Quaternary Structure Heterogeneity of Oligomeric Proteins: A SAXS and SANS Study of the Dissociation Products of Octopus vulgaris Hemocyanin

Octopus vulgaris hemocyanin shows a particular self-assembling pattern, characterized by a hierarchical organization of monomers. The highest molecular weight aggregate is a decamer, the stability of which in solution depends on several parameters. Different pH values, buffer compositions, H₂O/D₂O ratios and Hofmeister’s salts result in modifications of the aggregation state of Octopus vulgaris hemocyanin. The new QUAFIT method, recently applied to derive the structure of the decameric and the monomeric assembly from small-angle scattering data, is used here to model the polydisperse system that results from changing the solution conditions. A dataset of small-angle X-rays and neutron scattering curves is analysed by QUAFIT to derive structure, composition and concentration of different assemblies present in solution. According to the hierarchy of the association/dissociation processes and the possible number of different aggregation products in solution, each sample has been considered as a heterogeneous mixture composed of the entire decamer, the dissociated “loose” monomer and all the intermediate dissociation products. Scattering curves corresponding to given experimental conditions are well fitted by using a linear combination of single particle form factors. QUAFIT has proved to be a method of general validity to describe solutions of proteins that, even after purification processes, result to be intrinsically heterogeneous.     

Transgressive–regressive sequence stratigraphy of Pennsylvanian Donezella bioherms in a foreland basin (Lena Group, Cantabrian Zone, NW Spain)

A well-preserved Pennsylvanian (early Moscovian) succession including Donezella mounds, which accumulated in a highly subsiding foreland basin (Cantabrian Zone, NW of Spain), is described and discussed. This succession has been interpreted as one 3rd-order sequence reaching a thickness up to 815?m and recording?~2.3-My duration. It consists of four 4th-order transgressive–regressive (T-R) sequences (105–350?m thick and?~0.6-My duration), each subdivided into several 5th-order (~70-ky duration) meter-scale cycles (23-m average thickness). Nearly all the Donezella mounds are present in the second and third 4th-order sequences defined (Levinco Formation). They are up to?~90?m in thickness and several hundreds of meters wide, showing lenticular to domal morphologies with steep slopes up to 35–40°. Bioherms are composed of micritic boundstones with heterogeneous microfabrics and a diverse biotic community, including the microproblematic Donezella, calcitornellid foraminifers, bryozoans, agglutinated worm tubes, crinoids, and calcareous algae (red Komia/Ungdarella, beresellids, dasycladaceans and phylloids). According to the biotic assemblage and sedimentological features, these Donezella-rich bioherms thrived in a relatively shallow and low-energy environment (below fair-weather wave-base), and resulted from the baffling and binding ability of Donezella and associated biota, and the in situ-precipitation of microbial micrite. The upward evolution of the succession mainly resulted from the interplay between high tectonic subsidence rates and high-frequency moderate-amplitude glacioeustatic sea-level changes. The mounds growth mainly occurred during the transgressive phase of the 3rd-order sequence (Vereian), whereas during the regressive phase (Kashirian), deltaic siliciclastics prograding westward gradually buried and prevented the buildup development.     

Development of antithrombotic miniribozymes that target peripheral tryptophan hydroxylase

Transient receptor potential (TRP) proteins have been identified as cation channels that are activated by agonist–receptor coupling and mediate various cellular functions. TRPC7, a homologue of TRP channels, has been shown to act as a Ca²⁺ channel activated by G protein-coupled stimulation and to be abundantly expressed in the heart with an as-yet-unknown function. We studied the role of TRPC7 in G protein-activated signaling in HEK293 cells and cultured cardiomyocytes in vitro transfected with FLAG-tagged TRPC7 cDNA and in Dahl salt-sensitive rats with heart failure in vivo. TRPC7-transfected HEK293 cells showed an augmentation of carbachol-induced intracellular Ca²⁺ transient, which was attenuated under a Ca²⁺-free condition or in the presence of SK&F96365 (a Ca²⁺-permeable channel blocker). Upon stimulation with angiotensin II (Ang II), cultured neonatal rat cardiomyocytes transfected with TRPC7 exhibited a significant increase in apoptosis detected by TUNEL staining, accompanied with a decrease in the expression of atrial natriuretic factor and destruction of actin fibers, as compared with non-transfected cardiomyocytes. Ang II-induced apoptosis was inhibited by CV-11974 (Candesartan; Ang II type 1 [AT1] receptor blocker), SK&F96365, and FK506 (calcineurin inhibitor). In Dahl salt-sensitive rats, apoptosis and TRPC7 expression were increased in the failing myocardium, and a long-term treatment with temocapril, an angiotensin-converting enzyme inhibitor, suppressed both. Our findings suggest that TRPC7 could act as a Ca²⁺ channel activated by AT1 receptors, leading to myocardial apoptosis possibly via a calcineurin-dependent pathway. TRPC7 might be a key initiator linking AT1-activation to myocardial apoptosis, and thereby contributing to the process of heart failure.     

Dynamic metabolic control: towards precision engineering of metabolism

Advances in metabolic engineering have led to the synthesis of a wide variety of valuable chemicals in microorganisms. The key to commercializing these processes is the improvement of titer, productivity, yield, and robustness. Traditional approaches to enhancing production use the “push–pull-block” strategy that modulates enzyme expression under static control. However, strains are often optimized for specific laboratory set-up and are sensitive to environmental fluctuations. Exposure to sub-optimal growth conditions during large-scale fermentation often reduces their production capacity. Moreover, static control of engineered pathways may imbalance cofactors or cause the accumulation of toxic intermediates, which imposes burden on the host and results in decreased production. To overcome these problems, the last decade has witnessed the emergence of a new technology that uses synthetic regulation to control heterologous pathways dynamically, in ways akin to regulatory networks found in nature. Here, we review natural metabolic control strategies and recent developments in how they inspire the engineering of dynamically regulated pathways. We further discuss the challenges of designing and engineering dynamic control and highlight how model-based design can provide a powerful formalism to engineer dynamic control circuits, which together with the tools of synthetic biology, can work to enhance microbial production.     

