- Plasma Phospholipid Transfer Protein Promotes Platelet Aggregation. [Journal Article]
- THThromb Haemost 2018 Nov 12
- It remains unclear whether plasma phospholipid transfer protein (PLTP) is involved in hyper-coagulation or hypo-coagulation. This study investigated the direct effect of PLTP on platelet aggregation ...
It remains unclear whether plasma phospholipid transfer protein (PLTP) is involved in hyper-coagulation or hypo-coagulation. This study investigated the direct effect of PLTP on platelet aggregation and the underlying mechanism. Washed platelets from humans or mice and mouse platelet-rich plasma and human recombinant PLTP were isolated. PLTP is present in human platelets. We assessed adenosine diphosphate (ADP)-, collagen- and thrombin-induced platelet aggregation, phosphatidylserine externalization and photothrombosis-induced cerebral infarction in mice. PLTP over-expression increased platelet aggregation, while PLTP deficiency had the opposing reaction. Human recombinant PLTP increased both mouse and human platelet aggregation in a dose-dependent manner. Phosphatidylserine externalization provides a water/lipid surface for the interaction of coagulation factors, which accelerates thrombosis. Compared with wild-type controls, platelets from PLTP transgenic mice had significantly more phosphatidylserine on the exterior surface of the plasma membrane, whereas platelets from PLTP-deficient mice had significantly less phosphatidylserine on the surface, thus PLTP influences fibrinogen binding on the plasma membrane. Moreover, recombinant PLTP together with ADP significantly increased phosphatidylserine exposure on the plasma membrane of PLTP-deficient platelets, thereby increasing fibrinogen binding. PLTP over-expression significantly accelerated the incidence of photothrombosis-induced infarction in mice, whereas PLTP deficiency significantly reduced the frequency of infarction. We concluded that PLTP promotes phosphatidylserine externalization at the plasma membrane of platelets and accelerates ADP- or collagen-induced platelet aggregation. This effect plays an important role in the initiation of thrombin generation and platelet aggregation under sheer stress conditions. Thus, PLTP is involved in hyper-coagulation. Therefore, PLTP inhibition could be a novel approach for countering thrombosis.
- Allosteric regulation of pyruvate kinase from Mycobacterium tuberculosis by metabolites. [Journal Article]
- BBBiochim Biophys Acta Proteins Proteom 2018 Nov 09
- Mycobacterium tuberculosis (Mtb) causes both acute tuberculosis and latent, symptom-free infection that affects roughly one-third of the world's population. It is a globally important pathogen that p...
Mycobacterium tuberculosis (Mtb) causes both acute tuberculosis and latent, symptom-free infection that affects roughly one-third of the world's population. It is a globally important pathogen that poses multiple dangers. Mtb reprograms its metabolism in response to the host niche, and this adaptation contributes to its pathogenicity. Knowledge of the metabolic regulation mechanisms in Mtb is still limited. Pyruvate kinase, involved in the late stage of glycolysis, helps link various metabolic routes together. Here, we demonstrate that Mtb pyruvate kinase (Mtb PYK) predominantly catalyzes the reaction leading to the production of pyruvate, but its activity is influenced by multiple metabolites from closely interlinked pathways that act as allosteric regulators (activators and inhibitors). We identified allosteric activators and inhibitors of Mtb PYK originating from glycolysis, citrate cycle, nucleotide/nucleoside inter-conversion related pathways that had not been described so far. Enzyme was found to be activated by fructose-1,6-bisphosphate, ribose-5-phosphate, adenine, adenosine, hypoxanthine, inosine, L-2-phosphoglycerate, l-aspartate, glycerol-2-phosphate, glycerol-3-phosphate. On the other hand thiamine pyrophosphate, glyceraldehyde-3-phosphate and L-malate were identified as inhibitors of Mtb PYK. The detailed kinetic analysis indicated a morpheein model of Mtb PYK allosteric control which is strictly dependent on Mg2+ and substantially increased by the co-presence of Mg2+ and K+.
- Epigenetic Hallmarks of Fetal Early Atherosclerotic Lesions in Humans. [Journal Article]
- JCJAMA Cardiol 2018 Nov 07
- CONCLUSIONS: The present study provides a stringent quantitative estimate of the magnitude of the association of maternal cholesterol levels during pregnancy with fetal aortic lesions and reveals the epigenetic response of fetal aortic SREBP2 to maternal cholesterol level. The role of maternal cholesterol level during pregnancy and epigenetic signature in offspring in cardiovascular primary prevention warrants further long-term causal relationship studies.
