The RNA-dependent RNA polymerase VP1 of infectious pancreatic necrosis virus (IPNV) is a single polypeptide responsible for both viral RNA transcription and genome replication. Sequence analysis ...identifies IPNV VP1 as having an unusual active site topology. We have purified, crystallized and solved the structure of IPNV VP1 to 2.3 Å resolution in its apo form and at 2.2 Å resolution bound to the catalytically-activating metal magnesium. We find that recombinantly-expressed VP1 is highly active for RNA transcription and replication, yielding both free and polymerase-attached RNA products. IPNV VP1 also possesses terminal (deoxy)nucleotide transferase, RNA-dependent DNA polymerase (reverse transcriptase) and template-independent self-guanylylation activity. The N-terminus of VP1 interacts with the active-site cleft and we show that the N-terminal serine residue is required for formation of covalent RNA:polymerase complexes, providing a mechanism for the genesis of viral genome:polymerase complexes observed in vivo.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
This study examined the hypothesis that superficial femoral artery (SFA) subintimal angioplasty (SI-PTA) can maintain limb salvage with minimal complications in patients with symptomatic occlusive ...arterial disease.
From March 1, 2004, until April 28, 2006, 78 patients with rest pain (62.2%), gangrene (25.6%), or severe progressive claudication (12.2%) were treated consecutively with 82 SFA SI-PTAs (4 bilateral). The mean age was 59 ± 1.2 years, and 21 (27%) of the patients were female. All patients were treated in the operating room under local anesthesia by using fluoroscopic guidance, and the percentage SFA that was occluded was measured during the diagnostic portion of the procedure. Selective stent placement was performed after successful recanalization of the occluded arterial segments. Patients were treated with chronic aspirin and clopidogrel bisulfate for 3 months and followed up at 30 days and then every 3 months with physical examination and arterial duplex scan.
Of the 82 SFA SI-PTA attempts, 76 (92%) were initially successful, with an increase in the ankle-brachial index from 0.46 ± 0.02 to 0.88 ± 0.01 (P < .001). Five of the six patients with a failed SFA SI-PTA were female, two of the six had had previous bypass attempts, and one of the six had had a previous SFA SI-PTA attempt by another physician. Forty-nine (64%) of the 76 initially successful SFA SI-PTAs required placement of a stent, and 43 (56.5%) of the successful 76 SFA SI-PTAs required additional PTA of 1 or more arterial segments. The group treated with a successful SFA SI-PTA had 42.5% ± 3.5% SFA occlusion, compared with 82% ± 10% (P < .05) in the group with a failed attempt at SFA SI-PTA. Two of the six patients with initial SI-PTA failure underwent leg amputation within 30 days, three were treated with successful leg bypass surgery, and one was lost to follow-up. Of the 76 successful SFA SI-PTAs, 5 (6.5%) failed within 90 days, and the patients were treated successfully with leg bypass surgery. Of the 71 limbs with patent SI-PTAs at 90 days, 68 have remained patent with a mean follow-up 10.4 ± 0.7 months (range, 2-24 months). Three of the 71 SFA SI-PTAs failed between 4 and 7 months (mean, 5 ± 0.7 months): 1 patient was treated with successful bypass surgery, 1 patient is currently considering further intervention, and 1 patient was treated with amputation. Ten (14%) of the 71 successful SFA SI-PTAs required limited PTA for asymptomatic restenosis, as identified by the arterial duplex scan (7.4 ± 1.4 months; range, 2-16 months). There were no perioperative deaths, and three patients have died during follow-up with patent SFA SI-PTAs (9.3 ± 1.4 months).
These data suggest that SFA SI-PTA can be successfully used for limb salvage with minimal morbidity and mortality in a group of patients with severe lower extremity occlusive vascular disease.
The loss of renal function continues to be a frequent complication of the iodinated contrast agents used to perform diagnostic angiography and endovascular procedures. This study examined the ...hypothesis that contrast-induced renal injury is partly due to a decrease in cortical and medullary microvascular blood flow after the downregulation of endogenous renal cortical and medullary nitric oxide (NO) synthesis.
Anesthetized male Sprague-Dawley rats (300 g) had microdialysis probes or laser Doppler fibers inserted into the renal cortex to a depth of 2 mm and into the renal medulla to a depth of 4 mm. Laser Doppler blood flow was continuously monitored, and the microdialysis probes were connected to a syringe pump and perfused in vivo at 3 μL/min with lactated Ringer’s solution. Dialysate fluid was collected at time zero (basal) and 60 minutes after infusion of either saline or Conray 400 (6 mL/kg). Both groups were treated with saline carrier, N
ω-nitro-L-arginine methyl ester hydrochloride (L-NAME, 30 mg/kg), L-arginine (400 mg/kg), or superoxide dismutase (10,000 U/kg), an oxygen-derived free radical scavenger. Dialysate was analyzed for total NO and eicosanoid synthesis. The renal cortex and medulla were analyzed for inducible NO synthase (iNOS), cyclooxygenase-2 (COX2), prostacyclin synthase, and prostaglandin E
2 (PGE
2) synthase content by Western blot analysis.
