Inhibitory mechanisms of low concentrations of oxidized low-density lipoprotein on platelet aggregation

Summary The intracellular mechanisms underlying oxidized low-density lipoprotein (oxLDL)-signaling pathways in platelets are not yet completely understood. Therefore, the aim of this study was to further examine the effects of oxLDL in prevention of platelet aggregation. In this study, oxLDL concentration-dependently (40–120 g/ml) inhibited platelet aggregation in human platelet-rich plasma stimulated by agonists. Moreover, oxLDL (40 and 80 g/ml) markedly decreased the fluorescence intensity of platelet membranes tagged with diphenylhexatriene. Rapid phosphorylation of a protein of Mr 47,000 (P47), a marker of protein kinase C activation, was triggered by PDBu (150 nM). This phosphorylation was markedly inhibited by oxLDL (40 and 80 g/ml) in phosphorus-32-labeled platelets. In addition, oxLDL (40 and 80 g/ml) markedly increased levels of cyclic AMP and cyclic AMP-induced vasodilator-stimulated phosphoprotein (VASP) Ser¹⁵⁷ phosphorylation. The thrombin-evoked increase in pHi was inhibited in the presence of oxLDL (40 and 80 g/ml). These results indicate that the antiplatelet activity of oxLDL may involve the following pathways. (1) oxLDL may initially induce conformational changes in platelet membranes, leading to inhibition of the activation of protein kinase C, followed by inhibition of P47 protein phosphorylation, and intracellular Ca²⁺ mobilization. (2) oxLDL also activated formation of cyclic AMP and cyclic AMP-induced VASP Ser¹⁵⁷ phosphorylation, resulting in inhibition of the Na⁺/H⁺exchanger; this leads to reduced intracellular Ca²⁺ mobilization, and ultimately to inhibition of platelet aggregation. This study further provides new insights concerning the effects of low concentrations of oxLDL on platelet aggregation.     

Development of N,O-(carboxymethyl)chitosan/collagen matrixes as a wound dressing

In an attempt to accelerate wound healing by stimulating the recruitment of fibroblasts and improve the mechanical properties of collagen matrixes, N,O-(carboxymethyl)chitosan (NOCC) was incorporated into the backbone of a collagen (COL) matrix without or with chondroitin sulfate (CS) or an acellular dermal matrix (ADM). The result of a cell migration study demonstrated that the migration of fibroblasts was significantly enhanced by NOCC in a concentration-dependent manner. In the analysis with a dynamic mechanical analyzer, NOCC/CS/COL matrixes presented higher tensile strengths than did NOCC/ADM/COL matrixes. Skin fibroblasts cultured on the matrixes containing NOCC showed increased proliferation and secretion of three kinds of cytokines compared with the control. Results of the in vivo wound healing study showed that matrixes incorporating NOCC showed markedly enhanced wound healing compared with the control. Therefore, the above results clearly suggest that NOCC/COL matrixes containing CS or ADM can be potential wound dressings for clinical applications.     

Cdc7-Dbf4 phosphorylates MCM proteins via a docking site-mediated mechanism to promote S phase progression

Origins of DNA replication are licensed in G1 by recruiting the minichromosome maintenance (MCM) proteins to form a prereplicative complex (pre-RC). Prior to initiation of DNA synthesis from each origin, a preinitiation complex (pre-IC) containing Cdc45 and other proteins is formed. We report that Cdc7-Dbf4 protein kinase (DDK) promotes assembly of a stable Cdc45-MCM complex exclusively on chromatin in S phase. In this complex, Mcm4 is hyperphosphorylated. Studies in vitro using purified DDK and Mcm4 demonstrate that hyperphosphorylation occurs at the Mcm4 N terminus. However, the DDK substrate specificity is conferred by an adjacent DDK-docking domain (DDD), sufficient for facilitating efficient phosphorylation of artificial phosphoacceptors in cis. Genetic evidence suggests that phosphorylation of Mcm4 by DDK is important for timely S phase progression and for cell viability upon overproduction of Cdc45. We suggest that DDK docks on and phosphorylates MCM proteins at licensed origins to promote proper assembly of pre-IC.     

