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Thrombin Activity Associated with Neuronal Damage during Acute Focal Ischemia
Mechanisms of ischemic neuronal and vascular injury remain obscure. Here we test the hypothesis that thrombin, a blood-borne coagulation factor, contributes to neurovascular injury during acute focal ischemia. Stroke was induced in adult Sprague Dawley rats by occluding the middle cerebral artery. Intra-arterial thrombin infusion during ischemia significantly increased vascular disruption and cellular injury. Intravenous infusion of argatroban, a direct thrombin inhibitor, alleviated neurovascular injury. Immunostaining showed thrombin on neurons in the ischemic core.
Using an activatable cell-penetrating peptide engineered to detect thrombin activity, we discovered that thrombin proteolytic activity was specifically associated with neuronal damage during ischemia. Protease activated receptor-1, the presumptive thrombin receptor, appeared to mediate ischemic neurovascular injury. Furthermore, rats receiving thrombin during ischemia showed cognitive deficit, whereas rats receiving argatroban retained intact learning and memory. These results suggest a potential role for thrombin contributing to neurovascular injury and several potential avenues for neuroprotection.
The Journal of Neuroscience, 30 May 2012, 32(22): 7622-7631; doi: 10.1523/JNEUROSCI.0369-12.2012
PubMed citation
Articles by Chen, B.
Articles by Lyden, P. D.
DNA–enzyme conjugate with a weak inhibitor that can specifically detect thrombin in a homogeneous medium
Abstract
We present the DNA-assisted control of enzymatic activity for the detection of a target protein using a new type of DNA–enzyme conjugate. The conjugate is composed of an enzyme inhibitor to regulate enzyme activity and a DNA aptamer to be responsive toward the analyte protein. Glutathione S-transferase (GST) and thrombin were selected as a model enzyme and an analyte protein. A hexahistidine tag was genetically attached to the C terminus of the GST, and the 5′ end of an oligonucleotide was conjugated with nitrilotriacetic acid (NTA) for the site-specific conjugation of the DNA with the GST based on a Ni2+ complex interaction. We found that fluorescein acted as a weak inhibitor of GST and succeeded in the regulation of GST activity by increasing the local concentration of the weak inhibitor by the hybridization of a 3′-end fluorescein-modified DNA. The catalytic activity of the DNA aptamer–enzyme conjugate showed a dose-dependent response to thrombin, indicating that the GST activity was clearly recovered by the binding of the DNA aptamer to thrombin. The current system enables the sensitive and specific detection of thrombin simply by measuring the enzymatic activity in a homogeneous medium.
Analytical Biochemistry
Volume 414, Issue 1, 1 July 2011, Pages 103-108
We present the DNA-assisted control of enzymatic activity for the detection of a target protein using a new type of DNA–enzyme conjugate. The conjugate is composed of an enzyme inhibitor to regulate enzyme activity and a DNA aptamer to be responsive toward the analyte protein. Glutathione S-transferase (GST) and thrombin were selected as a model enzyme and an analyte protein. A hexahistidine tag was genetically attached to the C terminus of the GST, and the 5′ end of an oligonucleotide was conjugated with nitrilotriacetic acid (NTA) for the site-specific conjugation of the DNA with the GST based on a Ni2+ complex interaction. We found that fluorescein acted as a weak inhibitor of GST and succeeded in the regulation of GST activity by increasing the local concentration of the weak inhibitor by the hybridization of a 3′-end fluorescein-modified DNA. The catalytic activity of the DNA aptamer–enzyme conjugate showed a dose-dependent response to thrombin, indicating that the GST activity was clearly recovered by the binding of the DNA aptamer to thrombin. The current system enables the sensitive and specific detection of thrombin simply by measuring the enzymatic activity in a homogeneous medium.
Analytical Biochemistry
Volume 414, Issue 1, 1 July 2011, Pages 103-108
Researchers invent inkjet that prints out living skin
If you’ve ever seen the lesser-known Sam Raimi movie Darkman, you probably remember that the plot involved the main character, Dr. Westlake, trying to figure out a way to “print” liquid skin to help burn victims. Westlake never did figure out how to keep the synthetic skin from destabilizing past the 98 minute mark, but luckily, Wake Forest Instititute for Regenerative Medicine researchers seem to have mastered it, showing off their amazing skin printer that uses living cells instead of ink.
As the researcher note, “any loss of full-thickness skin of more than 4 cm in diameter will not heal by itself.” Enter their device, which allows a modified inkjet printer to produce reams of fresh skin which can be used to patch up victims of skin trauma. They’ve already tested it on mice, with extremely positive results.
How does the printer work? It has two heads: one dispenses skin cells mixed with a blood coagulent and type I collagen, and the other pumps out thrombin, which is another coagulate. Sprayed together, these chemicals create a reaction and form fibrin, which again helps to clot blood. On top of that, the printer then adds a layer of outer surface skin. Voila!
There’s still testing to be done — the next stage is on pigs, then human trials — but so far, this looks promising… if not to replace burned off skin, then at least to print out some life-like Halloween masks.
As the researcher note, “any loss of full-thickness skin of more than 4 cm in diameter will not heal by itself.” Enter their device, which allows a modified inkjet printer to produce reams of fresh skin which can be used to patch up victims of skin trauma. They’ve already tested it on mice, with extremely positive results.
How does the printer work? It has two heads: one dispenses skin cells mixed with a blood coagulent and type I collagen, and the other pumps out thrombin, which is another coagulate. Sprayed together, these chemicals create a reaction and form fibrin, which again helps to clot blood. On top of that, the printer then adds a layer of outer surface skin. Voila!
There’s still testing to be done — the next stage is on pigs, then human trials — but so far, this looks promising… if not to replace burned off skin, then at least to print out some life-like Halloween masks.
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