Activated protein C (APC) is a systemic anti-coagulant and anti-inflammatory factor. It reduces organ damage in animal models of sepsis, ischemic injury and stroke and substantially reduces mortality in patients with severe sepsis. It was not known whether APC acts as a direct cell survival factor or whether its neuroprotective effect is secondary to its anti-coagulant and anti-inflammatory effects. We report that APC directly prevents apoptosis in hypoxic human brain endothelium through transcriptionally dependent inhibition of tumor suppressor protein p53, normalization of the pro-apoptotic Bax/Bcl-2 ratio and reduction of caspase-3 signaling. These mechanisms are distinct from those involving upregulation of the genes encoding the anti-apoptotic Bcl-2 homolog A1 and inhibitor of apoptosis protein-1 (IAP-1) by APC in umbilical vein endothelial cells. Cytoprotection of brain endothelium by APC in vitro required endothelial protein C receptor (EPCR) and protease-activated receptor-1 (PAR-1), as did its in vivo neuroprotective activity in a stroke model of mice with a severe deficiency of EPCR. This is consistent with work showing the direct effects of APC on cultured cells via EPCR and PAR-1 (ref. 9). Moreover, the in vivo neuroprotective effects of low-dose mouse APC seemed to be independent of its anti-coagulant activity. Thus, APC protects the brain from ischemic injury by acting directly on brain cells.

Activated protein C (APC), a serine protease with anticoagulant and anti-inflammatory activities, exerts direct cytoprotective effects on endothelium via endothelial protein C receptor-dependent activation of protease activated receptor 1 (PAR1). Here, we report that APC protects mouse cortical neurons from two divergent inducers of apoptosis, N-methyl-D-aspartate (NMDA) and staurosporine. APC blocked several steps in NMDA-induced apoptosis downstream to nitric oxide, i.e., caspase-3 activation, nuclear translocation of apoptosis-inducing factor (AIF), and induction of p53, and prevented staurosporine-induced apoptosis by blocking caspase-8 activation upstream of caspase-3 activation and AIF nuclear translocation. Intracerebral APC infusion dose dependently reduced NMDA excitotoxicity in mice. By using different anti-PARs antibodies and mice with single PAR1, PAR3, or PAR4 deletion, we demonstrated that direct neuronal protective effects of APC in vitro and in vivo require PAR1 and PAR3. Thus, PAR1 and PAR3 mediate anti-apoptotic signaling by APC in neurons, which may suggest novel treatments for neurodegenerative disorders.

Brain hemorrhage is a serious complication of tissue plasminogen activator (tPA) therapy for ischemic stroke. Here we report that activated protein C (APC), a plasma serine protease with systemic anticoagulant, anti-inflammatory and antiapoptotic activities, and direct vasculoprotective and neuroprotective activities, blocks tPA-mediated brain hemorrhage after transient brain ischemia and embolic stroke in rodents. We show that APC inhibits a pro-hemorrhagic tPA-induced, NF-kappaB-dependent matrix metalloproteinase-9 pathway in ischemic brain endothelium in vivo and in vitro by acting through protease-activated receptor 1. The present findings suggest that APC may improve thrombolytic therapy for stroke, in part, by reducing tPA-mediated hemorrhage.

Although thrombolytic effects of tissue plasminogen activator (tPA) are beneficial, its neurotoxicity is problematic. Here, we report that tPA potentiates apoptosis in ischemic human brain endothelium and in mouse cortical neurons treated with N-methyl-D-aspartate (NMDA) by shifting the apoptotic pathways from caspase-9 to caspase-8, which directly activates caspase-3 without amplification through the Bid-mediated mitochondrial pathway. In vivo, tPA-induced cerebral ischemic injury in mice was reduced by intracerebroventricular administration of caspase-8 inhibitor, but not by caspase-9 inhibitor, in contrast to controls in which caspase-9 inhibitor, but not caspase-8 inhibitor, was protective. Activated protein C (APC), a serine protease with anticoagulant, anti-inflammatory and antiapoptotic activities, which is neuroprotective during transient ischemia and promotes activation of antiapoptotic mechanisms in brain cells by acting directly on endothelium and neurons, blocked tPA vascular and neuronal toxicities in vitro and in vivo. APC inhibited tPA-induced caspase-8 activation of caspase-3 in endothelium and caspase-3-dependent nuclear translocation of apoptosis-inducing factor in NMDA-treated neurons and reduced tPA-mediated cerebral ischemic injury in mice. Data suggest that tPA shifts the apoptotic signal in stressed brain cells from the intrinsic to the extrinsic pathway which requires caspase-8. APC blocks tPA's neurovascular toxicity and may add substantially to the effectiveness of tPA therapy for stroke.

