Conserved noncanonical residue Gly-126 confers instability to the middle part of the tropomyosin molecule

Tropomyosin (Tm) is a two-stranded α-helical coiled-coil protein with a well established role in regulation of actin cytoskeleton and muscle contraction. It is believed that many Tm functions are enabled by its flexibility whose nature has not been completely understood. We hypothesized that the well conserved non-canonical residue Gly-126 causes local destabilization of Tm. To test this, we substituted Gly-126 in skeletal muscle α-Tm either with an Ala residue, which should stabilize the Tm α-helix, or with an Arg residue, which is expected to stabilize both α-helix and coiled-coil structure of Tm. We have shown that both mutations dramatically reduce the rate of Tm proteolysis by trypsin at Asp-133. Differential scanning calorimetry was used for detailed investigation of thermal unfolding of the Tm mutants, both free in solution and bound to F-actin. It was shown that a significant part of wild type Tm unfolds in a non-cooperative manner at low temperature, and both mutations confer cooperativity to this part of the Tm molecule. The size of the flexible middle part of Tm is estimated to be 60-70 amino acid residues, about a quarter of the Tm molecule. Thus, our results show that flexibility is unevenly distributed in the Tm molecule and achieves the highest extent in its middle part. We conclude that the highly conserved Gly-126, acting in concert with the previously identified non-canonical Asp-137, destabilizes the middle part of Tm, resulting in a more flexible region that is important for Tm function.     

Association study of the calcitonin gene-related polypeptide-alpha (CALCA) and the receptor activity modifying 1 (RAMP1) genes with migraine

Migraine is a common neurovascular brain disorder characterised by recurrent attacks of severe headache that may be accompanied by various neurological symptoms. Migraine is thought to result from activation of the trigeminovascular system followed by vasodilation of pain-producing intracranial blood vessels and activation of second-order sensory neurons in the trigeminal nucleus caudalis. Calcitonin gene-related peptide (CGRP) is a mediator of neurogenic inflammation and the most powerful vasodilating neuropeptide, and has been implicated in migraine pathophysiology. Consequently, genes involved in CGRP synthesis or CGRP receptor genes may play a role in migraine and/or increase susceptibility. This study investigates whether variants in the gene that encodes CGRP, calcitonin-related polypeptide alpha (CALCA) or in the gene that encodes a component of its receptor, receptor activity modifying protein 1 (RAMP1), are associated with migraine pathogenesis and susceptibility. The single nucleotide polymorphisms (SNPs) rs3781719 and rs145837941 in the CALCA gene, and rs3754701 and rs7590387 at the RAMP1 locus, were analysed in an Australian Caucasian population of migraineurs and matched controls. Although we find no significant association of any of the SNPs tested with migraine overall, we detected a nominally significant association (p = 0.031) of the RAMP1 rs3754701 variant in male migraine subjects, although this is non-significant after Bonferroni correction for multiple testing.     

Rio Plus Ten: The Evolution of International Marine Fisheries Governance

The 1992 United Nations Conference on Environment and Development (UNCED) held at Rio de Janeiro was a seminal event that addressed the interplay of economic development and human use of natural resources with the need for protection of the natural environment. The general principles embodied in the Rio Declaration and the provisions included in the comprehensive Agenda 21 expressed the expectations of the international community and set the stage for national and international policy and legal developments in a host of different areas, including the governance of the ocean's living resources. Chapter 17 of Agenda 21 focused on the world's oceans and seas and crystallized growing world community concern with problems such as unregulated fishing, fishing vessel reflagging, overcapitalization of the fishing industry, inadequate fisheries enforcement, and insufficient cooperation among states. Further, the documentation of UNCED evidenced interest in new governance frameworks and concepts such as ecosystem-based management and precaution. In retrospect, it is clear that the ideas discussed at Rio have been important and have been implemented by a variety of governments and international organizations as they exercise their responsibilities for fisheries management. This article examines the principles and approaches suggested by UNCED as they relate to world fisheries and considers how they have been reflected in global fishery agreements such as the FAO Compliance Agreement and the United Nations Fish Stocks Agreement, in the work of international fishery bodies such as the FAO Committee on Fisheries, and in the efforts of a number of regional fishery commissions. It also addresses the significance and ramifications of changes suggested by Agenda 21 for the future conduct and management of world marine fisheries.     

