Kenichi Fujise

Preferential Interaction of Sentrin with a Ubiquitin-conjugating Enzyme, Ubc9

Sentrin is a ubiquitin-like molecule that has been shown to interact with the death domains of Fas and tumor necrosis factor receptor 1 (TNFR1), PML, Rad51, Rad52, and RanGAP1. We have reported previously that sentrin can be conjugated to other proteins in a manner analogous to protein ubiquitination (Kamitani, T., Nguyen, H. P., and Yeh, E. T. H. (1997) J. Biol. Chem. 272, 14001–14004). Furthermore, the conserved C-terminal Gly-Gly residues are required for sentrinization to occur. To identify enzymes which play a role in sentrinization, the yeast two-hybrid system was used to screen a human placenta cDNA library using sentrin as bait. A strong positive interacting clone was found to contain a cDNA insert encoding the ubiquitin-conjugating enzyme, Ubc9. The interaction between sentrin and Ubc9 required the ubiquitin domain and the C-terminal Gly-Gly residues of sentrin. This interaction appears to be specific because sentrin could only interact weakly with UbcH5B, but could not interact with HHR6B, UbcH6 nor E2-EPF. In vitro translated sentrin could be precipitated by a GST-Ubc9 fusion protein, but not by glutathione S-transferase. A β-mercaptoethanol-sensitive Ubc9-sentrin conjugate could also be identified in the in vitro binding assay. Substitution of the conserved cysteine residue of Ubc9 by serine abolished the formation of the Ubc9-sentrin conjugate. Taken together, Ubc9 is a strong candidate to be the key conjugating enzyme in the sentrinization pathway.

Regulation of Apoptosis and Cell Cycle Progression by MCL1 DIFFERENTIAL ROLE OF PROLIFERATING CELL NUCLEAR ANTIGEN

MCL1 (ML1 myeloid cellleukemia 1), a Bcl-2 (B-cell lymphoma-leukemia 2) homologue, is known to function as an anti-apoptotic protein. Here we show in vitro and in vivo that MCL1 interacts with the cell cycle regulator, proliferatingcell nuclear antigen (PCNA). This finding prompted us to investigate whether MCL1, in addition to its anti-apoptotic function, has an effect on cell cycle progression. A bromodeoxyuridine uptake assay showed that the overexpression of MCL1 significantly inhibited the cell cycle progression through the S-phase. The S-phase of the cell cycle is also known to be regulated by PCNA. A mutant of MCL1 that lacks PCNA binding (MCL1Δ 4A) could not inhibit cell cycle progression as effectively as wild type MCL1. In contrast, MCL1Δ 4A retained its anti-apoptotic function in HeLa cells when challenged by Etoposide. In addition, the intracellular localization of MCL1Δ 4A was identical to that of wild type MCL1. An in vitro pull-down assay suggested that MCL1 is the only Bcl-2 family protein to interact with PCNA. In fact, MCL1, not other Bcl-2 family proteins, contained the PCNA-binding motif described previously. Taken together, MCL1 is a regulator of both apoptosis and cell cycle progression, and the cell cycle regulatory function of MCL1 is mediated through its interaction with PCNA.

Intracoronary adenosine administered during rotational atherectomy of complex lesions in native coronary arteries reduces the incidence of no-reflow phenomenon.

Rotational atherectomy (RA) of complex, calcified lesions has been associated with a high incidence of no reflow ranging from 6%–15% and concomitant myocardial necrosis with adverse prognostic implications. There are no uniform strategies for preventing this complication. The role of intracoronary adenosine for the prevention of this phenomenon during RA has not been fully evaluated. We studied the procedural outcome of 122 patients who underwent RA of complex native coronary artery lesions. Fifty‐two patients received no adenosine but a variety of other agents. Seventy patients received intracoronary adenosine boluses (24 to 48 μg prior to and after each RA run). There was no difference in the type of lesion studied, run time, or Burr to artery ratio (0.6–0.7) between the two groups. Six patients without adenosine experienced no reflow (11.6%), with resultant infarction in the target artery territory, while only 1 of 70 patients (1.4%, P − 0.023) in the adenosine group experienced no reflow. No untoward complications were observed during adenosine infusion. Intracoronary adenosine bolus administered during rotational atherectomy is easy, safe, and may significantly reduce the incidence of no reflow, which may improve the 30‐day outcome of this procedure. Cathet. Cardiovasc. Intervent. 48:275–278, 1999.