Conray caused a marked decrease in cortical and medullary blood flow with a concomitant decrease in endogenous cortical NO, PGE
2, and medullary NO synthesis. The addition of L-NAME to the Conray further decreased cortical and medullary blood flow and NO synthesis, which were restored toward control by L-arginine. Neither L-NAME nor L-arginine (added to the Conray) altered cortical or medullary eicosanoids release. Medullary PGE
2 synthesis decreased when superoxide dismutase was added to the Conray treatment, suggesting that oxygen-derived free radicals had a protective role in maintaining endogenous medullary PGE
2 synthesis after Conray treatment. Conray did not significantly alter iNOS, COX-2, prostacyclin synthase, or PGE
2 synthase content.
These findings suggest that the downregulation of renal cortical and medullary NO synthesis contributes to the contrast-induced loss of renal cortical and medullary microvascular blood flow. Preservation of normal levels of renal cortical and medullary NO synthesis may help prevent or lessen contrast-induced renal vasoconstriction and lessen contrast-induced renal injury found after diagnostic and therapeutic endovascular procedures.
This study suggests that clinically relevant cortical and medullary vasodilators (NO and vasodilator prostanoids) are required to maintain microvascular renal cortical and medullary blood flow. This study combines in vivo techniques of microdialysis and laser Doppler flow probes to show that endogenous NO synthesis is important in serving a protective role to prevent vasoconstriction at the local cortical and medullary levels after radiocontrast administration. Understanding the mechanisms of contrast-induced nephropathy will help develop treatment regimens to either minimize or eliminate this important complication of contrast agents.
Objective Renal insufficiency continues to be a complication that can affect patients after treatment for suprarenal aneurysms and renal artery occlusive disease. To our knowledge, no data are ...available showing that suprarenal aortic clamping and reperfusion (SRACR) above the renal arteries (renal-SRACR) preserves renal function compared with SRACR above the superior mesenteric artery (SMA-SRACR). This study examined the hypothesis that SMA-SRACR–induced downregulation of renal blood flow and function is more severe than renal-SRACR owing to the addition of systemic oxygen-derived free radical (ODFR) release. Methods Male Sprague-Dawley rats (about 350 g) were anesthetized and microdialysis probes or laser Doppler fibers were inserted into the renal cortex (depth of 2 mm) and into the renal medulla (depth of 4 mm). Laser Doppler blood flow was continuously monitored, and the microdialysis probes were connected to a syringe pump and perfused in vivo at 3 μL/min with lactated Ringer’s solution. Results SMA-SRACR and Renal-SRACR decreased medullary and cortical blood flow and nitric oxide (NO) synthesis. SMA-SRACR downregulated cortical inducible NO synthase, whereas renal-SRACR did not. The cortex and medulla responded to the decreased blood flow and NO synthesis by increasing in prostaglandin E2 synthesis, which was due to increased cyclooxygenase-2 content. Superoxide dismutase restored SMA-SRACR (but not renal-SRACR) cortical and medullary NO synthesis, suggesting that ODFRs generated during mesenteric ischemia–reperfusion were one of the systemic mechanisms contributing to decreased renal NO synthesis in the SMA-SRACR model. The 90% decrease in creatinine clearance after SMA-SRACR was greater than the 60% decrease after renal-SRACR. Conclusions These data show that NO is important in maintaining renal cortical and medullary blood flow and NO synthesis after renal and SMA-SRACR. These data also suggest that in addition to the renal ischemia–reperfusion caused by both models, SMA SRACR induces mesenteric ischemia–reperfusion, resulting in the generation of ODFRs, which contribute to decreased renal cortical and medullary NO synthesis. Maintaining splanchnic blood flow or attempting to keep SRACR below the SMA level may be helpful in developing strategies to minimize the renal injury after SRACR.
