A synthetic peptide reproducing the mitochondrial targeting motif of AKAP121: a conformational study

The conformational features of a peptide derived by the 10-30 sequence of the mitochondrial domain of AKAP121 [Ac-1XKKPLALPGMLALLGWWWFFSRKKX25-NH2 (X=beta-Ala)] in water and in a water/trifluoroethanol (TFE) mixture at 298 K have been determined by NMR and CD spectroscopy. Backbone clustering analysis of NMR-derived structures led to the identification of a single representative structure in water/TFE. The structure of the peptide consists mainly of an alpha-helix, whose core is the region 7-23, with a less ordered N-terminal part. These data are confirmed by CD analysis. It is noteworthy that the high hydrophobic Trp16-Phe20 segment, that might also mediate interaction with tubulin, is organized in an alpha-helical wheel. Our conformational data can be the starting point for the development of highly selective peptides that interfere with the biological function of the Protein Kinase A scaffold protein AKAP121.     

Oxidative pathways in response to polyunsaturated aldehydes in the marine diatom Skeletonema marinoi (Bacillariophyceae)

Polyunsaturated aldehydes (PUA) have recently been shown to induce reactive oxygen species (ROS) and possibly reactive nitrogen species (RNS, e.g., peroxynitrite) in the diatom Skeletonema marinoi (S. marinoi), which produces high amounts of PUA. We now are attempting to acquire better understanding of which reactive molecular species are involved in the oxidative response of S. marinoi to PUA. We used flow cytometry, the dye dihydrorhodamine 123 (DHR) as the main indicator of ROS (but which is also known to partially detect RNS), and different scavengers and inhibitors of both nitric oxide (NO) synthesis and superoxide dismutase activity (SOD). Both the scavengers Tempol (for ROS) and uric acid (UA, for peroxynitrite) induced a lower DHR‐derived green fluorescence in S. marinoi cells exposed to the PUA, suggesting that both reactive species were produced. When PUA‐exposed S. marinoi cells were treated with the NO scavenger 2‐4‐carboxyphenyl‐4,4,5,5‐tetramethylimidazoline‐1‐oxyl‐3‐oxide (cPTIO), an opposite response was observed, with an increase in DHR‐derived green fluorescence. A higher DHR‐derived green fluorescence was also observed in the presence of sodium tungstate (ST), an inhibitor of NO production via nitrate reductase. In addition, two different SOD inhibitors, 2‐methoxyestradiol (2ME) and sodium diethyldithiocarbamate trihydrate (DETC), had an effect, with DETC inducing the strongest inhibition after 20 min. These results indicate the involvement of O₂^? generation and SOD activity in H₂O₂ formation (with downstream ROS generation dependent from H₂O₂) in response to PUA exposure. This is relevant as it refines the biological impact of PUA and identifies the specific molecules involved in the response. It is speculated that in PUA‐exposed S. marinoi cells, beyond a certain threshold of PUA, the intracellular antioxidant system is no longer able to cope with the excess of ROS, thus resulting in the observed accumulation of both O₂^?? and H₂O₂. This might be particularly relevant for population dynamics at sea, during blooms, when cell lysis increases and PUA are released. It can be envisioned that in the final stages of blooms, higher local PUA concentrations accumulate, which in turn induces intracellular ROS generation that ultimately leads to cell death and bloom decay.     

Haplotype affinities resolve a major component of goat (Capra hircus) MtDNA D-loop diversity and reveal specific features of the Sardinian stock

