Search

Aims: Before the discovery of contact-force guidance, eliminating pacing capture along the pulmonary vein (PV) isolation line had been reported to improve PV isolation durability and rhythm outcomes. DRAGON (UMIN-CTR, UMIN000015332) aimed to elucidate the efficacy of pace-capture-guided ablation following contact-force-guided PV isolation ablation in paroxysmal atrial fibrillation (AF) patients., Methods and Results: A total of 156 paroxysmal AF patients with AF-trigger ectopies from any of the four PVs induced by isoproterenol were randomly assigned to undergo pace-capture-guided ablation along a contact-force-guided isolation line around AF-trigger PVs (PC group, n = 76) or contact-force-guided PV isolation ablation alone (control group, n = 80). Follow-up of at least 1 year commenced with serial 24 h Holter and symptom-triggered ambulatory monitoring. There was no significant difference in acute PV reconnection rates during a 20 min waiting period after the last ablation or adenosine infusion testing between the PC and the control groups (per patient, 21% vs. 27%, P = 0.27; per AF-trigger PV, 5.9% vs. 7.3%, P = 0.70; and per non-AF-trigger PV, 7.1% vs. 7.4%, P = 0.92). Atrial tachyarrhythmia-free survival rates off antiarrhythmic drugs after the initial session were comparable at 19.3 ± 6.2 months between the two groups (82% vs. 80%, P = 0.80). Among 22 patients who required a second ablation procedure, there was no difference between the PC and the control groups in the PV reconnection rates at both previously AF-trigger (29% vs. 43%, P = 0.70) and non-AF-trigger PVs (18% vs. 19%, P = 0.88)., Conclusions: Pace-capture-guided ablation performed after contact-force-guided PV isolation demonstrated no improvement in PV isolation durability or rhythm outcome.

Background: Detection of yellow plaques (YP) by coronary angioscopy (CAS) 1 year after 1st-generation drug-eluting stent (DES) implantation has been related to future coronary events. However, the association between CAS findings and clinical outcomes following 2nd-generation DES implantation has not been investigated. Methods and Results: This study included a total of 248 2nd-generation DES in 179 patients, who were examined by CAS 9±2 months after implantation. Angioscopic evaluation included dominant neointimal coverage (NIC) grade, heterogeneity of NIC, presences of YP and intrastent thrombus. The outcome measure was major adverse cardiac events (MACE) defined as a composite of cardiac death, acute myocardial infarction and any coronary revascularization. The association between the CAS findings and MACE was evaluated using the Kaplan-Meier method. A Cox proportional hazards model was used to assess the predictors of MACE. The mean follow-up duration was 1,367±843 days. Dominant NIC grade (P=0.98), heterogeneity of NIC (P=0.20) and YP (P=0.53) were not associated with the incidence of MACE. However, intrastent thrombus was significantly associated with MACE (P=0.033). Intrastent thrombus (adjusted hazard ratio: 2.22; 95% confidence interval [CI]: 1.12-4.39), acute coronary syndrome (2.83; 95% CI: 1.42-5.67) and B2/C lesion (2.13; CI: 1.12-4.05) were independent predictors of MACE., Conclusions: Subclinical intrastent thrombus observed by CAS at 9 months after 2nd-generation DES implantation was independently associated with poor clinical outcome.

Background: Wood basic density (WBD), the biomass of plant cell walls per unit volume, is an important trait for elite tree selection in kraft pulp production. Here, we investigated the correlation between WBD and wood volumes or wood properties using 98 open-pollinated, 2.4 to 2.8 year-old hybrid Eucalyptus (Eucalyptus urophylla x E. grandis). Transcript levels of lignocellulose biosynthesis-related genes were studied., Results: The progeny plants had average WBD of 516 kg/m 3 with normal distribution and did not show any correlations between WBD and wood volume or components of α-cellulose, hemicellulose and Klason lignin content. Transcriptomic analysis of two groups of five plants each with high (570-609 kg/m 3 ) or low (378-409 kg/m 3 ) WBD was carried out by RNA-Seq analysis with total RNAs extracted from developing xylem tissues at a breast height. Lignocellulose biosynthesis-related genes, such as cellulose synthase, invertase, cinnamate-4-hydroxylase and cinnamoyl-CoA reductase showed higher transcript levels in the high WBD group. Among plant cell wall modifying genes, increased transcript levels of several expansin and xyloglucan endo-transglycosylase/hydrolase genes were also found in high WBD plants. Interestingly, strong transcript levels of several cytoskeleton genes encoding tubulin, actin and myosin were observed in high WBD plants. Furthermore, we also found elevated transcript levels of genes encoding NAC, MYB, basic helix-loop-helix, homeodomain, WRKY and LIM transcription factors in the high WBD plants. All these results indicate that the high WBD in plants has been associated with the increased transcription of many genes related to lignocellulose formation., Conclusions: Most lignocellulose biosynthesis related genes exhibited a tendency to transcribe at relatively higher level in high WBD plants. These results suggest that lignocellulose biosynthesis-related genes may be associated with WBD.

