subcutaneous-angiotensin-ii-infusion-using-osmotic-pumps-induces

Subcutaneous Angiotensin II Infusion using Osmotic Pumps Induces Aortic Aneurysms in Mice

Osmotic pumps continuously deliver compounds at a constant rate into small animals.  This article introduces a standard protocol used to induce aortic ...

University of Kentucky

Heme oxygenase catalyzes the first and rate-controlling step of heme catabolism. Induction of heme oxygenase-1 can be caused by numerous factors, including heme, other metalloporphyrins, transition metal ions, heat shock, ultraviolet light, phorbol esters, sodium arsenite, and phenylarsine oxide (PAO). Induction of this enzyme may protect cells from oxidative damage. Using heme oxygenase-1 promoter/reporter gene constructs, we have previously reported that the sodium arsenite-mediated induction of heme oxygenase-1 in chick embryo liver cells and chicken hepatoma (LMH) cells involves an AP-1 element. We have now investigated whether the PAO-mediated induction of heme oxygenase-1 also involves an AP-1 element. Primary cultures of chick embryo liver cells were transiently transfected with heme oxygenase-1 promoter/reporter gene constructs, treated with PAO, and reporter gene activities were measured. We found that the PAO-mediated increase in reporter gene activity was dose- and time-dependent. This activity was decreased by prior treatment with N-acetylcysteine. Studies with mutated constructs showed that both an AP-1 element and a metal responsive element are involved in the PAO-mediated induction of the heme oxygenase-1 reporter construct. Electrophoretic mobility shift assays showed that nuclear proteins from PAO-treated cells had increased binding to an AP-1 probe, and that this increase was abrogated by N-acetylcysteine. These findings support the hypothesis that the PAO-mediated induction of heme oxygenase-1 is caused by activation of AP-1 and MRE/cMyc elements and may involve nuclear proteins whose states of phosphorylation determine binding to regulatory elements, and thus the level of expression of heme oxygenase-1.

Effects of phenylarsine oxide on expression of heme oxygenase-1 reporter constructs in transiently transfected cultures of chick embryo liver cells.

Compared to simulated moving bed (SMB) chromatography, fractional crystallization is a simple and economical method for enantioseparation. Therefore, the coupling of SMB chromatography and fractional crystallization is suggested for enantioseparation of racemic compounds. In this work, a nonsteroidal antiinflammatory drug, ketoprofen (Kp), was chosen to be studied. Kp was verified as a racemic compound by FTIR, PXRD, and thermodynamic calculations. To derive the condition where pure (S)-Kp could be crystallized from a solution, which was previously enantiomerically enriched, the binary melting phase diagram and the ternary solubility phase diagram in the mixed solvent of ethanol and water over a temperature range of 15-30 degrees C were obtained. From these phase diagrams the eutectic point was determined as 91.6% mole percent (S)-Kp from the binary phase diagram and 91% from the ternary phase diagram. The results may provide valuable experiment data for the possibility of coupling fractional crystallization with SMB for Kp separation.

Physicochemical properties, binary and ternary phase diagrams of ketoprofen.

Heparin-induced thrombocytopenia with thrombosis (HITT) is the most severe side effect of heparin administration. Patients with HITT may die or have permanent sequelae such as stroke or limb amputation. Contaminated heparin is associated with anaphylactic reactions and deaths by activating the contact system. It is also associated with high incidence of HIT via a yet unknown mechanism. This study showed that although oversulfated heparin byproduct induced thrombin activities in both normal and HIT patient plasmas through the contact system activation, authentic heparin induced thrombin activities only in HIT patient plasmas containing autoantibodies against protein/ heparin complex. These data suggest that the negatively charged immunoglobulin G (IgG)/platelet factor 4 (PF4)/heparin complex activate the contact system and produce thrombin in human plasma, and thrombin partially activates the platelets allowing subsequent platelet activation through IgG/Fc receptor II signaling. The newly discovered mechanism of heparin-induced thrombin activity could explain the increased incidence of HIT in patients exposed to contaminated heparin. Furthermore, the assays used in these studies would be valuable for HIT diagnosis, prevention, and treatment.