Goat mtDNA haplogroup A is a poorly resolved lineage absorbing most of the overall diversity and is found in locations as distant as Eastern Asia and Southern Africa. Its phylogenetic dissection would cast light on an important portion of the spread of goat breeding. The aims of this work were 1) to provide an operational definition of meaningful mtDNA units within haplogroup A, 2) to investigate the mechanisms underlying the maintenance of diversity by considering the modes of selection operated by breeders and 3) to identify the peculiarities of Sardinian mtDNA types. We sequenced the mtDNA D-loop in a large sample of animals (1,591) which represents a non-trivial quota of the entire goat population of Sardinia. We found that Sardinia mirrors a large quota of mtDNA diversity of Western Eurasia in the number of variable sites, their mutational pattern and allele frequency. By using bayesian analysis, a distance-based tree and a network analysis, we recognized demographically coherent groups of sequences identified by particular subsets of the variable positions. The results showed that this assignment system could be reproduced in other studies, capturing the greatest part of haplotype diversity.We identified haplotype groups overrepresented in Sardinian goats as a result of founder effects. We found that breeders maintain diversity of matrilines most likely through equalization of the reproductive potential. Moreover, the relevant amount of inter-farm mtDNA diversity found does not increase proportionally with distance. Our results illustrate the effects of breeding practices on the composition of maternal gene pool and identify mtDNA types that may be considered in projects aimed at retrieving the maternal component of the oldest breeds of Sardinia.     

Does disturbance affect bud bank size and belowground structures diversity in Brazilian subtropical grasslands?

Brazilian Campos grasslands are ecosystems under high frequency of disturbance by grazing and fires. Absence of such disturbances may lead to shrub encroachment and loss of plant diversity. Vegetation regeneration after disturbance in these grasslands occurs mostly by resprouting from belowground structures. We analyzed the importance of bud bank and belowground bud bearing organs in Campos grasslands. We hypothesize that the longer the intervals between disturbances are, the smaller the size of the bud bank is. Additionally, diversity and frequency of belowground organs should also decrease in areas without disturbance for many years. We sampled 20 soil cores from areas under different types of disturbance: grazed, exclusion from disturbance for two, six, 15 and 30 years. Belowground biomass was sorted for different growth forms and types of bud bearing organs. We found a decrease in bud bank size with longer disturbance intervals. Forbs showed the most drastic decrease in bud bank size in the absence of disturbance, which indicates that they are very sensitive to changes in disturbance regimes. Xylopodia (woody gemmiferous belowground organs with hypocotyl-root origin) were typical for areas under influence of recurrent fires. The diversity of belowground bud bearing structures decreased in the absence of disturbance. Longer intervals between disturbance events, resulting in decrease of bud bank size and heterogeneity of belowground organs may lead to the decline and even disappearance of species that relay on resprouting from the bud bank upon disturbance.     

Comparison of spontaneous ascites filtration and reinfusion with total paracentesis with intravenous albumin infusion in cirrhotic patients with tense ascites.

OBJECTIVE--To compare the effectiveness and safety of spontaneous ascites filtration and reinfusion and total paracentesis plus intravenous albumin infusion in cirrhotic patients with tense ascites. DESIGN--Randomised trial of the two treatments. SETTING--Teaching hospital and district general hospital in Milan. PATIENTS--45 consecutive cirrhotic patients with recurrent tense ascites and urinary sodium excretion rate less than 20 mmol/day. 35 fulfilled admission criteria and completed the study. 17 received spontaneous ascites filtration and 18 paracentesis plus albumin infusion. MAIN OUTCOME MEASURES--Body weight; urinary volume; serum and urinary electrolyte, serum fibrinogen, and plasma aldosterone concentrations; and plasma renin activity before the procedure and 24 hours and eight days afterwards. RESULTS--Both procedures were effective in all patients. Weight decreased in both groups and showed no substantial increase after eight days. In patients receiving ascites filtration, values decreased significantly (p less than 0.01) after 24 hours for platelet count (mean relative change 0.92; 99% confidence interval 0.86 to 0.98) and serum fibrinogen concentration (0.92; 0.88 to 0.98) but returned to pretreatment values after eight days; no laboratory and clinical signs of disseminated intravascular coagulation were noted. Three patients in this group had fever, which receded spontaneously. One patient in each group had dilutional hyponatraemia. CONCLUSIONS--Spontaneous ascites filtration and reinfusion is an effective treatment for tense ascites. Reinfusion of the patient''s concentrated proteins provides savings without compromising safety.     

The role of oxidative stress in anxiety disorder: cause or consequence?