Aims: An elevated left atrial pressure has been reported to play an important role in the development of atrial remodelling in atrial fibrillation (AF) patients. The study aimed at elucidating the association between the diastolic early transmitral flow velocity/mitral annular velocity (E/e', a non-invasive surrogate of left atrial pressure) and left atrial low-voltage-area existence, and the prognostic impact of the E/e' on procedural outcomes in patients undergoing AF ablation., Methods and Results: Total of 215 consecutive patients were divided into 3 groups based on the estimated left atrial pressure: normal (E/e' < 8.0, n = 58), undetermined (E/e' = 8.0-14.0, n = 114), and elevated (E/e' > 14.0, n = 43). Left atrial endocardial voltage mapping was performed following pulmonary vein isolation. Patients with a high E/e' more frequently had low-voltage areas (E/e' < 8.0, 31%, E/e' = 8.0-14.0, 35%; E/e' > 14.0, 67%; P = 0.0001). After adjusting for other correlates, a high E/e' was an independent predictor of low-voltage-area existence (HR = 1.11, 95% CI = 1.02-1.21, P = 0.017). During a mean follow-up period of 12 ± 6 months, recurrent atrial tachyarrhythmias occurred in 22 (10%) patients after multiple (1.4 ± 0.5) procedures. Patients with an E/e' > 14 had more frequent recurrent atrial tachyarrhythmias after multiple ablation procedures than those with an E/e' ≤ 14 (23% vs. 7%, P = 0.001)., Conclusion: A high E/e' obtained by pre-ablation echocardiography was associated with a left atrial arrhythmogenic substrate in patients undergoing AF ablation. Furthermore, a high E/e' predicted poor procedural outcomes after pulmonary vein isolation.

Background: Although stent-to-vessel (S/V) diameter ratio has been described as a restenotic factor after superficial femoral artery (SFA) stenting, the reference vessel diameter is commonly measured distally at a healthy site. It remains unclear whether S/V ratio assessed at the lesion site would be more predictive than that assessed distally at a healthy site.Methods and Results:A total of 117 patients (mean age, 73±7 years; 74% male) who underwent successful nitinol stent implantation in SFA lesions (mean lesion length, 172±104 mm) on intravascular ultrasound (IVUS) were retrospectively analyzed. S/V ratio at the proximal and distal healthy site, and at the smallest lesion site, was evaluated on IVUS. One-year restenosis predictors were evaluated on multivariate analysis. Mean S/V diameter ratio on IVUS at proximal and distal healthy sites, and at the lesion site, was 0.98±0.11, 1.02±0.11 and 1.15±0.16, respectively. One-year primary patency was 77%. On multivariate analysis, lesion length (OR, 1.06 per 10-mm increment; P=0.046) and S/V ratio measured at the lesion site (OR, 1.34 per 0.1 increment; P=0.032), but not that at the distal healthy site (OR, 1.05 per 0.1 increment; P=0.705), were significantly associated with 1-year restenosis., Conclusions: S/V ratio assessed on IVUS at the lesion site, but not at the distal healthy site, was independently associated with 1-year restenosis after SFA stenting.

