Conformational Activation of Antithrombin by Heparin Involves an Altered Exosite Interaction with Protease

Heparin allosterically activates antithrombin as an inhibitor of factors Xa and IXa by enhancing the initial Michaelis complex interaction of inhibitor with protease through exosites. Here, we investigate the mechanism of this enhancement by analyzing the effects of alanine mutations of six putative antithrombin exosite residues and three complementary protease exosite residues on antithrombin reactivity with these proteases in unactivated and heparin-activated states. Mutations of antithrombin Tyr²⁵³ and His³¹⁹ exosite residues produced massive 10–200-fold losses in reactivity with factors Xa and IXa in both unactivated and heparin-activated states, indicating that these residues made critical attractive interactions with protease independent of heparin activation. By contrast, mutations of Asn²³³, Arg²³⁵, Glu²³⁷, and Glu²⁵⁵ exosite residues showed that these residues made both repulsive and attractive interactions with protease that depended on the activation state and whether the critical Tyr²⁵³/His³¹⁹ residues were mutated. Mutation of factor Xa Arg¹⁴³, Lys¹⁴⁸, and Arg¹⁵⁰ residues that interact with the exosite in the x-ray structure of the Michaelis complex confirmed the importance of all residues for heparin-activated antithrombin reactivity and Arg¹⁵⁰ for native serpin reactivity. These results demonstrate that the exosite is a key determinant of antithrombin reactivity with factors Xa and IXa in the native as well as the heparin-activated state and support a new model of allosteric activation we recently proposed in which a balance between attractive and repulsive exosite interactions in the native state is shifted to favor the attractive interactions in the activated state through core conformational changes induced by heparin binding.     

Design,synthesis and biological evaluation of 6-(benzyloxy)-4-methylquinolin-2(1H)-one derivatives as PDE3 inhibitors

Selective PDE3 (phosphodiesterase 3) inhibitors improve cardiac contractility and may be used in congestive heart failure. However, their proarrhythmic potential is the most important side effect. In this work ten new synthetic compounds (3-[(4-methyl-2-oxo-1,2-dihydro-6-quinolinyl)oxy]methylbenzamide analogs: 4a–j) were designed, synthesized and tested for the inhibitory activity against human PDE3A and PDE3B. The strategy of the design was based on the structure of vesnarinone (a selective PDE3 inhibitor) and its docking analysis results. The synthetic compounds showed better PDE3 inhibitory activity in comparison with vesnarinone. Using docking analysis, a common binding model of each compound toward PDE3 was suggested. In the next step the potential cardiotonic activity of the best PDE3A inhibitors (4b, IC₅₀ = 0.43 ± 0.04 μM) was evaluated by using the spontaneously beating atria model. In the experiment, atrium of reserpine-treated rat was isolated and the contractile and chronotropic effects of the synthetic compound were assessed. That was carried out in comparison with vesnarinone. The best pharmacological profile was obtained for the compound 4b, which displayed selectivity for increasing the force of contraction (46 ± 3% change over the control) rather than the frequency rate (16 ± 4% change over the control) at 100 μM.     

The impact of a 6-year comprehensive community trial on the awareness, treatment and control rates of hypertension in Iran: experiences from the Isfahan healthy heart program

Background

Acute mental stress may contribute to the cardiovascular disease progression via autonomic nervous system controlled negative effects on the endothelium. The joint effects of stress-induced sympathetic or parasympathetic activity and endothelial function on atherosclerosis development have not been investigated. The present study aims to examine the interactive effect of acute mental stress-induced cardiac reactivity/recovery and endothelial function on the prevalence of carotid atherosclerosis.

Methods

Participants were 81 healthy young adults aged 24-39 years. Preclinical atherosclerosis was assessed by carotid intima-media thickness (IMT) and endothelial function was measured as flow-mediated dilatation (FMD) using ultrasound techniques. We also measured heart rate, respiratory sinus arrhythmia (RSA), and pre-ejection period (PEP) in response to the mental arithmetic and speech tasks.

Results

We found a significant interaction of FMD and cardiac RSA recovery for IMT (p = 0.037), and a significant interaction of FMD and PEP recovery for IMT (p = 0.006). Among participants with low FMD, slower PEP recovery was related to higher IMT. Among individuals with high FMD, slow RSA recovery predicted higher IMT. No significant interactions of FMD and cardiac reactivity for IMT were found.

Conclusions

Cardiac recovery plays a role in atherosclerosis development in persons with high and low FMD. The role of sympathetically mediated cardiac activity seems to be more important in those with impaired FMD, and parasympathetically mediated in those with relatively high FMD. The development of endothelial dysfunction may be one possible mechanism linking slow cardiac recovery and atherosclerosis via autonomic nervous system mediated effect.     