Phlegmasia cerulea dolens (PCD) is limb-threatening. Traditional treatments are very morbid. We examine the efficacy of percutaneous treatment of PCD. Between May 2005 and September 2008, we treated ...21 limbs in 20 patients with lower extremity PCD who were candidates for thrombolysis. Diagnosis was by clinical examination and duplex ultrasound. Catheter access to the deep venous system was obtained through a popliteal vein. Therapy used pulse spray thrombolysis with tissue plasminogen activator (tPA). Infusion catheters and adjunctive percutaneous techniques were used as indicated. Postoperatively, patients were treated with systemic anticoagulation, compression hose, and interval follow-up. Limbs were graded according to the CEAP classification. Twenty patients (13 male) were treated with a mean age of 55.8 years. Nine patients had hypercoagulable states, four May Thurner syndrome, three a history of cancer, one postcolon resection, one acute myocardial infarction, and one postfemoral vein puncture. All patients had resolution of PCD without the need for open surgery. The initial tPA dose was 19.5 mg with pulse spray thrombolysis. Infusion catheters were required in 18 patients and used for 16.1 hours (range, 8 to 36 hours) until complete thrombolysis. Venous angioplasty was necessary in 14 patients with nine of these requiring venous stents. One patient required above-knee amputation despite successful treatment of her PCD. Mean follow-up was 10.7 months (range, 1 to 39 months). All patients demonstrated no or minimal residual thrombus and intact valvular function and a mean clinical CEAP score of 2.4. Percutaneous treatment of PCD produced excellent results with minimal morbidity.
A 65-year-old female presented with upper gastrointestinal hemorrhage thirty years following an aorta-to-right renal artery bypass constructed with saphenous vein. Upper endoscopy demonstrated a ...duodenal ulcer, and a CAT scan demonstrated aneurysmal degeneration of her renal artery bypass with duodenal impingement. Laparotomy demonstrated erosion of the aneurysm through the posterior wall of the duodenum; extra-anatomic renovascular reconstruction and primary duodenal repair was performed. Although aneurysmal degeneration of intraabdominal saphenous vein grafts is well described and rupture likewise reported, this report represents the first description of an intraabdominal autogenous vein graft aneurysm presenting with gastrointestinal erosion and fistula.
Renal insufficiency continues to be complication that can affect patients after treatment for suprarenal aneurysms and renal artery occlusive disease. One proposed mechanism of renal injury after ...suprarenal aortic clamping (above the superior mesenteric artery) and reperfusion (SMA-SRACR) is the loss of microvascular renal blood flow with subsequent loss of renal function. This study examines the hypothesis that the loss of medullary and cortical microvascular blood flow following SMA-SRACR is due to oxygen-derived free radical down-regulation of endogenous medullary and cortical nitric oxide synthesis.
Anesthetized male Sprague-Dawley rats (about 350 g) either had microdialysis probes or laser Doppler fibers inserted into the renal cortex (depth of 2 mm) and into the renal medulla (depth of 4 mm). Laser Doppler blood flow was continuously monitored. The microdialysis probes were connected to a syringe pump and perfused in vivo at 3 μL/min with lactated Ringer’s solution. The animals were subjected to SMA-SRACR (or sham) for 30 minutes, followed by 60 minutes of reperfusion. Laser Doppler blood flow after the 30 minutes of SMA-SRACR followed by 60 minutes of reperfusion was compared with the time zero (basal) and with the corresponding sham group and reported as percent change compared with the time zero baseline. The microdialysis fluid was collected at time zero (basal) and compared with the dialysis fluid collected after 30 minutes of SMA-SRACR followed by 60 minutes of reperfusion as well as the corresponding sham group. The microdialysis dialysate was analyzed for total nitric oxide (μM) and prostaglandin E
2 (PGE
2), 6-keto-PGF
1α (PGI
2 metabolite), and thromboxane B
2 synthesis. The data are reported as percent change compared with the baseline time zero. The laser Doppler blood flow and microdialysis groups were treated with either saline carrier, N
ω-nitro-L-arginine methyl ester hydrochloride (L-NAME) (30 mg/kg, nitric oxide synthesis inhibitor), L-arginine (400 mg/kg, nitric oxide precursor), superoxide dismutase (SOD, 10,000 U/kg, oxygen-derived free radical scavenger), L-NAME + SOD, or L-arginine + SOD. SOD was given 30 minutes before the reperfusion, and the other drugs were given 15 minutes before reperfusion. The renal cortex and medulla were separated and analyzed for inducible nitric oxide synthase (iNOS), cyclooxygenase-2, prostacyclin synthase, and PGE
2 synthase content by Western blot.
Superior mesenteric artery-SRACR caused a marked decrease in medullary and cortical blood flow with a concomitant decrease in endogenous medullary and cortical nitric oxide synthesis. These changes were further accentuated by L-NAME treatment but restored toward sham levels by L-arginine treatment after SMA-SRACR. The kidney appeared to compensate for these changes by increasing cortical and medullary PGE
2 synthesis and release. SOD treatment restored renal cortical and medullary nitric oxide synthesis and blood flow in the ischemia-reperfusion group and in the ischemia-reperfusion group treated with L-NAME.