Background: Association between the presence of left atrial low-voltage areas and atrial fibrillation (AF) recurrence after pulmonary vein isolation (PVI) has been shown mainly in persistent AF patients. We sought to compare the AF recurrence rate in paroxysmal AF patients with and without left atrial low-voltage areas., Methods: This prospective observational study included 147 consecutive patients undergoing initial ablation for paroxysmal AF. Voltage mapping was performed after PVI during sinus rhythm, and low-voltage areas were defined as regions where bipolar peak-to-peak voltage was <0.50mV., Results: Left atrial low-voltage areas after PVI were observed in 22 (15%) patients. Patients with low-voltage areas were significantly older (72±6 vs. 66±10, p<0.0001), more likely to be female (68% vs. 32%, p=0.002), and had higher CHA 2 DS 2 -VASc score (2.5±1.5 vs. 1.8±1.3, p=0.028). During a mean follow-up of 22 (18, 26) months, AF recurrence was observed in 24 (16%) and 16 (11%) patients after the single and multiple ablation procedures, respectively. AF recurrence rate after multiple ablations was higher in patients with low-voltage areas than without (36% vs. 6%, p<0.001). Low-voltage areas were independently associated with AF recurrence even after adjustment for the other related factors (Hazard ratio, 5.89; 95% confidence interval, 2.16 to 16.0, p=0.001)., Conclusion: The presence of left atrial low-voltage areas after PVI predicts AF recurrence in patients with paroxysmal AF as well as in patients with persistent AF., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Whether arterial repair following implantation of drug-eluting stents (DES) of the second generation differs among stent types remains unknown. We examined 41 DES placed in 28 patients (age 72 ± 7 years, male 89%) presenting with stable angina pectoris due to de novo lesions in native coronary arteries. Coronary angioscopy was performed 4 ± 1 months after stent implantation. Patients were divided into two groups based on the DES types: 22 cobalt-chrome everolimus-eluting stents (CoCr-EES) in 13 patients and 19 slow-release zotarolimus-eluting stents (R-ZES) in 15 patients. Neointimal coverage (NIC) was graded as: grade 0, stent struts exposed; grade 1, struts bulging into the lumen, although covered; grade 2, struts embedded in the neointima, but translucent; grade 3, struts fully embedded and invisible. NIC was defined as heterogeneous when the NIC grade variation was ≥1. Presence of thrombus was also investigated. Distribution of dominant NIC grade (CoCr-EES: grade 0, 9%; grade 1, 77%; grade 2, 9%; grade 3, 5%; R-ZES: grade 0, 16%; grade 1: 47%; grade 2, 37%; grade 3, 0%, P = 0.38) and heterogeneity of NIC (P = 0.43) were similar between CoCr-EES and R-ZES groups. Existence of thrombus was not significantly different in CoCr-EES and R-ZES (18 versus 42%, P = 0.17). Arterial repair occurred without significant differences between CoCr-EES and R-ZES 4 months after implantation.

Aim: We investigated 2-year clinical outcomes after implantaton of Epic TM self-expanding nitinol stents for patients with peripheral artery disease (PAD) due to the aortoiliac occlusive disease (AIOD)., Methods: This study was a multicenter and retrospective study. From February 2013 through October 2014, 292 lesions (chronic total occlusion, 21%; TASC Ⅱ C/D, 35%) in 217 consecutive patients (74±8 years; male, 81%; diabetes mellitus, 47%; dialysis, 21%; critical limb ischemia, 29%) who had undergone endovascular therapy (EVT) with Epic TM self-expanding nitinol stents for PAD with AIOD were analyzed. The primary endpoints were 2-year primary patency and target lesion revascularization (TLR)-free rate. The primary patency and freedom from TLR were determined by Kaplan-Meier analysis. Additionally, predictors for loss of patency were estimated by Cox proportional hazard model., Results: The mean follow-up duration was 19.1±8.5 months. Primary patency was 87.3% at 2 years. Freedom from TLR rate was 94.1% at 2 years. Multivariate analysis revealed that the presence of diabetes mellitus was associated with a loss of patency., Conclusion: The Epic TM self-expanding nitinol stent was demonstrated to be safe and effectivene for AIOD when tested for two years in patients with PAD.