Simultaneous determination of 11 phytoestrogens in human serum using a 2 min liquid chromatography/tandem mass spectrometry method

A rapid 2 min liquid chromatography–tandem mass spectrometry (LC–MS/MS) method operating in multiple reaction ion monitoring mode was developed and validated that allows for the characterization and simultaneous quantification of 11 phytoestrogen metabolites with mass transitions m/z 241/119 (equol), 253/132 (daidzein), 255/149 (dihydrodaidzein), 257/108 (O-desmethylangolesin), 269/133 (genistein), 283/184 (glycitein), 267/191 (formononetin), 289/109 (biochanin A), 267/91 (coumestrol), enterodiol (301/253), and enterolactone (297/253). The method was demonstrated to be specific and sensitive, and a linear response for each phytoestrogen was observed over a range of 1–5000 ng/mL in human serum with the exception of dihydrodaidzein, whose lower limit of quantification was 2 ng/mL. The separation was carried out on a Synergi Polar-RP 2.5 micron (50 mm × 2.0 mm i.d.) column at 50 °C with water and acetonitrile (both containing 10 mM ammonium acetate) as the mobile phase under gradient conditions at a flow rate of 0.75 mL/min. This LC–MS/MS method is very useful for high-throughput analysis of phytoestrogens and proved to be simple, sensitive, reproducible, and reliable.     

Proteome analysis of brain in murine experimental autoimmune encephalomyelitis

Multiple sclerosis is considered a prototype inflammatory autoimmune disorder of the CNS. Experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein is one of the best‐characterized animal models of multiple sclerosis. Comprehensive understanding of gene expression in EAE can help identify genes that are important in drug response and pathogenesis. We applied a 2‐DE‐based proteomics approach to analyze the protein expression pattern of the brain in healthy and EAE samples. Of more than 1000 protein spots we analyzed, 70 showed reproducible and significant changes in EAE compared to controls. Of these, 42 protein spots could be identified using MALDI TOF‐TOF‐MS. They included mitochondrial and structural proteins as well as proteins involved in ionic and neurotransmitter release, blood barriers, apoptosis, and signal transduction. The possible role of these proteins in the responses of mice to animal models of multiple sclerosis is discussed.     

Sciatic nerve regeneration by transplantation of menstrual blood-derived stem cells

This is the first study demonstrating the efficacy of menstrual blood-derived stem cell (MenSC) transplantation via a neural guidance conduit, for peripheral nerve regeneration. The synthesized poly (?-caprolactone)/Gelatin conduit, filled with collagen type I and seeded with 3?×?10⁴ MenSCs, was implanted into a rat’s 10 mm sciatic nerve defect. The results of hot plate latency, sciatic functional index and weight-loss percentage of wet gastrocnemius muscle demonstrated that the MenSC transplantation had comparable nerve regeneration outcome to autograft, as the gold standard of nerve bridging. The transplantation of MenSCs via a synthetic conduit could ameliorate the functional recovery of sciatic nerve-injured rats which make them a potential candidate for cell therapy of peripheral nervous system disorders.     

Long-term storage of sexual and asexual Trichogramma brassicae (Hymenoptera: Trichogrammatidae)

The effect of exposure duration to pre-storage (representing fall conditions) and storage (representing winter conditions) temperatures on the emergence rate in sexual and asexual Trichogramma brassicae Bezdenko (Hymenoptera: Trichogrammatidae) was studied under controlled laboratory conditions. Asexual T. brassicae was infected by Wolbachia, an endosymbiont bacterium. We considered 0, 30, 40, 50 and 60 days as exposure duration at 10°C and 0, 30, 60, 90 followed by 180 days as exposure duration at 4°C for both T. brassicae strains. Emergence rate was significantly affected by the presence of Wolbachia, the duration of exposure to 10°C, the duration of storage at 4°C and their interactions, but not by the interaction between strain × storage duration at 4°C. Emergence rate of adults was positively correlated with exposure duration at 10°C, but it gradually decreased as the cold storage duration (4°C) increased in both strains. In conclusion, we are able to store both asexual and sexual strains for longer periods of time at 4°C, but the emergence rate of sexual T. brassicae was greater than asexual in all the treatments. The presence of Wolbachia could have a negative effect on fitness of T. brassicae and reduces its emergence rate after long-term storage.     

The critically endangered Asiatic cheetah <Emphasis Type="Italic">Acinonyx jubatus venaticus</Emphasis> in Iran: a review of recent distribution,and conservation status

Considerable effort has been put into conservation of the critically endangered Asiatic cheetah Acinonyx jubatus venaticus in Iran during the past few decades, and a thorough review of the species’ status, demography, range and conservation is provided here. We collated a large dataset of all verified occurrence data, photographic records and mortality cases since 1980 throughout the species’ range in Iran. Currently, the cheetah is distributed throughout the arid landscapes of the eastern half of Iran, but the limits of its current and past range as well as population trends are uncertain. Surveys of nearly 40 different areas resulted in 18 localities with confirmed presence of cheetahs in recent years. Camera trapping has been an effective tool to provide evidence of presence and status of cheetahs, revealing the species’ extremely low density and long inter-reserve movements. Together with photographic records, a total of 82 different cheetahs were detected during the 2000s in Iran. Protection status in most areas has been elevated by the Iran government. Asiatic cheetahs are highly vulnerable to extinction, mainly due to causalities mediated by herder persecution, poaching and road collisions as well as prey and habitat loss. Some efforts have been made to address these threats, but range expansion in recent years is a result of greater survey effort, rather than population recovery. We suggest that, despite conservation investment of the last 15 years, the species remains critically endangered on the verge of extinction.