These data show that nitric oxide is important in maintaining renal cortical and medullary blood flow and nitric oxide synthesis. These data also support the hypothesis that the loss of medullary and cortical microvascular blood flow following SRACR is due in part to oxygen-derived free radical downregulation of endogenous medullary and cortical nitric oxide synthesis.
This study suggests that clinically relevant cortical and medullary vasodilators (nitric oxide and vasodilator prostanoids) are required to maintain microvascular renal cortical and medullary blood flow. This study combines in vivo techniques of microdialysis and laser Doppler flow probes to show that oxygen-derived free radicals are one of the mediators that downregulate endogenous cortical and medullary nitric oxide synthesis, contributing to decreased cortical and medullary blood flow that occurs in the SMA-SRACR model. Prevention or inhibition of oxygen-derived free radical production during SMA-SRACR should be one of the treatment strategies that could help maintain renal microvascular blood flow during the treatment of complex aortic pathology that requires SMA-SRACR.
This study examined the hypothesis that clamping the aorta above the superior mesenteric artery (SMA) followed by suprarenal aortic clamping and reperfusion (SRACR) decreases microvascular blood flow ...by loss of endogenous medullary and cortical nitric oxide (NO) and prostaglandin (PG) E2 synthesis.
Anesthetized male Sprague-Dawley rats (350 g) had either microdialysis probes or laser Doppler fibers inserted into the renal cortex to a depth of 2 mm and into the renal medulla at 4 mm. Laser Doppler blood flow was continuously monitored (data reported as percentage of change compared to basal), and the microdialysis probes were connected to a syringe pump and perfused in vivo at 3 μL/min with lactated Ringer solution. Dialysate fluid was collected at basal time zero, following 30 minutes of suprarenal aortic clamping (ischemia) followed by 60 minutes of reperfusion and compared to a sham operation. Both groups were treated with saline carrier, indomethacin (INDO) (10 mg/kg, a cyclooxygenase COX inhibitor), NG-nitro-l-arginine methyl ester (l-NAME) (20 mg/kg, a NO synthase NOS inhibitor), or l-arginine (200 mg/kg, an NO precursor). Dialysate was analyzed for total NO (μM) and PGE2 (pg/mL) synthesis. The renal cortex and medulla were analyzed for inducible NOS (iNOS) and COX-2 content by Western blot. All data are reported as mean ± SEM, N > 5 and analyzed by analysis of variance.
SRACR caused a marked decrease in medullary and cortical blood flow with a concomitant decrease in endogenous medullary and cortical NO synthesis. Treatment with l-NAME further decreased blood flow and NO synthesis in the medulla and cortex. l-Arginine restored medullary and cortical NO synthesis and blood flow in the cortex but not the medulla. SRACR did not alter renal medullary or cortical PGE2; however, addition of INDO, COX inhibitor, caused a concomitant decrease in medullary and cortical PGE2 synthesis and blood flow.
NO is an important endogenous renal vasodilator that, when maintained can help preserve cortical blood flow following SRACR. These data also suggest that avoidance of COX-2 inhibitors can help maintain endogenous renal cortical and medullary PGE2 synthesis and thus contribute to maintaining normal blood flow.
This study is the first to combine in vivo physiologic assays to simultaneously identify clinically relevant intrarenal vasodilators (cortical and medullary) that are required to maintain microvascular blood flow. Identification of endogenous renal cortical and medullary vasodilators responsible for maintaining renal microvascular blood flow will allow development of treatment strategies to preserve these vasodilators following SRACR. Successful preservation of endogenous intrarenal vasodilators will help maintain renal microvascular blood flow and renal function in the treatment of complex aortic pathology that requires SRACR.
A 58-year-old man with hypertension, severe abdominal pain, and pulseless extremities was diagnosed with an isolated abdominal intramural hematoma (IMH). The IMH extended from the distal descending ...thoracic aorta to just proximal to the renal arteries. β-Blockade treatment resolved the abdominal pain but induced progressive oliguria; decreasing β-blockade treatment increased urine output but caused return of abdominal pain. An axillobifemoral bypass allowed distal perfusion and retrograde visceral artery perfusion while maintaining normal blood pressure. The abdominal pain resolved, urine output increased, and the patient was discharged on day 7. Six months later the patient required an emergent thrombectomy of the axillobifemoral graft and normal antegrade aortic flow was found. A computed tomography scan showed resolution of the IMH.
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