Methodology To Probe Subunit Interactions in Ribonucleotide Reductases

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides, providing the monomeric precursors required for DNA replication and repair. <i>Escherichia coli</i> RNR is a 1:1 complex of two homodimeric subunits, α2 and β2. The interactions between α2 and β2 are thought to be largely associated with the C-terminal 20 amino acids (residues 356−375) of β2. To study subunit interactions, a single reactive cysteine has been introduced into each of 15 positions along the C-terminal tail of β2. Each cysteine has been modified with the photo-cross-linker benzophenone (BP) and the environmentally sensitive fluorophore dimethylaminonaphthalene (DAN). Each construct has been purified to homogeneity and characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS−PAGE) and electrospray ionization mass spectrometry (ESI-MS). Each BP-β2 has been incubated with 1 equiv of α2 and photolyzed, and the results have been analyzed quantitatively by SDS−PAGE. Each DAN-β2 was incubated with a 50-fold excess of α2, and the emission maximum and intensity were measured. A comparison of the results from the two sets of probes reveals that sites with the most extensive cross-linking are also associated with the greatest changes in fluorescence. Titration of four different DAN-β2 variants (351, 356, 365, and 367) with α2 gave a <i>K</i>_d ≈ 0.4 μM for subunit interaction. Disruption of the interaction of the α2–DAN-β2 complex is accompanied by a decrease in fluorescence intensity and can serve as a high-throughput screen for inhibitors of subunit interactions

The infarct volume measured by TTC staining.

<p>(<b>A</b>) TTC staining showed the infarct territory 24 hours after reperfusion in successfully reperfused mice (<b>a</b>) and mice with failed reperfusion (<b>b</b>). (<b>B</b>) Quantitative data of the infarct volume in heparin-treated mice, heparin-free mice with failed reperfusion and successfully reperfused heparin-free mice, suggesting that the infarct volume in heparin-free mice with failed reperfusion (n=4) was significantly larger than heparin-treated (n=6) and successfully reperfused heparin-free mice (n=4). Data were mean±SD, ***<i>p<0.001</i>, Bar= 2 mm.</p

Cortical collateral circulation after tMCAO detected by LSCI.

<p>Cortical collateral circulation opened after tMCAO in successfully reperfused heparin-free mice (<b>A</b>) and heparin-treated mice (<b>B</b>). LSCI before MCAO (<b>a</b>-<b>b</b>) showed a clear cortical vascular morphology. Collateral arteries opened at 10 (<b>c</b>) and 30 minutes (<b>d</b>) after reperfusion. Collateral arteries were missing at 10 (<b>e</b>) and 60 minutes (<b>f</b>) after reperfusion. Notably, collateral arteries opened again at 120 minutes after reperfusion (<b>g</b>). (<b>C</b>) Collateral circulation after tMCAO in heparin-free mice with failed reperfusion. LSCI before MCAO (<b>a</b>-<b>b</b>) showed a clear cortical vascular morphology. Collateral arteries opened at 10 (<b>c</b>) and 30 minutes (<b>d</b>) after suture withdrawal. Collateral arteries were still open at 10 (<b>e</b>), 60 (<b>f</b>) and 120 minutes (<b>g</b>) after suture withdrawal. Broken Circle indicates the selected areas of images <b>b</b> to <b>g</b>. Arrows indicate the collateral artery. Bar= 1 mm. A: anterior, L: left, LSCI: laser speckle contrast imaging, tMCAO: transient middle cerebral artery occlusion.</p

Thrombosis in vascular after reperfusion detected by HE-staining.

<p>(<b>A</b>) Stereomicroscopic imaging of brain was performed after 1 to 3 hours after reperfusion. No blood clotting was detected in the Circus Willis in successfully reperfused mice (<b>a</b>, <b>c</b>) while blood clotting was clearly detected in MCA, ACA and ICA territory in heparin-free mice with failed reperfusion (<b>b</b>, <b>d</b>). Arrowheads indicate thrombus in the MCA, ACA and ICA. (<b>B</b>) Schematic diagram of paraffin section indicating the location of ACA, MCA and ICA. (<b>C</b>) HE-staining shows the cross-sections of ICA (<b>a</b>), MCA (<b>b</b>) and ACA (<b>c</b>) in successfully reperfused mice with no thrombus in the lumen after reperfusion. In contrast, the cross-sections of ICA (<b>d</b>), MCA (<b>e</b>) and ACA (<b>f</b>) in mice with failed reperfusion have mixed thrombus in the lumen after suture withdrawal. Bar= 100 µm. ACA: anterior cerebral artery, ICA: internal carotid artery, MCA: middle cerebral artery.</p

Mouse cerebral vascular morphology after tMCAO and at various time after reperfusion detected by SRA.