Heterogeneity of lotus rhizome starch granules as revealed by α-amylase degradation

Lotus (Nelumbo nucifera Gaertn.) rhizome starch granules have an elongated oval shape with the hilum located at one end. The morphologic characteristics were used as a direction anchor to study the heterogeneity of molecular organization of starch granules using microscopy before and after partial digestion by bacterial α-amylase (Bacillus sp.) The partially digested granule showed a single, big eroded hole at the end distant from the hilum. The enzyme-attacked end was revealed to be the loosely packed end and to be the weak point for enzyme hydrolysis. The α-amylase hydrolyzed the loosely packed central part of the granule faster than the densely packed periphery, and left an empty shell with a fish-bone-like tunnel inside. The periphery was more resistant to amylase hydrolysis and had strong birefringence. For the whole starch granule, the selectivity of α-amylase hydrolysis was low for the crystalline and amorphous regions and for amylose and amylopectin molecules. This study elucidated that the molecular organization of lotus rhizome starch granules was heterogeneous.     

Metabolic impairment induces oxidative stress, compromises inflammatory responses, and inactivates a key mitochondrial enzyme in microglia

Microglial activation, oxidative stress, and dysfunctions in mitochondria, including the reduction of cytochrome oxidase activity, have been implicated in neurodegeneration. The current experiments tested the effects of reducing cytochrome oxidase activity on the ability of microglia to respond to inflammatory insults. Inhibition of cytochrome oxidase by azide reduced oxygen consumption and increased reactive oxygen species (ROS) production but did not affect cell viability. Azide also attenuated microglial activation, as measured by nitric oxide (NO.) production in response to lipopolysaccharide (LPS). It is surprising that the inhibition of cytochrome oxidase also diminished the activity of the alpha-ketoglutarate dehydrogenase complex (KGDHC), a Krebs cycle enzyme. This reduction was exaggerated when the azide-treated microglia were also treated with LPS. The combination of the azide-stimulated ROS and LPS-induced NO. would likely cause peroxynitrite formation in microglia. Thus, the possibility that KGDHC was inactivated by peroxynitrite was tested. Peroxynitrite inhibited the activity of isolated KGDHC, nitrated tyrosine residues of all three KGDHC subunits, and reduced immunoreactivity to antibodies against two KGDHC components. Thus, our data suggest that inhibition of the mitochondrial respiratory chain diminishes aerobic energy metabolism, interferes with microglial inflammatory responses, and compromises mitochondrial function, including KGDHC activity, which is vulnerable to NO. and peroxynitrite that result from microglial activation. Thus, activation of metabolically compromised microglia can further diminish their oxidative capacity, creating a deleterious spiral that may contribute to neurodegeneration.     

H3 domain of syntaxin 1A inhibits KATP channels by its actions on the sulfonylurea receptor 1 nucleotide-binding folds-1 and -2

The ATP-sensitive potassium (K(ATP)) channel in pancreatic islet beta cells consists of four pore-forming (Kir6.2) subunits and four regulatory sulfonylurea receptor (SUR1) subunits. In beta cells, the K(ATP) channel links intracellular metabolism to the dynamic regulation of the cell membrane potential that triggers insulin secretion. Syntaxin 1A (Syn-1A) is a SNARE protein that not only plays a direct role in exocytosis, but also binds and modulates voltage-gated K(+) and Ca(2+) channels to fine tune exocytosis. We recently reported that wild type Syn-1A inhibits rat islet beta cell K(ATP) channels and binds both nucleotide-binding folds (NBF-1 and NBF-2) of SUR1. However, wild type Syn-1A inhibition of rat islet beta cell K(ATP) channels seems to be mediated primarily via NBF-1. During exocytosis, Syn-1A undergoes a conformational change from a closed form to an open form, which would fully expose its active domain, the C-terminal H3 domain. Here, we show that the constitutively open form Syn-1A mutant (L165A/E166A) has a similar affinity to NBF-1 and NBF-2 as wild type Syn-1A and was equally effective in inhibiting the K(ATP) channels of rat pancreatic beta cells and a cell line (BA8) stably expressing SUR1/Kir6.2. Although dialysis of NBF-1 into BA8 and islet beta cells effectively blocked wild type and open form Syn-1A inhibition of the K(ATP) current, NBF-2 was also effective in blocking the open form Syn-1A inhibition. This prompted us to examine the specific domains within Syn-1A that would mediate its action on the K(ATP) channels. The C-terminal H3 domain of Syn-1A (Syn-1A-H3), but not the N-terminal H(ABC) domain (Syn-1A-H(ABC)), binds the SUR1 protein of BA8 cells, causing an inhibition of K(ATP) currents, and this inhibition was mediated via both NBF-1 and NBF-2. It therefore appears that the H3 domain of Syn-1A is the putative domain, which binds SUR1, but its distinct actions on the NBFs may depend on the conformation of Syn-1A occurring during exocytosis.     