L-type Ca2؉ channels in native tissues have been found to contain a pore-forming ␣ 1 subunit that is often truncated at the C terminus. However, the C terminus contains many important domains that regulate channel function. To test the hypothesis that C-terminal fragments may associate with and regulate C-terminaltruncated ␣ 1C (Ca V 1.2) subunits, we performed electrophysiological and biochemical experiments. The voltage-activated L-type Ca 2ϩ channels are heteromeric proteins minimally composed of a pore-forming ␣ 1 subunit and accessory ␣ 2 ␦ and ␤ subunits (1, 2). Each ␣ 1 subunit contains four repeated domains containing a total of 24 membranespanning domains as well as a long hydrophilic C terminus which contains important regulatory domains that contribute to channel regulation. For example, the C terminus of the ␣ 1C subunit constitutes ϳ30% of the total mass of the ␣ 1C subunit (3) and is critical for membrane targeting of the channels (4), the regulation of the channels by protein phosphorylation (5), and the binding of Ca 2ϩ -binding proteins such as calmodulin and sorcin (6 -8). In addition, the C terminus of ␣ 1C appears to contain inhibitory domains because deletion of up to ϳ70% of the C-terminal 665 amino acids leads to increased currents (9).A puzzling observation that has been made in several laboratories is that the C terminus of several L-type Ca 2ϩ channels appears to be truncated in many native tissues. For example, when the ␣ 1C subunit was isolated from cardiac myocytes, only 10 -15% of the total protein was a full-length 240-kDa ␣ 1C subunit, whereas the majority migrated on SDS gels as a ϳ190-kDa protein that was lacking the distal ϳ50 kDa of the C terminus (10). Similar observations have been made for the ␣ 1C subunit expressed in brain (11) and the ␣ 1S subunit isolated from skeletal muscle (12,13). If the C termini of these proteins were truly absent, this would have major implications for channel regulation. Although many protease inhibitors have been used to try to prevent this truncation, none has altered the proportion of full-length to truncated protein (14). In marked contrast to what has been observed in native systems, fulllength ␣ 1C subunits have been observed in heterologous expression systems (5,15,16). Despite the fact that ϳ90% of the ␣ 1C

No abstract

Iprodione [3-(3,5-dichlorophenyl) -isopropyl-2,4-dioxoimidazolidine-1-carboxamide] is a highly effective broad-spectrum dicarboxamide fungicide. Several bacteria with iprodione-degrading capabilities have been reported; however, the enzymes and genes involved in this process have not been characterized. In this study, an iprodione-degrading strain, sp. strain YJN-5, was isolated and characterized. Strain YJN-5 degraded iprodione through the typical pathway, with hydrolysis of its N-1 amide bond to -(3,5-dichlorophenyl)-2,4-dioxoimidazolidine as the initial step. The gene, encoding a novel amidase responsible for this step, was cloned from strain YJN-5 by the shotgun method. IpaH shares the highest similarity (40%) with an indoleacetamide hydrolase (IAHH) from USDA 110. IpaH displayed maximal enzymatic activity at 35°C and pH 7.5, and it was not a metalloamidase. The and of IpaH against iprodione were 22.42 s and 7.33 μM, respectively, and the catalytic efficiency value ( ) was 3.09 μM s IpaH has a Ser-Ser-Lys motif, which is conserved among members of the amidase signature family. The replacement of Lys82, Ser157, and Ser181 with alanine in IpaH led to the complete loss of enzymatic activity. Furthermore, strain YJN-5M lost the ability to degrade iprodione, suggesting that is the only gene responsible for the initial iprodione degradation step. The gene could also be amplified from another previously reported iprodione-degrading strain, sp. strain YJN-G. The sequence similarity between the two IpaHs at the amino acid level was 98%, indicating that conservation of IpaH exists in different strains. Iprodione is a widely used dicarboxamide fungicide, and its residue has been frequently detected in the environment. The U.S. Environmental Protection Agency has classified iprodione as moderately toxic to small animals and a probable carcinogen to humans. Bacterial degradation of iprodione has been widely investigated. Previous studies demonstrate that hydrolysis of its N-1 amide bond is the initial step in the typical bacterial degradation pathway of iprodione; however, enzymes or genes involved in iprodione degradation have yet to be reported. In this study, a novel gene encoding an amidase responsible for the initial degradation step of iprodione in sp. strain YJN-5 was cloned. In addition, the characteristics and key amino acid sites of IpaH were investigated. These findings enhance our understanding of the microbial degradation mechanism of iprodione.

Indoxyl sulfate (IS), a uremic toxin, causes chronic kidney disease (CKD) progression via its tubulotoxicity. After cellular uptake, IS directly induces apoptotic and necrotic cell death of tubular cells. Additionally, IS increases oxidative stress and decreases antioxidant capacity, which are associated with tubulointerstitial injury. Injured tubular cells are a major source of transforming growth factor-β1 (TGF-β1), which induces myofibroblast transition from residual renal cells in damaged kidney, recruits inflammatory cells and thereby promotes extracellular matrix deposition in renal fibrosis. Moreover, IS upregulates signal transducers and activators of transcription 3 phosphorylation, followed by increases in TGF-β1, monocyte chemotactic protein-1 and α-smooth muscle actin production, which participate in interstitial inflammation, renal fibrosis and, consequently, CKD progression. Clinically, higher serum IS levels are independently associated with renal function decline and predict all-cause mortality in CKD. The poor removal of serum IS in conventional hemodialysis is also significantly associated with all-cause mortality and heart failure incidence in end-stage renal disease patients. Scavenging the IS precursor by AST-120 can markedly reduce tubular IS staining that attenuates renal tubular injury, ameliorates IS-induced oxidative stress and rescues antioxidant glutathione activity in tubular epithelial cells, thereby providing a protective role against tubular injury and ultimately retarding renal function decline.