Lack of protection from ischemic injury of monoamine oxidase B-deficient mice following middle cerebral artery occlusion

Recently, it has been shown that a single leucine-to-tyrosine mutation in the agonist binding domains of the homomerically expressed alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors GluR3 and GluR1 is sufficient to completely block receptor desensitization. In the present study we tested heteromeric subunit combinations of AMPA receptors to demonstrate that the block of desensitization afforded by this mutation is dominant in heteromeric subunit complexes containing the leucine-to-tyrosine mutation in at least one of the subunits. In addition, by comparing mutated, desensitization-deficient forms of unedited GluR1 and GluR1 edited at the Q/R-site of the ion pore we demonstrate that the desensitization properties of AMPA receptors are not linked to the editing state of the ion pore domain and thus are independent of the permeability properties of the ion channel.     

Neuronal and endothelial nitric oxide synthase isoforms: quantification of protein and mRNA in the normal rat penis

Nitric oxide synthase (NOS) is an important enzyme for erection. We evaluated the content of neuronal (nNOS) and endothelial (eNOS) isoforms and their mRNA in the penis and major pelvic ganglion (MPG) of adult male rats by Western and Northern blot analysis. The cerebellum was evaluated as a control. nNOS protein and its mRNA were detected in abundance in the MPG, cerebellum, pelvic urethra and within the crura of the penis. In contrast, the penile urethra, neurovascular bundle and the shaft of penis contained smaller amounts of this protein. eNOS protein was most abundant in the penile and pelvic parts of the urethra, whereas a moderate level was found in the penile shaft, crura, neurovascular bundle, MPG and cerebellum. Similarly eNOS mRNA was abundant in the penile and pelvic parts of the urethra, MPG and cerebellum. Penile shaft, crura and neurovascular bundle showed moderate amounts of eNOS mRNA. In conclusion, nNOS and its mRNA are most abundant in the MPG and crura of penis whereas eNOS is most abundant in the urethra and to a lesser extent present in the penis. Importantly eNOS protein and mRNA were demonstrated in the MPG, where eNOS function has to be studied.     

Primary right atrial synovial sarcoma manifesting as transient ischemic attacks

Primary tumors of the heart are rare and most of them benign. The majority of benign cardiac tumors are myxomas while almost all malignant cardiac tumors are sarcomas. We present a case of primary right atrial synovial sarcoma, a form of sarcoma particularly rare in the heart. The tumor manifested clinically as transient ischemic attacks probably related to a patent foramen ovale allowing paradoxical tumor embolization.     

Development of a high-yield expression and purification system for platelet factor 4

Heparin-induced thrombocytopenia (HIT) is an adverse drug reaction characterized by IgG antibodies bound to complexes of platelet factor 4 (PF4) and heparin. The majority of diagnostic tests for HIT rely on an exogenous source of PF4 to identify anti-PF4/heparin antibodies. These include the PF4-dependent enhanced serotonin release assay (PF4-SRA) among others. Using a bacterial expression system, we developed a novel and efficient method of producing recombinant human PF4 (rhPF4) that is biochemically and antigenically similar to platelet-derived human PF4. rhPF4 was produced using the pET expression system in the BL21(DE3) strain of Escherichia coli. The system was optimized for protein expression using isopropyl β-D-1-thiogalactopyranoside at different induction temperatures and incubation times. rhPF4 solubility was improved by using different detergents during cell lysis and by purifying with heparin affinity and ion exchange chromatography. Biochemical characteristics of rhPF4 were investigated using mass spectrometry, SDS-PAGE analysis, and gel filtration chromatography and compared to platelet-derived PF4. Antigenic and functional characteristics of rhPF4 were studied using the anti-PF4/heparin EIA and the PF4-SRA. Using this method, we could produce 11.4 ± 0.6 mg of pure rhPF4 per liter of bacterial culture. Absorbance readings from the anti-PF4/heparin EIA using platelet-derived and rhPF4 were highly correlated (n = 194; r = 0.9545, p < 0.0001); and functional release of serotonin in the PF4-SRA induced by anti-PF4/heparin antibodies was similar to either platelet-derived or rhPF4 and heparin (r = 0.9597, p < 0.0001). Our method of rhPF4 production is efficient and does not rely on a source of platelets. The rhPF4 purification method described produces greater yields at a lower cost than other current methods. The application of this method can improve the efficiency of biochemical investigations and HIT diagnostic testing by supplying sufficient amounts of PF4.     

Hypoxia-Inducible Factor-1α and its Role in the Proliferation of Retinoblastoma Cells

In order to better understand the role of HIF-1α in the proliferation of the retinoblastoma cells, a siRNA knockdown of HIF-1α followed by a proliferation assay was performed. Further sequencing was then carried out in order to assess knockdown efficiency and expression of HIF-1α. Upregulation of HIF-1α gene expression in CoCl₂-treated retinoblastoma cells was demonstrated via melting curve analysis from PCR tests and was further analyzed using western blot and densitometry analysis. Reduction of HIF-1α expression in retinoblastoma, post HIF-1α knockdown, was observed after siRNA transfection into Y-79 cells. Knockdown of HIF-1α resulted in a significant decrease in proliferation thereby demonstrating that HIF-1α is involved in promoting survival and proliferation in retinoblastoma cells. Stabilization of HIF-1α in retinoblastoma cells using CoCl₂ was unsuccessful.