and antifungal properties. However, few studies on the antiproliferative profile of compound 1 and/or its derivatives have been reported up to date. Here, we describe the isolation of compound 1 from hexane extract of P. polygalaeflorus fruits as well as the semisynthesis of three lactone derivatives: 6α-hydroxyvouacapan-7β,17β-lactone (2), 6α-acetoxyvouacapan-7β,17β-lactone (3), and 6-oxovouacapan-7β,17β-lactone (4). Additionally, antiproliferative activity of these compounds against nine human cancer cell lines was investigated. Our results revealed that 6α-hydroxyvouacapan-7β,17β-lactone (2) was the most potent furanditerpene against all cancer cell lines studied. The presence of non-substituted hydroxyl group at C-6 and the presence of 7β,17β-lactone ring are important for the antiproliferative activity of these compounds.     

Synthesis and characterization of tetrairon-tungsten cluster complex containing a μ4, η-carbonyl ligand

Reaction of [(PPh₂C₅H₄)Cp₃Fe₄(CO)₄] (1) with (CO)₄W(CH₃CN)₂ at ambient temperature affords [(CO)₄W(PPh₂C₅H₄)Cp₃Fe₄(CO)₄] (2) as the major product, together with a small amount of [(CO)₅W(PPh₂C₅H₄)Cp₃Fe₄(CO)₄] (3). Compound 3 can be obtained in good yield by treating (CO)₅W(CH₃CN) with equal molar of 1, and reaction of 3 with Me₃NO in acetonitrile solvent produces 2 exclusively. The crystal structures of 2 and 3 have been determined by an X-ray diffraction study. Compound 2 contains an interesting μ₄, η²-CO ligand, where two electrons donated by the carbon atom are involved to bridge a Fe₃ face and two electrons from oxygen are donated to the tungsten(0) atom.     

On the Activation of Integrin αIIbβ3: Outside-in and Inside-out Pathways

Integrin αIIbβ3 is a member of the integrin family of transmembrane proteins present on the plasma membrane of platelets. Integrin αIIbβ3 is widely known to regulate the process of thrombosis via activation at its cytoplasmic side by talin and interaction with the soluble fibrinogen. It is also reported that three groups of interactions restrain integrin family members in the inactive state, including a set of salt bridges on the cytoplasmic side of the transmembrane domain of the integrin α- and β-subunits known as the inner membrane clasp, hydrophobic packing of a few transmembrane residues on the extracellular side between the α- and β-subunits that is known as the outer membrane clasp, and the key interaction group of the βA domain (located on the β-subunit head domain) with the βTD (proximal to the plasma membrane on the β-subunit). However, molecular details of this key interaction group as well as events that lead to detachment of the βTD and βA domains have remained ambiguous. In this study, we use molecular dynamics models to take a comprehensive outside-in and inside-out approach at exploring how integrin αIIbβ3 is activated. First, we show that talin’s interaction with the membrane-proximal and membrane-distal regions of integrin cytoplasmic-transmembrane domains significantly loosens the inner membrane clasp. Talin also interacts with an additional salt bridge (R734-E1006), which facilitates integrin activation through the separation of the integrin’s α- and β-subunits. The second part of our study classifies three types of interactions between RGD peptides and the extracellular domains of integrin αIIbβ3. Finally, we show that the interaction of the Arg of the RGD sequence may activate integrin via disrupting the key interaction group between K350 on the βA domain and S673/S674 on the βTD.     

Lens β-crystallins: The role of deamidation and related modifications in aging and cataract

Crystallins are the major proteins in the lens of the eye and function to maintain transparency of the lens. Of the human crystallins, α, β, and γ, the β-crystallins remain the most elusive in their structural significance due to their greater number of subunits and possible oligomer formations. The β-crystallins are also heavily modified during aging. This review focuses on the functional significance of deamidation and the related modifications of racemization and isomerization, the major modifications in β-crystallins of the aged human lens. Elucidating the role of these modifications in cataract formation has been slow, because they are analytically among the most difficult post-translational modifications to study. Recent results suggest that many amides deamidate to similar extent in normal aged and cataractous lenses, while others may undergo greater deamidation in cataract. Mimicking deamidation at critical structural regions induces structural changes that disrupt the stability of the β-crystallins and lead to their aggregation in vitro. Deamidations at the surface disrupt interactions with other crystallins. Additionally, the α-crystallin chaperone is unable to completely prevent deamidated β-crystallins from insolubilization. Therefore, deamidation of β-crystallins may enhance their precipitation and light scattering in vivo contributing to cataract formation.     