- Effect of metformin exposure on growth and photosynthetic performance in the unicellular freshwater chlorophyte, Chlorella vulgaris. [Journal Article]
- PlosPLoS One 2018; 13(11):e0207041
- Many pharmaceuticals have negative effects on biota when released into the environment. For example, recent work has shown that the commonly prescribed antidiabetic drug, metformin (N,N-dimethylbigua...
Many pharmaceuticals have negative effects on biota when released into the environment. For example, recent work has shown that the commonly prescribed antidiabetic drug, metformin (N,N-dimethylbiguanide), has endocrine disrupting effects on fish. However, effects of metformin on aquatic primary producers are poorly known. We exposed cultured isolates of a freshwater chlorophyte, Chlorella vulgaris, to a range of metformin concentrations (0-767.9 mg L-1) to test the hypothesis that exposure negatively affects photosynthesis and growth. A cessation of growth, increase in non-photochemical quenching (NPQ, NPQmax), and reduced electron transport rate (ETR) were observed 24 h after exposure to a metformin concentration of 767.8 mg L-1 (4.6 mM). By 48 h, photosynthetic efficiency of photosystem II (Fv/Fm), α, the initial slope of the ETR-irradiance curve, and Ek (minimum irradiance required to saturate photosynthesis) were reduced. At a lower concentration (76.8 mg L-1), negative effects on photosynthesis (increase in NPQ, decrease in ETR) were delayed, occurring between 72 and 96 h. No negative effects on photosynthesis were observed at an exposure concentration of 1.5 mg L-1. It is likely that metformin impairs photosynthesis either through downstream effects from inhibition of complex I of the electron transport chain or via activation of the enzyme, SnRK1 (sucrose non-fermenting-related kinase 1), which acts as a cellular energy regulator in plants and algae and is an ortholog of the mammalian target of metformin, AMPK (5' adenosine monophosphate-activated protein kinase).
- Inexpensive High-Throughput Screening of Kinase Inhibitors Using One-Step Enzyme-Coupled Fluorescence Assay for ADP Detection. [Journal Article]
- SDSLAS Discov 2018 Nov 12; :2472555218810139
- Protein kinases are attractive targets for both biological research and drug development. Several assay kits, especially for the detection of adenosine diphosphate (ADP), which is universally produce...
Protein kinases are attractive targets for both biological research and drug development. Several assay kits, especially for the detection of adenosine diphosphate (ADP), which is universally produced by kinases, are commercially available for high-throughput screening (HTS) of kinase inhibitors, but their cost is quite high for large-scale screening. Here, we report a new enzyme-coupled fluorescence assay for ADP detection, which uses just 10 inexpensive, commercially available components. The assay protocol is very simple, requiring only the mixing of test solutions with ADP detection solution and reading the fluorescence intensity of resorufin produced by coupling reaction. To validate the assay, we focused on CDC2-like kinase 1 (CLK1), a dual-specificity kinase that plays an important role in alternative splicing, and we used the optimized assay to screen an in-house chemical library of about 215,000 compounds for CLK1 inhibitors. We identified and validated 12 potent inhibitors of CLK1, including a novel inhibitory scaffold. The results demonstrate that this assay platform is not only simple and cost-effective, but also sufficiently robust, showing good reproducibility and giving similar results to those obtained with the widely used ADP-Glo bioluminescent assay.
- Simultaneous Observation of Kinesin-Driven Microtubule Motility and Binding of Adenosine Triphosphate Using Linear Zero-Mode Waveguides. [Journal Article]
- ANACS Nano 2018 Nov 12
- Single-molecule fluorescence observation of adenosine triphosphate (ATP) is a powerful tool to elucidate the chemomechanical coupling of ATP with a motor protein. However, in total internal reflectio...
Single-molecule fluorescence observation of adenosine triphosphate (ATP) is a powerful tool to elucidate the chemomechanical coupling of ATP with a motor protein. However, in total internal reflection fluorescence microscopy (TIRFM), available ATP concentration is much lower than that in the in vivo environment. To achieve single-molecule observation with a high signal-to-noise ratio, zero-mode waveguides (ZMWs) are utilized even at high fluorescent molecule concentrations in the micromolar range. Despite the advantages of ZMWs, the use of cytoskeletal filaments for single-molecule observation has not been reported because of difficulties in immobilization of cytoskeletal filaments in the cylindrical aperture of ZMWs. Here, we propose linear ZMWs (LZMWs) to visualize enzymatic reactions on cytoskeletal filaments, specifically kinesin-driven microtubule motility accompanied by ATP binding/unbinding. Finite element method simulation revealed excitation light confinement in a 100-nm-wide slit of LZMWs. Single-molecule observation was then demonstrated with up to 1 μM labeled ATP, which was 10-fold higher than that available in TIRFM. Direct observation of binding/unbinding of ATP to kinesins that propel microtubules enabled us to find that significant fraction of ATP molecules bound to kinesins were dissociated without hydrolysis. This highlights the advantages of LZMWs for single molecule observation of proteins that interact with cytoskeletal filaments such as microtubules, myosin filaments, or intermediate filaments.