<p>SRA after 90 minutes of MCAO and at 0 (<b>a</b>), 1 (<b>b</b>) and 3 hours (<b>c</b>) after reperfusion in heparin-treated mice shows that the CCA, ICA, PPA, PCA, MCA and ACA could be clearly detected. In contrast, SRA after 90 minutes of MCAO and at 0 (<b>d</b>), 1 (<b>e</b>) and 3 hours (<b>f</b>) after suture withdrawal in heparin-free mice shows that the CCA, ICA, PPA and PCA could be clearly detected while the MCA and ACA could only be partially detected (<b>d</b>) or undetected (<b>e</b>, <b>f</b>). Arrows indicate that MCA was well filled with contrast agent Ipamiro. Black arrowheads indicate the location of MCA, which was undetectable. White arrowhead indicate the location of MCA, which was partially detected. Bar= 1 mm. ACA: anterior cerebral artery, CCA: common carotid artery, ICA: internal carotid artery, MCAO: middle cerebral artery occlusion, PPA: pterygopalatine artery, PCA: posterior cerebral artery, SRA: synchrotron radiation microangiography.</p

Mouse cerebral vascular morphology imaged by SRA and blood flow measured by LSCI.

<p>(<b>A</b>) Cerebral vascular morphology imaged by SRA. (<b>a</b>) cerebral vascular morphology was obtained before middle cerebral artery occlusion (MCAO) in adult mice. CCA, ICA, PPA, PCA, MCA and ACA as well as their branches could be clearly detected. (<b>b</b>) SRA after MCAO showed that CCA, ICA, PPA and PCA could be detected while MCA was missing, suggesting that MCA was occluded. (<b>c</b>) SRA was performed 1 hour after reperfusion in heparin-treated mice, which showed CCA, ICA, PPA, PCA and MCA were well filled with contrast agent Ipamiro. (<b>d</b>) SRA was performed 1 hour after reperfusion in the heparin-free mice, which showed CCA, ICA, PPA and PCA could be detected while MCA was still undetectable. (<b>B</b>) CBF was measured by LSCI. (<b>a</b>) LSCI was recorded in normal mice showing that the CBF of ipsilateral hemisphere was normal, similar to that of the contralateral. (<b>b</b>) LSCI was recorded after MCAO showing that the CBF of the ipsilateral hemisphere decreased to 10% of the baseline. (<b>c</b>) LSCI was recorded 1 hour after reperfusion in heparin-treated mice showing that the CBF recovered to the baseline. (<b>d</b>) LSCI recorded 1 hour after reperfusion in the heparin-free mice showing that the CBF was still low. Arrows indicate that MCA was well filled with blood. Arrowheads indicate the location of MCA, which was undetectable. Broken Circle indicated the MCA territory in the cortex of ipsilateral hemisphere. Bar= 1 mm. Color bar: normalized relative CBF speed (0%~100%). ACA: anterior cerebral artery, CBF: cerebral blood flow, CCA: common carotid artery, ICA: internal carotid artery, LSCI: laser speckle contrast imaging, PCA: posterior cerebral artery, PPA: pterygopalatine artery, SRA: synchrotron radiation microangiography.</p

The neurological score at 24 hours after tMCAO.

<p>Quantitative data showed that the neurological score in heparin-free mice with failed reperfusion (n=4) was significantly higher than heparin-treated (n=6) and successfully reperfused heparin-free mice (n=4). Data were mean±SD, ***<i>p<0.001</i>. </p

Chrysanthemum indicum L.

原著和名: シマカンギク科名: キク科 = Compositae採集地: 和歌山県 日高郡 日ノ御埼 (紀伊 日高郡 日ノ岬)採集日: 1978/11/20採集者: 萩庭丈壽整理番号: JH026213国立科学博物館整理番号: TNS-VS-97621

Therapeutic Benefit of Bone Marrow–Derived Endothelial Progenitor Cell Transplantation after Experimental Aneurysm Embolization with Coil in Rats

<div><p>Aneurysm embolization with coil is now widely used clinically. However, the recurrence of aneurysms after embolization has always plagued neurosurgeons because the endothelial layer of the aneurysm neck loses its integrity after being embolized by coil. Bone marrow–derived endothelial progenitor cells (BM-EPCs) could be incorporated into injured endothelium and differentiate into mature endothelial cells during vascular repairing processes. The aim of our study is to explore the effects of BM-EPCs on aneurysm repairing and remodeling in a rat embolization model of abdominal aortic aneurysm. BM-EPC proliferation, migration and tube formation were not affected by super-paramagnetic iron oxide nanoparticle (SPIO) labeling compared to the controls (<i>p</i>>0.05). The number of SPIO-labeled cells greatly increased in EPC transplanted rats compared to that of phosphate buffered saline treated rats. SPIO-labeled EPC (SPIO-EPC) are mainly located in the aneurysm neck and surrounded by fibrous tissue. A histology study showed that the aneurysm orifice was closed with neointima and the aneurysm was filled with newly formed fibrous tissue. The SPIO-EPC accumulated in the aneurysm neck, which accelerated focal fibrous tissue remodeling, suggesting that BM-EPCs play a crucial role in repairing and remodeling the aneurysm neck orifice.</p></div

BM-EPCs involved in aneuryam repairing.

<p>HE staining showing that the tissue repairation of aneurysm neck in EPC transplantation group (<b>b</b>) is better than in HUVEC transplantation group (<b>c</b>) and PBS control group (<b>a</b>). Prussian blue staining showing SPIO-labeled cells located in the aneurysm neck of all rats in the EPC transplantation group (<b>e</b>), in contrast to the HUVEC transplantation group (<b>f</b>) and control group (<b>d</b>). Bar = 100 µm.</p