5-azacytidine alters TGF-beta and BMP signaling and induces maturation in articular chondrocytes

Maintenance of the articular surface depends on the function of articular chondrocytes (ACs) which produce matrix and are constrained from undergoing the maturation program seen in growth plate chondrocytes. Only during pathologic conditions, such as in osteoarthritis, are maturational constraints lost causing recapitulation of the process that occurs during endochondral ossification. With the aim of establishing a model to identify regulatory mechanisms that suppress AC hypertrophy, we examined the capability of 5-azacytidine (Aza) to have an impact on the maturational program of these cells. Primary ACs do not spontaneously express markers of maturation and are refractory to treatment by factors that normally regulate chondrocyte maturation. However, following exposure to Aza, ACs (i) were induced to express type X collagen (colX), Indian hedgehog, and alkaline phosphatase and (ii) showed altered colX and AP expression in response to bone morphogenetic protein-2 (BMP-2), transforming growth factor-beta (TGF-beta), and parathyroid hormone-related protein (PTHrP). Since Aza unmasked responsiveness of ACs to BMP-2 and TGF-beta, we examined the effect of Aza treatment on signaling via these pathways by assessing the expression of the TGF-beta Smads (2 and 3), the BMP-2 Smads (1 and 5), and the Smad2 and 3-degrading ubiquitin E3 ligase Smurf2. Aza-treated ACs displayed less Smad2 and 3 and increased Smad1, 5, and Smurf2 protein and showed a loss of TGF-beta signaling on the P3TP-luciferase reporter. Suggesting that Aza-induction of Smurf2 may be responsible for the loss of Smad2 and 3 protein via this pathway, immunoprecipitation and metabolic labeling experiments confirmed that Aza accelerated the ubiquitination and degradation of these targets. Overall, Aza-treated ACs represent a novel model for the study of mechanisms that regulate maturational potential of articular cartilage, with the data suggesting that maturation of these cells may be due to up-regulation of Smad1 and 5 coupled with a Smurf2-dependent degradation of Smad2 and 3 and loss of TGF-beta signaling.     

Graves' disease presented as painful goiter

Pain in the thyroid gland is rarely present in Graves' disease. We describe a 32-year-old female hyperthyroid Graves' disease patient with an initial manifestation of painful goiter. On physical examination, the thyroid gland was diffusely enlarged and tender. The laboratory examinations showed high serum thyroid hormone and low thyrotropin values. Serum inflammatory markers, including C-reactive protein and erythrocyte sedimentation rate, were elevated. Thyroid ultrasound revealed multiple focal hypoechoic areas. All these findings gave an initial impression of an acute inflammatory and destructive process in the thyroid gland. However, subsequent thyroid scintigraphy demonstrated a diffuse radioactive iodide uptake pattern with positive serum thyrotropin receptor antibodies. Fine-needle aspiration cytology showed only the presence of lymphocytes. She was diagnosed as having Graves' disease and was treated with propylthiouracil, and prednisolone was given for neck pain. Within a few days, the thyroid tenderness dramatically improved, and the erythrocyte sedimentation rate progressively normalized. However, follow-up thyroid function tests still showed high serum thyroid hormone levels. The possible etiologies of a painful thyroid gland in Graves' disease will be discussed.     

Alterations in Ca2+-channels during the development of diabetic cardiomyopathy

In order to examine the status of Ca²⁺ channels in heart sarcolemma during the development of diabetes, rats were injected intravenously with 65 mg/kg streptozotocin and hearts were removed 1, 3 and 8 weeks later. Crude membranes from the ventricular muscle were prepared and the specific binding of ³H-nitrendipine was studied by employing different concentrations of this Ca ²⁺-antagonist. A significant decrease in both dissociation constant and maximal number of 3H-nitrendipine binding was observed in 3 and 8 weeks diabetic preparations. No such alterations were evident in diabetic brain membranes. Treatment of diabetic animals with insulin prevented the occurrence of these changes in the myocardium. The altered ³H-nitrendipine binding characteristics in diabetic heart membranes may not be due to the high levels of circulating catecholamines in this experimental model because no such changes were seen upon injecting a high dose (40 mg/kg) of isoproterenol in rats for 24 hr. The reduced number of ³H-nitrendipine binding sites may decrease Ca²⁺-influx through voltage sensitive Ca²⁺ channels and partly explain the depressed cardiac contractile force development in chronic diabetes whereas the increased affinity of Ca²⁺ channels may partly explain the increased sensitivity of diabetic heart to Ca ²⁺.