Heparin and oversulfated heparin byproduct induce thrombin generation through contact system activation in plasma of patients with HIT.

Thrombin generation is thought to be mediated predominantly by the tissue factor or ''extrinsic'' coagulation pathway. An alternate pathway to thrombin generation (the ''intrinsic'' pathway or contact system) has been observed when blood or plasma comes in contact with artificial surfaces. Here we present evidence for a new route to thrombin formation that begins with the activation of the contact system protein prekallikrein by oversulfated heparin (OS-HB). Kallikrein, instead of activated factor X, cleaves prothrombin to form thrombin. Thrombin then cleaves fibrinogen to form fibrin clots. Moreover, we show that OS-HB by-products induce kallikrein- and thrombin-like activities in normal human plasma and in human plasma devoid of coagulation factor X or downstream contact system components factor IX or factor XI. Oversulfated heparin by-product-induced thrombin generation may have had a role in the adverse reactions associated with the recent clinical use of contaminated heparin.

Oversulfated heparin by-products induce thrombin generation in human plasmas through contact system activation.

Contaminated heparin-affected patient plasmas are associated with activated contact systems.

Diffusion tensor imaging (DTI) is a sensitive technique for studying cerebral white matter. Only a few studies have examined the association between changes in extratemporal integrity and cognition in temporal lobe epilepsy (TLE), especially in Chinese patients.

Changes in extratemporal integrity and cognition in temporal lobe epilepsy: a diffusion tensor imaging study.

We used diffusion tensor imaging to characterize microstructural changes and their associations with cognition in Chinese patients with frontal lobe epilepsy (FLE). We examined 18 adult patients with FLE and 20 healthy controls. Compared with normal controls, patients with FLE had increased mean diffusivity (MD) in the right frontal lobe and decreased fractional anisotropy (FA) in both thalami. Patients with FLE also had decreased FA in the right frontal lobe that correlated with patient age at seizure onset and increased MD in the left thalamus that correlated with duration of epilepsy. Patients with FLE performed significantly worse on nearly all cognitive tasks, and there was a positive correlation between Mini-Mental Status Examination scores and FA in the left frontal lobe and the left thalamus. Our results suggest that the thalamus might be an important extrafrontal structure involved in FLE and that a longer duration of epilepsy might result in more abnormalities in the thalamus. Our results also support the hypothesis that the left frontal lobe white matter and the thalamus contribute to cognitive impairment in patients with FLE.

Changes in extrafrontal integrity and cognition in frontal lobe epilepsy: a diffusion tensor imaging study.

Adenovirus serotype 5 (Ad5) vectors are well suited for gene therapy. However, tissue-selective transduction by systemically administered Ad5-based vectors is confounded by viral particle sequestration in the liver. Hexon-modified Ad5 expressing reporter gene under transcriptional control by the immediate/early cytomegalovirus (CMV) or the Roundabout 4 receptor (Robo4) enhancer/promoter was characterized by growth in cell culture, stability in vitro, gene transfer in the presence of human coagulation factor X, and biodistribution in mice. The obtained data demonstrate the utility of the Robo4 promoter in an Ad5 vector context. Substitution of the hypervariable region 7 (HVR7) of the Ad5 hexon with HVR7 from Ad serotype 3 resulted in decreased liver tropism and dramatically altered biodistribution of gene expression. The results of these studies suggest that the combination of liver detargeting using a genetic modification of hexon with an endothelium-specific transcriptional control element produces an additive effect in the improvement of Ad5 biodistribution.

Hong Lu

Saha Cardiovascular Research Center

University of Kentucky

Subcutaneous Angiotensin II Infusion using Osmotic Pumps Induces Aortic Aneurysms in Mice