Effects of a Four-Year Ecological Cultivation on Spider Coenoses (Arachnida,Araneae) on the "Ökohof Seeben" In Halle (Saale)

From 1994 to 1997 investigations have been carried out on fields of the "Ökohof Seeben" in Halle (Saale), refering to long-term effects of ecological agriculture on fauna, flora and soil. For the sampling of the epigeic fauna, pitfall traps were used. The purpose was to examine the effect of the changes in management on spiders for example. The influence of field size, edge-structures and different crops on species- and individual-numbers was studied. The question was, whether ecological agriculture results in an increasing biodiversity overviewed over years. In 4 years, on 6 fields, 190 species of spiders (82,202 adults spiders) could be proved, among them 32 species endangered in Sachsen-Anhalt and 28 species endangered according to the Red-Data-Books of Germany. The species numbers in the edge-biotops were nearly twice the numbers in the fields. The activity density of pioneer species (for example Oedothorax apicatus ) decreased rapidly on all fields. As a whole species numbers, and also the number of seldom species, were higher on the small fields than on the large fields lying in a poor structured environment. On the large fields some species, especially some Gnaphosidae and Lycosidae were missing in the first two years, but could be proved in the later years. Increasing numbers of species bigger in size and also more balanced dominance structures could be observed in the later years of investigation. In the fields the Linyphiidae species O. apicatus, E. atra, E. dentipalpis, M. rurestris, L. tenuis, P. microphthalmum, A. humilis and the Lycosidae P. agrestis and P. prativaga were dominant. Other species, like D. concolor, B. gracilis, H. signifer, P. juncea, T. ruricola, D. pusillus, X. kochi, O. claveata, P. degeeri, P. palustris, R. lividus prefered the adjacent boundaries, but were represented in the field as well. Several species, such as A. cuneata, A. pulverulenta, T. terricola, X. miniata, X. nemoralis, A. albimana, P. mirabilis, Ch. campestre, X. acerbus, Z. spinimana, P. festivus, D. praeficus and D. lutetianus could be proved only in the adjacent boundaries. Altogether positive effects on the dominance structure of spiders could be shown. An increasing biodiversity may be expected.     

Lack of amino acids in mouse hepatocytes in culture induces the selection of preneoplastic cells

Protein malnutrition occurs when there is insufficient protein to meet metabolic demands. Previous works have indicated that cycles of protein fasting/refeeding enhance the incidence of early lesions during chemical carcinogenesis in rat liver. The general objective of this work was to study the effect of aminoacids (Aa) deprivation on the proliferation and survival of hepatocytes, to understand its possible involvement in the generation of pre-neoplastic stages in the liver. Lack of Aa in the culture medium of an immortalized mice hepatocyte cell line induced loss in cell viability, correlating with apoptosis. However, a subpopulation of cells was able to survive, which showed a more proliferative phenotype and resistance to apoptotic stimuli. Escaping to Aa deprivation-induced death is coincident with an activated mTOR signaling and higher levels of phospho-AKT and phospho-ERKs, which correlated with increased activation of EGFR/SRC pathway and overexpression of EGFR ligands, such as TGF-α and HB-EGF. Lack of Aa induced a rapid increase in reactive oxygen species (ROS) production. However, cells that survived showed an enhancement in the levels of reduced glutathione and a higher expression of γ-GCS, the regulatory enzyme of glutathione synthesis, which can be interpreted as an adaptation of the cells to counteract the oxidative stress. In conclusion, results presented in this paper indicate that it is possible to isolate a subpopulation of hepatocytes that are able to grow in the absence of Aa, showing higher capacity to proliferate and survive, reminiscent of a preneoplastic phenotype.     