Purification and some properties of Δ2-isopentenylpyrophosphate:5′AMP Δ2-isopentenyltransferase from the cellular slime mold Dictyostelium discoideum

Δ²-Isopentenylpyrophosphate:5′AMP Δ²-isopentenyltransferase, which catalyzes the formation of isopentenyl-AMP from Δ²-isopentenylpyrophosphate and 5′AMP, was purified 6800-fold from the fruiting body of the cellular slime mold Dictyostelium discoideum using several separation procedures including 5′AMP^ox-redAH-Sepharose 4B affinity column chromatography. The final preparation was very unstable and lost its activity in a day. Various properties of the 1000-fold-purified enzyme preparation were examined. The molecular mass was 40,000 ± 2000 Da, as determined by Sephadex G-100 superfine gel filtration. The divalent metal ions Mn²⁺, Zn²⁺, and Mg²⁺ profoundly affected the enzymatic activity depending on their concentration, and also altered the optimum pH and temperature. Of the compounds tested, 5′AMP was the best acceptor of the isopentenyl group and, interestingly, ADP also served as a substrate, being 60–80% as effective as 5′AMP. Adenine, adenosine, and ATP were not substrates for this enzyme. Under the optimum assay conditions (pH 7.0, 1 mm Zn²⁺, and 25 °C) the K_m values for 5′AMP and Δ²-isopentenylpyrophosphate were 1.0 × 10^?7m and 2.2 × 10^?6m, respectively.     

Fast and Forceful Refolding of Stretched α-Helical Solenoid Proteins

Anfinsen's thermodynamic hypothesis implies that proteins can encode for stretching through reversible loss of structure. However, large in vitro extensions of proteins that occur through a progressive unfolding of their domains typically dissipate a significant amount of energy, and therefore are not thermodynamically reversible. Some coiled-coil proteins have been found to stretch nearly reversibly, although their extension is typically limited to 2.5 times their folded length. Here, we report investigations on the mechanical properties of individual molecules of ankyrin-R, β-catenin, and clathrin, which are representative examples of over 800 predicted human proteins composed of tightly packed α-helical repeats (termed ANK, ARM, or HEAT repeats, respectively) that form spiral-shaped protein domains. Using atomic force spectroscopy, we find that these polypeptides possess unprecedented stretch ratios on the order of 10-15, exceeding that of other proteins studied so far, and their extension and relaxation occurs with minimal energy dissipation. Their sequence-encoded elasticity is governed by stepwise unfolding of small repeats, which upon relaxation of the stretching force rapidly and forcefully refold, minimizing the hysteresis between the stretching and relaxing parts of the cycle. Thus, we identify a new class of proteins that behave as highly reversible nanosprings that have the potential to function as mechanosensors in cells and as building blocks in springy nanostructures. Our physical view of the protein component of cells as being comprised of predominantly inextensible structural elements under tension may need revision to incorporate springs.     

α-Rhamnosidase and β-glucosidase expressed by naringinase immobilized on new ionic liquid sol-gel matrices: Activity and stability studies

Novel ionic liquid (IL) sol-gel materials development, for enzyme immobilization, was the goal of this work. The deglycosylation of natural glycosides were performed with α-l-rhamnosidase and β-d-glucosidase activities expressed by naringinase. To attain that goal ILs with different structures were incorporated in TMOS/Glycerol sol-gel matrices and used on naringinase immobilization.The most striking feature of ILs incorporation on TMOS/Glycerol matrices was the positive impact on the enzyme activity and stability, which were evaluated in fifty consecutive runs. The efficiency of α-rhamnosidase expressed by naringinase TMOS/Glycerol@ILs matrices increased with cation hydrophobicity as follows: [OMIM] > [BMIM] > [EMIM] > [C₂OHMIM] > [BIM] and [OMIM] ≈ [E₂-MPy] ? [E₃-MPy]. Regarding the imidazolium family, the hydrophobic nature of the cation resulted in higher α-rhamnosidase efficiencies: [BMIM]BF₄ ? [C₂OHMIM]BF₄ ? [BIM]BF₄. Small differences in the IL cation structure resulted in important differences in the enzyme activity and stability, namely [E₃-MPy] and [E₂-MPy] allowed an impressive difference in the α-rhamnosidase activity and stability of almost 150%. The hydrophobic nature of the anion influenced positively α-rhamnosidase activity and stability. In the BMIM series the more hydrophobic anions (PF₆⁻, BF₄⁻ and Tf₂N⁻) led to higher activities than TFA. SEM analysis showed that the matrices are shaped lens with a film structure which varies within the lens, depending on the presence and the nature of the IL.The kinetics parameters, using naringin and prunin as substrates, were evaluated with free and naringinase encapsulated, respectively on TMOS/Glycerol@[OMIM][Tf₂N] and TMOS/Glycerol@[C₂OHMIM][PF₆] and on TMOS/Glycerol. An improved stability and efficiency of α-l-rhamnosidase and β-glucosidase expressed by encapsulated naringinase on TMOS/Glycerol@[OMIM][Tf₂N] and TMOS/Glycerol@[C₂OHMIM][PF₆] were achieved. In addition to these advantageous, with ILs as sol-gel templates, environmental friendly processes can be implemented.