Objectives: The aim of this study was to assess the use of wave front propagation speed on a right ventricular map for determining the earliest activation site as the origin of outflow tract ventricular arrhythmias (VAs)., Background: VAs with centrifugal right ventricular outflow tract (RVOT) activation can be from an RVOT focus or a focus outside the RVOT., Methods: This prospective observational study included 23 patients with idiopathic outflow tract VAs. Mapping of the RVOT was performed using a new ultra-high-resolution electroanatomic mapping system. The wave front propagation speed was estimated from the area surrounded by a propagated wave front at 5, 10, 15, and 20 ms after the earliest activation., Results: VAs disappeared following ablations in the RVOT in 15 patients (RVOT origin). The remaining 8 patients had VAs of non-RVOT origin determined by ablation success at another site or ablation failure. The areas surrounded by a propagated wave front were significantly smaller in VAs of RVOT origin than non-RVOT VAs at 5 ms (1.0 [0.7 to 1.1] cm 2  vs. 2.2 [1.6 to 4.4] cm 2 ), 10 ms (1.9 [1.4 to 2.2] cm 2 vs. 4.5 [3.2 to 5.8] cm 2 ), 15 ms (3.2 [2.3 to 4.4] cm 2 vs. 7.1 [6.3 to 9.8] cm 2 ), and 20 ms (5.0 [3.0 to 6.6] cm 2 vs. 9.8 [9.3 to 14.8] cm 2 ). A propagated area of <5.0 cm 2 at 15 ms predicted RVOT VAs with 87% sensitivity, 100% specificity, and 91% predictive accuracy., Conclusions: VAs with slow wave front propagation speed on the right ventricular map indicate an RVOT origin., (Copyright © 2018 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Background: Differentiation of atrial fibrillation (AF) trigger ectopy from other ectopy is often difficult. The purpose of this study was to compare the origin and coupling intervals (CI) between AF-trigger and non-AF-trigger ectopy., Methods: This study consisted of 120 patients with AF who underwent an initial ablation. Isoproterenol was infused up to 20μg/min to provoke ectopy and AF. We measured the CI of all ectopy provoked by an isoproterenol infusion. The %CI was calculated as the CI of the ectopy/P-P interval of the preceding 2 beats., Results: A total of 117 patients had at least one ectopy, and AF was induced in 56 (47%) patients. Of the 276 ectopies observed in this study, 211 (76%) originated from pulmonary veins and 77 (28%) were AF-trigger ectopy. AF-trigger ectopy more frequently originated from pulmonary veins (PVs) (74 vs. 3, p<0.001) and had a significantly shorter CI (201±70ms vs. 365±147ms, p<0.001) and lower %CI (29±11% vs. 55±14%, p<0.001) than that of non-AF-trigger ectopy. A receiver operating characteristics analysis revealed that a %CI of 40% was the best cut-off value for differentiating whether it was an AF-trigger or not. The identified trigger group, including patients with provoked AF-trigger ectopy or ectopy with a low %CI (<40%), had a significantly better AF recurrence-free survival rate than the other group (88% vs. 65%, p=0.004)., Conclusions: AF-trigger ectopy predominantly originated from PVs and had a short CI. These findings may be useful for estimating whether ectopies are an AF-trigger or not., (Copyright © 2017 Japanese College of Cardiology. Published by Elsevier Ltd. All rights reserved.)

Background: The reconnection of left atrial-pulmonary vein (LA-PV) conduction after the initial procedure of pulmonary vein (PV) isolation is not rare, and is one of the main cause of atrial fibrillation (AF) recurrence after PV isolation., Objective: We investigated feasibility of a new ultrahigh-resolution mapping system using a 64-pole small basket catheter for the identification of LA-PV conduction gaps., Methods: This prospective study included 31 consecutive patients (20 with persistent AF) undergoing a second ablation after a PV isolation procedure with LA-PV reconnected conduction at any of the 4 PVs. An LA-PV map was created using the mapping system, and ablation was performed at the estimated gap location., Results: The propagation map identified 54 gaps from 39 ipsilateral PV pairs, requiring manual electrogram reannotation for 23 gaps (43%). Gaps at the anterior and carinal regions of left and right ipsilateral PVs required manual electrogram reannotation more frequently than the other regions. The voltage map could identify the gap only in 19 instances (35%). Electrophysiological properties of the gaps (multiple gaps in the same ipsilateral PVs, conduction time, velocity, width, and length) did not differ between those needing and not needing manual electrogram reannotation. During the gap ablation, either the activation sequence alteration or elimination of PV potentials was observed using a circular catheter placed in the PV, suggesting that all the identified gaps were correct., Conclusion: This new electroanatomic mapping system visualized all the LA-PV gaps in patients undergoing a second AF ablation., (Copyright © 2017 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)

The impact of the severity of coronary artery disease (CAD) and left ventricular ejection fraction (LVEF) on the prognosis of patients with peripheral artery disease (PAD) has not been systematically studied. We retrospectively analysed 622 patients with PAD (intermittent claudication (IC): n = 446; critical limb ischaemia (CLI): n = 176). The association of SYNTAX score and LVEF with mortality was analysed using the Cox proportional hazard model. In patients with IC, a high SYNTAX score was significantly associated with mortality, whereas reduced LVEF was significantly associated with mortality in patients with CLI. The prognostic impact of CAD and LVEF appears different between patients with IC and CLI., (© 2017 Royal Australasian College of Physicians.)

Purpose: To report histological examination of a Viabahn stent-graft implanted in the superficial femoral artery (SFA) for nearly 2 years., Case Report: A 78-year-old man with peripheral artery disease was treated successfully with a 6.0×250-mm Viabahn self-expanding stent-graft in the right SFA, relieving his lower limb claudication. The patient died suddenly due to acute myocardial infarction 23 months later. Histological evaluation of the stent-graft implantation site revealed moderate neointimal proliferation at both proximal and distal edges of the device. In the middle part of the stent, significant macrophages and multinucleated foreign body giant cells had accumulated, although the stent was entirely patent. Furthermore, no endothelial cell coverage was found., Conclusion: Judging from these features, it might be necessary to continue dual antiplatelet therapy after stent-graft implantation over the long term to prevent thrombosis and subsequent restenosis or reocclusion.