Antioxidant and detoxifying fish enzymes as biomarkers of river pollution

The activity of several antioxidant and detoxifying enzymes, superoxide dismutase SOD , GSH peroxidase GSHPx , GSSG reductase GSR and GSH S transferase GST , the contents of thiobarbituric acid reactive substances TBARS , and the SOD and GST isoenzyme patterns were studied in the livers of chubs Leuciscus cephalus from reference river areas and polluted urban sites. Livers of polluted fish contained higher concentrations of transition metals, especially copper and iron. Total GSHPx activity was 1.8 fold higher in the polluted fish than in reference animals, while the SOD and GSR activities and the TBARS content were not significantly changed. Three new SOD isoforms pI 4.45, 5.1, 5.2 and a higher intensity of the band pI 4.2 were observed after isoelectrofocusing of polluted fish extracts. Total GST activity was higher in fish from polluted areas. The GST isoenzyme pattern was studied using subunit specific substrates DCNB, EPNP, EA, NPB, NBC and by Western blot using antibodies specific to rat GST subunits 1, 8 Alpha class , 3 Mu class and 7 Pi class . Reference and polluted fish lacked cross reactivity towards Alpha class GSTs. Reference fish displayed weaker cross reactivity towards CST 7 and 2.3 fold lower activity with EA, while higher cross reaction with GST 3 was observed in polluted fish.     

Coping style in primary care adult patients with abridged somatoform disorders

Background and objective Empirical data are scarce regarding the adaptive response to stress for patients with somatoform disorders. Our objective was to identify the preferred coping strategies of patients with abridged somatisation, a common condition in primary care. Because of the functional impairment associated with somatisation, we predicted a preference for less effective, emotion-focused coping strategies over more effective, problem-focused adaptations.Design We conducted a cross-sectional, observational study of physician referred primary care patients who presented with persistent, medically unexplained, physical symptoms. Patients were classified into two abridged somatisation groups by symptom frequency and duration, as determined by the Diagnostic Interview Schedule. The groups were compared with each other and with a non-clinical reference group; outcome variables were eight emotion- and problem-focused strategies, as measured by the Ways of Coping Questionnaire.Results Of the 72 eligible individuals, 48 participated in the study. Median age was 48 years and 75% of patients were women; 26 had somatic syndrome and 22 had a subthreshold somatising level. Patients with abridged somatisation disorders preferred emotion-focused coping strategies - typically detachment and impact minimisation, wishful thinking and problem avoidance.Conclusions Patients with abridged somatising disorder responded to stress with predominantly emotion-focused strategies, which may be associated with a lower level of positive adaptive outcome. Our findings suggest that patients with abridged somatising disorders might benefit from emphasis on problem-focused coping strategies, delivered through primary care, to improve quality of life and decrease healthcare utilisation costs.     

Calculating Free Energies Using a Scaled-Force Molecular Dynamics Algorithm

We propose and test a family of methods to calculate the free energy along a generalized coordinate, , based on computing the force acting on this coordinate. First, we derive a formula that connects the free energy in unconstrained simulations with the force of constraint that can be readily calculated numerically. Then, we consider two methods, which improve the efficiency of the free energy calculation by yielding uniform or nearly uniform sampling of . Both rely on modifying the force acting on . In one method, this force is replaced by a force with zero mean and is advanced quasistatically. In the second method, the force is augmented adaptively by a biasing force. We provide formulas for calculating the free energy of the unmodified system from the forces acting in these modified, non-Hamiltonian systems. Using conformational transitions in 1,2-dichloroethane as a test case, we show that both methods perform very well.     

The chemiluminescence response of bivalve haemocytes: utility in screening for immunomodulators and as a biomarker

Resistance to infectious diseases in bivalves depends primarily on the vigour and efficacy of haemocyte-dependent antimicrobial defence mechanisms. Like other phagocytes, haemocytes seem to rely on oxygen-independent (lysosomal hydrolases, lysozyme) and oxygen-dependent (reactive oxygen species) mechanisms to destroy ingested microorganisms. The generation of cytotoxic oxyradicals by haemocytes can be precisely quantified by means of a simple chemiluminescence (CL) assay using luminol or other CL probes. Tributyltin (TBT), and other environmental contaminants, at sublethal levels, will produce dose-dependent suppression of CL activity in haemocytes exposed in short-term, in vitro assays. Presumably, this suppression would find expression as impaired host defence capability. In fact, TBT has been shown to exacerbate progression and lethality of Perkinsus marinus infections in the oyster, Crassostrea virginica. This suggests that CL assays on haemocytes exposed in vitro to single agents or complex mixtures might be useful in screening for aquatic immunomodulators. Statistically significant alterations in CL responses of haemocytes withdrawn from bivalves exposed to xenobiotics in the laboratory or field are more difficult to identify because of high inter-animal variation; however, the use of haemocyte CL as a biomarker of effect merits further investigation.