- Sex-specific influence of the vacuolar adenosine triphosphatase a2 isoform on outcome in twin pregnancies. [Journal Article]
- AJAm J Reprod Immunol 2018 Nov 12; :e13071
- CONCLUSIONS: We conclude that the a2V-mediated regulation of maternal immunity during twin pregnancies is influenced by fetal sex. This article is protected by copyright. All rights reserved.
- An Update On The Neurochemistry Of Essential Tremor. [Journal Article]
- CMCurr Med Chem 2018 Nov 11
- CONCLUSIONS: The most consistent data regarding neurochemistry of ET are related with the GABAergic and glutamatergic systems, with a lesser contribution of adenosine and dopaminergic and adrenergic systems, while there is not enough evidence of a definite role of other neurotransmitter systems in ET. The improvement of harmaline-induced tremor in rodent models achieved with T-type calcium channel antagonists, cannabinoid 1 receptor, sphingosine-1-phosphate receptor agonists, and gap-junction blockers, suggests a potential role of these structures in the pathogenesis of ET.
- Enzymatic Reaction Generates Biomimic Nanominerals with Superior Bioactivity. [Journal Article]
- SSmall 2018 Nov 12; :e1804321
- In vivo mineralization is a multistep process involving mineral-protein complexes and various metastable compounds in vertebrates. In this complex process, the minerals produced in the mitochondrial ...
In vivo mineralization is a multistep process involving mineral-protein complexes and various metastable compounds in vertebrates. In this complex process, the minerals produced in the mitochondrial matrix play a critical role in initiating extracellular mineralization. However, the functional mechanisms of the mitochondrial minerals are still a mystery. Herein, an in vitro enzymatic reaction strategy is reported for the generation of biomimic amorphous calcium phosphate (EACP) nanominerals by an alkaline phosphatase (ALP)-catalyzed hydrolysis of adenosine triphosphate (ATP) in a weakly alkalescent aqueous condition (pH 8.0-8.5), which is partially similar to the mitochondrial environment. Significantly, the EACP nanomineral obviously promotes autophagy and osteogenic differentiation of human bone marrow-derived mesenchymal stem cells by activating an AMPK related pathway, and displays a high performance in promoting bone regeneration. These results provide in vitro evidence for the effect of ATP on the formation and stabilization of the mineral in the mineralization process, demonstrating a potential strategy for the preparation of the biomimic mineral for treating bone related diseases.
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- Ecto-5'-nucleotidase (CD73) is a potential target of hepatocellular carcinoma. [Journal Article]
- JCJ Cell Physiol 2018 Nov 11
- High expression of ecto-5'-nucleotidase (CD73) has been reported in a number of epithelium origin malignancies. Here, we hypothesize that CD73 promotes hepatocellular carcinoma (HCC) growth and metas...
High expression of ecto-5'-nucleotidase (CD73) has been reported in a number of epithelium origin malignancies. Here, we hypothesize that CD73 promotes hepatocellular carcinoma (HCC) growth and metastasis and that the effect is mediated by epithelial growth factor receptor (EGFR). HCC cells with different malignancies and Tissue microarrays of the tumor and peritumoral liver tissues from 30 independent patients were used to examine CD73 and EGFR expression. Then, MTT and Ki67 detection, together with cell adhesion, invasion, and migration assays were used to evaluate the effects of CD73 on cell growth and metastasis. The expression of EGFR in HCC cells was also tested after suppressing or overexpressing CD73. Lastly, tumor tissues from nude mice, which had been injected subcutaneously with HCC cells, were transplanted subcutaneously into CD73-/- and wild-type (WT) C57 mice. CD73 expression was higher in HCC cells with greater metastatic potentials and tumor tissues compared with low metastatic cells and peritumor tissues. CD73 and EGFR were coexpressed and positively correlated in tumor and peritumor liver tissues in HCC tissue microarrays. Up-regulationof CD73 by plasmid transfection or by pharmacological agents promoted EGFR expression in HCC cells, whereas suppression of CD73 inhibited these effects. The growth of transplanted tumor tissues was dramatically slower in CD73-/- mice than in WT type mice in the in vivo experiments. CD73 promotes HCC growth and metastasis and upregulated the expression of EGFR in HCC. Thus, CD73 and EGFR are potential targets in the treatment of HCC.