Although stent implantation has become widespread for the treatment of patients with peripheral artery disease with femoropopliteal (FP) lesions, in-stent restenosis, especially in-stent occlusion (ISO), remains as a major concern for refractory recurrence. Furthermore, the mechanisms of ISO in FP lesions have not been well elucidated. We performed angioscopy for 6 lesions (bare-metal stent: 3, drug-eluting stent: 3) from 5 patients (mean age 74 ± 10 years, male 40 %) with ISO in the FP artery immediately after wire-passing or thrombus aspiration. The presence of thrombus as well as the presence and location of organic stenosis were evaluated. Median duration from stent implantation to angioscopic evaluation was 1099.5 (514.5-2272.5) days, while the duration from recurrence of symptoms to angioscopic evaluation was 45 (5.75-60) days. Mixed thrombi were observed in all stents. Organic stenosis was detected at the proximal edge of the stents in 5 lesions. Organic stenosis was observed at the overlapping segment of the stent in one lesion. The distal edge of the stents could be evaluated in 3 lesions, and all of them showed organic stenosis at the site. Mixed thrombi and organic stenosis were observed in all stents. Partial development of organic stenosis in a stent followed by thrombus formation may be the potential mechanism of the development of ISO in the FP artery though the sample size of this study was small and it had no serial angioscopic data so that we should consider it as preliminary one at best.

A bipolar voltage reflects a thick musculature where formation of a transmural lesion may be hard to achieve. The purpose of this study was to explore the association between local bipolar voltage and conduction gap in patients with persistent atrial fibrillation (AF) who underwent atrial roof or septal linear ablation. This prospective observational study included 42 and 36 consecutive patients with persistent AF who underwent roof or septal linear ablations, respectively. After pulmonary vein isolation, left atrial linear ablations were performed, and conduction gap sites were identified and ablated after first-touch radiofrequency application. Conduction gap(s) after the first-touch roof and septal linear ablation were observed in 13 (32%) and 19 patients (53%), respectively. Roof and septal area voltages were higher in patients with conduction gap(s) than in those without (roof, 1.23 ± 0.77 vs 0.73 ± 0.42 mV, p = 0.010; septal, 0.96 ± 0.43 vs 0.54 ± 0.18 mV, p = 0.001). Trisected regional analyses revealed that the voltage was higher at the region with a conduction gap than at the region without. Complete conduction block across the roof and septal lines was not achieved in 3 (7%) and 6 patients (17%), respectively. Patients in whom a linear conduction block could not be achieved demonstrated higher ablation area voltage than those with a successful conduction block (roof, 1.91 ± 0.74 vs 0.81 ± 0.51 mV, p = 0.001; septal, 1.15 ± 0.56 vs 0.69 ± 0.31 mV, p = 0.006). In conclusion, a high regional bipolar voltage predicts failure to achieve conduction block after left atrial roof or septal linear ablation. In addition, the conduction gap was located at the preserved voltage area., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Purpose: To investigate the angiographic dissection patterns after balloon angioplasty for superficial femoral artery (SFA) lesions, the clinical outcome associated with each dissection pattern, and the predictive factors for severe dissection., Methods: A retrospective, multicenter analysis examined 621 patients (mean age 72.8±9.5 years; 414 men) with 748 symptomatic de novo SFA lesions treated with endovascular therapy. Vessel dissection after the initial balloon angioplasty procedure was graded into 7 types according to a modified version of the coronary artery classification types A to F. Severe vessel dissection patterns were defined as type C or higher. Nitinol stent implantation was performed in 555 (74.2%) lesions for residual stenosis >30% or flow-limiting dissection; 193 lesions (25.8%) were treated with balloon angioplasty only. To determine the clinical outcomes associated with each dissection pattern and identify predictive factors for severe dissection, 2-year follow-up data for the 193 lesions treated with balloon angioplasty only were analyzed for primary patency and clinically driven target lesion revascularization (TLR)., Results: No dissection was found in 16% (120/748) of lesions, and types A and B dissections were seen in 19% (142/748) and 23% (172/748), respectively. Dissection grades above type C were observed in 42% of cases, most frequently type D (180/748, 24%) and less often type C (37/748, 5%), type E (67/748, 9%), and type F (30/748, 4%). The bailout stent implantation rate increased according to dissection severity. At up to 2 years, the severe dissection group (types C-F) showed a significantly lower patency rate (p<0.001) and higher clinically driven TLR (p<0.001) compared to the nonsevere group (no dissection and types A and B dissections). Severe dissection was a significant risk factor for restenosis, which rose progressively from types C to F. Multivariate analysis identified a small reference vessel diameter <5 mm (p=0.001), lesion length >15 cm (p=0.001), and chronic total occlusion (p<0.001) as independent predictors of severe dissection. In subgroup analysis, vessels with a small reference diameter and TASC II C and D lesions had a higher prevalence of severe dissection., Conclusion: Severe dissection was found in 42% of cases after PTA. A small vessel diameter and/or TASC II C/D lesions were related to a high incidence of dissection. Severe dissection during procedures employing balloon angioplasty only could affect long-term patency.