Anxiety disorders are the most common mental illness in the USA affecting 18% of the population. The cause(s) of anxiety disorders is/are not completely clear, and research in the neurobiology of anxiety at the molecular level is still rather limited. Although mounting clinical and preclinical evidence now indicates that oxidative stress may be a major component of anxiety pathology, whether oxidative stress is the cause or consequence remains elusive. Studies conducted over the past few years suggest that anxiety disorders may be characterised by lowered antioxidant defences and increased oxidative damage to proteins, lipids, and nucleic acids. In particular, oxidative modifications to proteins have actually been proposed as a potential factor in the onset and progression of several psychiatric disorders, including anxiety and depressive disorders. Oxidised proteins are normally degraded by the proteasome proteolytic complex in the cell cytoplasm, nucleus, and endoplasmic reticulum. The Lon protease performs a similar protective function inside mitochondria. Impairment of the proteasome and/or the Lon protease results in the accumulation of toxic oxidised proteins in the brain, which can cause severe neuronal trauma. Recent evidence points to possible proteolytic dysfunction and accumulation of damaged, oxidised proteins as factors that may determine the appearance and severity of psychotic symptoms in mood disorders. Thus, critical interactions between oxidative stress, proteasome, and the Lon protease may provide keys to the molecular mechanisms involved in emotional regulation, and may also be of great help in designing and screening novel anxiolytics and antidepressants.     

Properties of some variants of human beta2-microglobulin and amyloidogenesis

Three variants of human beta(2)-microglobulin (beta(2)-m) were compared with wild-type protein. For two variants, namely the mutant R3Abeta(2)-m and the form devoid of the N-terminal tripeptide (DeltaN3beta(2)-m), a reduced unfolding free energy was measured compared with wild-type beta(2)-m, whereas an increased stability was observed for the mutant H31Ybeta(2)-m. The solution structure could be determined by (1)H NMR spectroscopy and restrained modeling only for R3Abeta(2)-m that showed the same conformation as the parent species, except for deviations at the interstrand loops. Analogous conclusions were reached for H31Ybeta(2)-m and DeltaN3beta(2)-m. Precipitation and unfolding were observed over time periods shorter than 4-6 weeks with all the variants and, sometimes, with wild-type protein. The rate of structured protein loss from solution as a result of precipitation and unfolding always showed pseudo-zeroth order kinetics. This and the failure to observe an unfolded species without precipitation suggest that a nucleated conformational conversion scheme should apply for beta(2)-m fibrillogenesis. The mechanism is consistent with the previous and present results on beta(2)-m amyloid transition, provided a nucleated oligomeric species be considered the stable intermediate of fibrillogenesis, the monomeric intermediate being the necessary transition step along the pathway from the native protein to the nucleated oligomer.     

The cytokine interleukin-1beta reduces the docking and fusion of insulin granules in pancreatic beta-cells, preferentially decreasing the first phase of exocytosis

The prediabetic period in type I diabetes mellitus is characterized by the loss of first phase insulin release. This might be due to islet infiltration mediated by mononuclear cells and local release of cytokines, but the mechanisms involved are unknown. To determine the role of cytokines in insulin exocytosis, we have presently utilized total internal reflection fluorescence microscopy (TIRFM) to image and analyze the dynamic motion of single insulin secretory granules near the plasma membrane in live beta-cells exposed for 24 h to interleukin (IL)-1beta or interferon (IFN)-gamma. Immunohistochemistry observed via TIRFM showed that the number of docked insulin granules was decreased by 60% in beta-cells treated with IL-1beta, while it was not affected by exposure to IFN-gamma. This effect of IL-1beta was paralleled by a 50% reduction in the mRNA and the number of clusters of SNAP-25 in the plasma membrane. TIRF images of single insulin granule motion during a 15-min stimulation by 22 mm glucose in IL-1beta-treated beta-cells showed a marked reduction in the fusion events from previously docked granules during the first phase insulin release. Fusion from newcomers, however, was well preserved during the second phase of insulin release of IL-1beta-treated beta-cells. The present observations indicate that IL-1beta, but not IFN-gamma, has a preferential inhibitory effect on the first phase of glucose-induced insulin release, mostly via an action on previously docked granules. This suggests that beta-cell exposure to immune mediators during the course of insulitis might be responsible for the loss of first phase insulin release.