Differential responses of five cherry tomato varieties to water stress: changes on phenolic metabolites and related enzymes

Different tomato cultivars (Solanum lycopersicum L.) with differences in tolerance to drought were subjected to moderate water stress to test the effects on flavonoids and caffeoyl derivatives and related enzymes. Our results indicate that water stress resulted in decreased shikimate pathway (DAHP synthase, shikimate dehydrogenase, phenylalanine ammonium lyase, cinnamate 4-hydroxylase, 4-coumarate CoA ligase) and phenolic compounds (caffeoylquinic acid derivatives, quercetin and kaempferol) in the cultivars more sensitive to water stress. However, cv. Zarina is more tolerant, and registered a rise in querc-3-rut-pent, kaempferol-3-api-rut, and kaempferol-3-rut under the treatment of water stress. Moreover, this cultivar show increased activities of flavonoid and phenylpropanoid synthesis and decreased in degradation-related enzymes. These results show that moderate water stress can induce shikimate pathway in tolerant cultivar.     

2-O-β-d-Glucopyranosyl-sn-glycerol based analogues of sulfoquinovosyldiacylglycerols (SQDG) and their role in inhibiting Epstein-Barr virus early antigen activation

New sulfoquinovosyldiacylglycerols derived from 2-O-β-d-glucopyranosyl-sn-glycerol, carrying acyl chains of various length on the glycerol moiety, were prepared through a convenient synthetic procedure in which a sulfonate is introduced at the C-6 position of glucose by oxidation of a thioacetate in the presence of the unprotected secondary hydroxyl groups, and tested for their anti-tumor-promoting activity using a short-term in vitro assay for Epstein-Barr virus early antigen (EBV-EA) activation. Our study has allowed to ascertain the role of the 6′-sulfonate group and the need of a free hydroxyl group on the glycerol moiety in inhibiting the EBV activation promoted by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA).     

Trait-based analyses for the detection of linkage between marker loci and quantitative trait loci in crosses between inbred lines

Summary Methods are presented for determining linkage between a marker locus and a nearby locus affecting a quantitative trait (quantitative trait locus=QTL), based on changes in the marker allele frequencies in selection lines derived from the F-2 of a cross between inbred lines, or in the high and low phenotypic classes of an F-2 or BC population. The power of such trait-based (TB) analyses was evaluated and compared with that of methods for determining linkage based on the mean quantitative trait value of marker genotypes in F-2 or BC populations [marker-based (MB) analyses]. TB analyses can be utilized for marker-QTL linkage determination in situations where the MB analysis is not applicable, including analysis of polygenic resistance traits where only a part of the population survives exposure to the Stressor and analysis of marker-allele frequency changes in selection lines. TB analyses may be a useful alternative to MB analyses when interest is centered on a single quantitative trait only and costs of scoring for markers are high compared with costs of raising and obtaining quantitative trait information on F-2 or BC individuals. In this case, a TB analysis will enable equivalent power to be obtained with fewer individuals scored for the marker, but more individuals scored for the quantitative trait. MB analyses remain the method of choice when more than one quantitative trait is to be analyzed in a given population.Contribution from the ARO, Bet Dagan, Israel. No. 1698-E, 1986 series     

<Emphasis Type="Italic">Corynosoma australe</Emphasis> Johnston, 1937 and <Emphasis Type="Italic">C. cetaceum</Emphasis> Johnston &; Best, 1942 (Acanthocephala: Polymorphidae) from marine mammals and fishes in Argentinian waters: allozyme markers and taxonomic status

Genetic and morphological studies were carried out on acanthocephalans belonging to Corynosoma Lühe, 1904 and referable to the species C. cetaceum Johnston & Best, 1942 and C. australe Johnston, 1937, which were recovered from both definitive and intermediate hosts in Argentinian waters. The aims were to estimate the level of genetic differentiation between the two taxa at any stage of their life-cycle, to provide genetic (allozyme) markers for their recognition and to analyse the systematic status of both taxa. Acanthocephalans were collected from the stomach and intestine of Arctocephalus australis (Zimmerman), the intestine of Mirounga leonina (Linnaeus) and the stomach of Pontoporia blainvillei Gervais & D’Orbigny (definitive hosts) in Argentinian waters. Alternative alleles at all the 13 enzymatic loci studied were observed for C. australe and C. cetaceum. The specimens from the stomach of both P. blainvillei and A. australis were identified, on the basis of the great number of diagnostic loci found, as C. cetaceum; those from intestine of both A. australis and M. leonina as C. australe. A high level of genetic differentiation (D_Nei=∞: I_Nei = 0.00) between the two taxa was found, suggesting a generic distinction between the two species. Cystacanths of the two species from the body-cavity of the fish Cynoscion guatucupa (Cuvier) collected from the same geographical area were identified genetically. Morphological patterns, such as the number of hooks and hook rows on the proboscis, the distribution of somatic and genital armature, and other morphometric and meristic differences, in addition to ecological data, enabled the identification of these two species at cystacanth, juvenile and adult stages. However, a number of morphological and morphometric features of the Argentinian material were different to those of C. australe and C. cetaceum described from other regions of the world.