Background: The efficacy of low-voltage-guided ablation in addition to pulmonary vein (PV) isolation for atrial fibrillation (AF) has been reported with voltage mapping being performed during sinus rhythm (SR) or AF. The study aimed to compare the left atrial voltage between SR and AF in association with the electrogram waveform., Methods: This prospective observational study included 30 consecutive patients with persistent AF. After completion of PV isolation, electrogram points were taken during both SR and AF at the identical locations evenly throughout the left atrium. Electrograms were divided into two types: normal (sharp electrogram with ≤3 peaks or duration <50 ms) and fractionated (>3 peaks and duration ≥50 ms)., Results: During SR, 14 (47%) patients had low-voltage (0.5 mV) substrate with an area of 6.8 ± 4.5 cm 2 . In a total of 1,063 point pairs, 135 (13%) demonstrated a fractionated electrogram during SR and 483 (45%) during AF. The locations with fractionated electrograms during AF more frequently showed fractionation during SR compared to those with normal electrograms during AF (23% vs 5%, P < 0.0001), and had lower amplitude during SR (1.47 ± 1.29 mV vs 2.03 ± 1.19 mV, P < 0.0001). Electrogram amplitude was higher during SR than that during AF (1.77 ± 1.27 mV vs 0.96 ± 0.77 mV, P < 0.0001) with a weak correlation (r = 0.56, P < 0.0001). Subgroup analyses revealed that the correlation was relatively strong (r = 0.73, P < 0.0001) among the electrogram amplitudes with normal waveform during SR and AF., Conclusions: Significant differences in electrogram voltage and fractionation degree may exist between SR and AF at the same locations in patients with persistent AF., (© 2017 Wiley Periodicals, Inc.)

Purpose: To investigate whether the severity of lesion calcification assessed by the novel peripheral artery calcification scoring system (PACSS) was associated with clinical outcomes after endovascular therapy (EVT) for superficial femoral artery (SFA) lesions., Methods: A retrospective analysis was conducted of 394 consecutive patients (mean age 72±8 years; 290 men) with intermittent claudication [223 (57%) with diabetes, 81 (21%) on hemodialysis] who underwent successful EVT for de novo SFA lesions [length 152.1±95.7 mm; 199 (50%) TransAtlantic Inter-Society Consensus II class C/D] between January 2010 and December 2013. The patients were retrospectively categorized using the PACSS classification (grades 0-4: no visible calcification of the target lesion, unilateral wall calcification <5 cm, unilateral calcification ≥5 cm, bilateral wall calcification <5 cm, and bilateral calcification ≥5 cm, respectively). The main outcome was primary patency, while the secondary outcome measures were mortality and major adverse limb events [MALE: any intervention (repeat EVT or surgical revision) or major (above ankle) amputation]. Cox proportional hazards analysis was used to explore whether the PACSS classification was an independent predictor of clinical outcomes. Results are presented as the hazard ratio (HR) and 95% confidence interval (CI)., Results: The distribution of PACSS grades was 0 in 54%, grade 1 in 16%, grade 2 in 12%, grade 3 in 9%, and grade 4 in 9%. The 2-year primary patency rates in these grades, respectively, were 70.0%, 66.6%, 72.1%, 55.6%, and 36.3% (p<0.001). After multivariate analysis, PACSS grade 4 (HR 2.74, 95% CI 1.56 to 4.83, p<0.001), diabetes (HR 1.52, 95% CI 1.06 to 2.20, p=0.022), lesion length (HR 1.04, 95% CI 1.01 to 1.07, p=0.006), and vessel diameter (HR 0.80, 85% CI 0.65 to 0.98, p=0.038) were associated with loss of primary patency. PACSS grade 4 was also associated with MALE and mortality (p=0.048 and 0.011, respectively). Bare metal stent use (HR 0.47, 95% CI 0.30 to 0.73, p<0.001) was positively associated with primary patency., Conclusion: PACSS grade 4 calcification was independently associated with clinical outcomes after EVT for de novo SFA lesions., (© The Author(s) 2016.)