Vascular access site complications with the use of closure devices in patients treated with platelet glycoprotein IIb/IIIa inhibitors during rescue angioplasty

The objective of this study was to evaluate the effectiveness of two different closure devices in patients undergoing rescue percutaneous coronary intervention (PCI) using IIb/IIIa inhibitors and to compare it with patients undergoing elective PCI. One hundred sixty‐two patients undergoing rescue PCI treated with IIb/IIIa inhibitors underwent vascular access site closure (6 Fr Perclose, n = 92, or 6 Fr Angioseal, n = 70). Vascular complications were compared with a sex‐ and age‐matched group (n = 100) of patients undergoing manual compression after sheath removal and a similar group of patients undergoing elective PCI (n = 196). The incidence of access site complications was not significantly different between the three groups undergoing rescue PCI and was not higher than in patient receiving GP IIb/IIIa inhibitors without fibrinolysis (RR = 0.95; 95% CI = 0.88–1.01). In patients undergoing rescue PCI and receiving IIb/IIIa inhibitors, closure devices allow early sheath removal and are associated with similar outcomes compared with manual compression and elective PCI regardless of the type of closure device used. Catheter Cardiovasc Interv 2004;63:284–289.

A tissue plasminogen activator/P-selectin fusion protein is an effective thrombolytic agent.

BACKGROUND P-selectin is expressed on the surface of activated endothelial cells and platelets. We hypothesized that a tissue plasminogen activator (TPA)/P-selectin fusion protein would have not only thrombolytic activity but also might target TPA to the thrombi. In addition, it seemed possible that this chimeric protein would competitively inhibit the binding of native P-selectin on endothelial cells and platelets to leukocytes and thus further promote thrombolysis. METHODS AND RESULTS The full-length, plasminogen activator inhibitor-1-resistant form of TPA (TPAIR) together with two TPAIR/P-selectin fusion constructs (P280IR and P121IR) were expressed with the use of baculovirus vectors. After infection of Sf21 cells with the recombinant baculovirus, recombinant TPAIR and P-selectin/TPAIR fusion proteins were purified with the use of metal ion chromatography. The intact protease activity of TPAIR and the ligand binding capability of P-selectin were confirmed through indirect chromogenic and cell binding assays, respectively. These molecules were assessed both in vitro and in vivo for thrombolytic activity. In vitro clot lysis assays indicated equal efficacy of TPAIR, P280IR, and P121IR (P > .5). The in vivo efficacy was tested in a cyclic flow variation model with the use of the rat mesenteric artery. Compared with saline control treatment, reduction in cyclic flow variations was significant (P < .05) and similar (P > .5) among TPAIR, P280IR, and P121IR. No significant bleeding was noted among treated animals. CONCLUSIONS Chimeric proteins P280IR and P121IR have clot lysis activities that are similar to TPAIR both in vitro and in vivo. These chimeric proteins also bind to P-selectin ligand in vitro. Thus, these proteins may provide an efficient method of targeting TPA to the thrombotic region. Further experimental analysis with the use of larger animal coronary occlusion models should help determine the future value of these proteins as clinical therapeutic agents.

Differential Effects of Endothelin Receptor Activation on Cyclic Flow Variations in Rat Mesenteric Arteries

BACKGROUND Cyclic flow variations (CFVs) represent repetitive cycles of platelet adherence-aggregation and vasoconstriction, followed by dislodgment of platelet thrombi and restoration of blood flow at the site of vascular injury. Although activation of endothelin A (ETA) and endothelin B (ETB) receptors leads to vasoconstriction and nitric oxide release, respectively, the roles of endogenous endothelin-1 (ET-1) and its receptors in CFVs are unknown. METHODS AND RESULTS A side branch of a mesenteric artery of male Wistar rats was cannulated and a short segment of the artery was mechanically injured to induce CFVs. After 20 minutes of saline infusion, either saline (negative control), BQ-123 (ETA receptor antagonist, 10 microg/min), BQ-788 (ETB receptor antagonist, 10 microg/min), or sarafotoxin S6c (ETB receptor agonist, 10 ng/min) was infused for 20 minutes from the side branch into the injured arterial segment. Percent (%) luminal stenosis as well as proximal and distal vessel diameters were observed and quantitatively measured every minute using intravital video microscopy and a micrometer-calibrated video screen. Both BQ-123 and sarafotoxin S6c significantly reduced CFVs represented by the mean luminal stenosis (BQ-123=29+/-13% and sarafotoxin S6c=27+/-11% reduction, respectively; P<.05 for both, compared with saline). In contrast, BQ-788 significantly increased CFVs (33+/-6% increase, P<.05 compared with saline). Moreover, the inhibitory effect of sarafotoxin S6c on CFVs was completely abolished in the presence of N(omega)-nitro-L-arginine methyl ester (L-NAME) (a nitric oxide synthase inhibitor, 10(-5) mol/L) in superfusate over the arteries (16.1+/-5% increase, P=NS compared with saline in the presence of L-NAME). In addition, BQ-123 caused a significant increase in the diameter of the vessel distal to the injured segment (12+/-4% increase, P<.05 compared with saline). CONCLUSIONS Endogenous ET-1 release from sites of vascular injury contributes to CFVs and vasomotor tone in the rat mesenteric artery CFV model. ETA and ETB receptors have differential roles in CFVs: ETA receptor antagonism and ETB receptor stimulation reduce CFVs, the latter at least partially through increased nitric oxide formation.