Purpose: The influence of left atrial (LA) enlargement on the efficacy of steerable sheath usage during pulmonary vein (PV) isolation is poorly understood. Here, we compared the catheter-tissue contact force (CF) and clinical outcomes between steerable and non-steerable sheaths among patients with different severities of LA enlargement., Methods: In this prospective observational study, 90 consecutive atrial fibrillation patients scheduled for PV isolation (paroxysmal, 67 %) were enrolled and divided into tertiles of the LA volume (LA1, <124 cm 3 ; LA2, 124 to 145 cm 3 ; LA3, ≥146 cm 3 ). An ipsilateral PV isolation was completed using either a steerable (n = 57) or non-steerable sheath (n = 33)., Results: The average CF of each ipsilateral PV was lower in the LA3 group (right 13.8 ± 5.8 g; left 9.7 ± 5.2 g) than LA1 (right 15.6 ± 6.8 g, p = 0.005; left 12.5 ± 5.6 g, p < 0.0001) or LA2 (right 15.2 ± 7.3 g, p = 0.04; left 12.6 ± 5.3 g, p < 0.0001) groups. In total, the average CF around the left PVs was significantly higher with steerable sheaths than without (12.7 ± 5.5 vs. 10.2 ± 4.9 g, p = 0.03). Subgroup analysis revealed that a tendency toward higher CF around the left PVs was observed in the LA3 group (12.3 ± 7.3 vs. 8.5 ± 3.9 g, p = 0.08) but was not in the LA1 and LA2 groups. Further, in the LA3 group, steerable sheath usage showed better clinical outcomes: shorter procedural (55 ± 29 min vs. 81 ± 39 min, p = 0.04) and fluoroscopic times (27 ± 11 min vs. 37 ± 17 min, p = 0.045) and a tendency toward a lower 1-year AF recurrence rate (39 vs. 12 %, p = 0.09)., Conclusions: The beneficial effects of using steerable sheaths were more prominent in patients with severe LA enlargement.

Eucalyptus species constitutes the most widely planted hardwood trees in temperate and subtropical regions. In this study, we compared the transcript levels of genes involved in lignocellulose formation such as cellulose, hemicellulose and lignin biosynthesis in two selected 3-year old hybrid Eucalyptus (Eucalyptus urophylla × Eucalyptus grandis) genotypes (AM063 and AM380) that have different lignin content. AM063 and AM380 had 20.2 and 35.5% of Klason lignin content and 59.0 and 48.2%, α-cellulose contents, respectively. We investigated the correlation between wood properties and transcript levels of wood formation-related genes using RNA-seq with total RNAs extracted from developing xylem tissues at a breast height. Transcript levels of cell wall construction genes such as cellulose synthase (CesA) and sucrose synthase (SUSY) were almost the same in both genotypes. However, AM063 exhibited higher transcript levels of UDP-glucose pyrophosphorylase and xyloglucan endotransglucoxylase than those in AM380. Most monolignol biosynthesis-related isozyme genes showed higher transcript levels in AM380. These results indicate monolignol biosynthesis-related genes may regulate wood composition in Eucalyptus. Flavonoids contents were also observed at much higher levels in AM380 as a result of the elevated transcript levels of common phenylpropanoid pathway genes, phenylalanine ammonium lyase, cinnamate-4-hydroxylase (C4H) and 4-coumarate-CoA ligase (4CL). Secondary plant cell wall formation is regulated by many transcription factors. We analyzed genes encoding NAC, WRKY, AP2/ERF, and KNOX transcription factors and found higher transcript levels of these genes in AM380. We also observed increased transcription of some MYB and LIM domain transcription factors in AM380 compared to AM063. All these results show that genes related to monolignol biosynthesis may regulate the wood composition and help maintain the ratio of cellulose and lignin contents in Eucalyptus plants.

Background: Recurrent atrial tachyarrhythmias occur as a result of residual atrial arrhythmogenic substrates after atrial fibrillation (AF) ablation. In patients with AF, electrograms with reduced amplitudes indicate diseased myocardium., Objective: The purpose of this study was to investigate the association between the distribution of low-voltage areas and the type of induced atrial tachyarrhythmias., Methods: Our prospective observational study enrolled 152 consecutive AF patients scheduled for an initial ablation (46% persistent AF). After pulmonary vein isolation, voltage mapping was performed during sinus rhythm, and regions with reduced electrogram amplitudes (<0.5 mV) were defined as low-voltage areas. Burst pacing was performed to investigate the inducibility of atrial tachyarrhythmias., Results: Low-voltage areas were more frequently observed in patients with persistent AF than paroxysmal AF (50% vs. 34%, P = .048). A higher proportion of patients with low-voltage areas presented with inducibility of atrial tachyarrhythmias than those without, as follows: AF 70% vs. 16% (P = .0001); perimitral macroreentrant atrial tachycardia (AT) 18% vs. 0% (P = .0001); and roof-dependent macroreentrant AT 13% vs. 0% (P = .01). Investigation into the regional distribution of low-voltage areas revealed that patients with perimitral macroreentrant AT more frequently coincided with low-voltage areas than those without in the septal (100% vs. 18%, P <.0001) and anterior regions (55% vs. 11%, P = .001), and those with roof-dependent AT in the roof (75% vs. 15%, P <.0001) and posterior regions (75% vs. 15%, P = .0001)., Conclusion: Low-voltage areas are associated with high inducibility of atrial tachyarrhythmias after pulmonary vein isolation. In addition, the distribution of low-voltage areas is specific for each type of macroreentrant AT., (Copyright © 2016 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)

Efficacy of endovascular therapy (EVT) with nitinol stents for femoropopliteal (FP) lesions is limited by restenosis. Oral cilostazol reduces angiographic restenosis rate; however, treatment duration remains unclear. In a retrospective analysis of a multicenter database of 3471 consecutive limbs in 2737 patients (mean age: 72 ± 9 years; 61% diabetic; and 26% on regular dialysis) undergoing EVT for FP lesions between January 2004 and December 2011, we compared Kaplan-Meier estimated primary patency after EVT followed or not by cilostazol treatment. We used Cox hazard regression analysis to assess temporal association between cilostazol treatment and post-EVT restenosis. Five-year primary patency was higher in the cilostazol group than in the noncilostazol group (57% vs 47%, P < .0001). Cilostazol treatment was inversely associated with restenosis for the first 2 years following EVT (P < .05); however, no significant association was observed thereafter. Cilostazol use therefore appears efficacious in preventing restenosis up to 2 years after EVT for FP lesions., (© The Author(s) 2014.)

Fabry disease is an X-linked recessive glycosphingolipid storage disorder caused by a deficiency of lysosomal enzyme α-galactosidase A. It is recognized that Fabry disease patients often have ventricular arrhythmias. Although the effectiveness of implantable cardioverter-defibrillator (ICD) therapy in patients with ventricular fibrillation is established, there is little evidence regarding ICD therapy for Fabry disease. Here, we report the case of patient with Fabry disease who was treated with an ICD and presented with high defibrillation thresholds.

We report recombinase-mediated cassette exchange (RMCE), which can permit integration of transgenes into pre-defined chromosomal loci with no co-expressed marker gene by using Agrobacterium -mediated transformation. Transgenic tobacco plants which have a single copy of negative marker genes ( codA ) at target loci in heterozygous and homozygous conditions were used for gene exchange by the RMCE method. By negative selection, we were able to obtain five heterozygous and four homozygous transgenic plants in which the genes were exchanged from 64 leaf segments of heterozygous and homozygous target plants, respectively. Except for one transgenic plant with an extra copy, the other eight plants had only a single copy of marker-free transgenes, and no footprint of random integrated copies was detected in half of the eight plants. The RMCE re-transformation frequencies were calculated as 6.25 % per explant and were approximately the same as the average percentage of intact single-copy transformation events for standard tobacco Agrobacterium -mediated transformation.

Arterial repair differences between paclitaxel-coated nitinol drug-eluting stent (DES) vs. bare-metal stent (BMS) up to 1 year after implantation in the superficial femoral artery (SFA) have not been well characterized. We compared angioscopic findings 1 year after implantation in the SFA of 22 DES in 18 patients and 12 BMS in 9 patients (follow-up period 354 ± 49 vs. 383 ± 81 days, P = 0.20). Neointimal coverage (NIC) was graded as: 0, stent struts exposed; 1, struts bulged into the lumen, although covered; 2, struts embedded by the neointima, but translucent; and 3, struts fully embedded and invisible. NIC was defined as heterogeneous when NIC grade variation was ≥1. Presence of thrombus and yellow plaques (YP) was assessed. Baseline patient and lesion characteristics were similar between the groups. In terms of procedural characteristics, stent diameter was smaller in DES than in BMS (6.5 ± 0.5 vs. 7.4 ± 0.8 mm, P = 0.0020). Both DES (grade 2 5 %, grade 3 95 %) and BMS (grade 3 100 %) revealed dominant NIC grades of 2 or 3 (P = 0.46), however, DES showed greater NIC heterogeneity than BMS (91 vs. 42 %, P = 0.004). Both thrombus and YP were more frequently observed in DES than in BMS (64 vs. 17 %, P = 0.001; 95 vs. 67 %, P = 0.042, respectively). In conclusion, although arterial repair was limited after DES implantation in the SFA, it was extensive at 1 year.

Background: Several non-invasive methods for measuring pulmonary vascular resistance (PVR) have been proposed to date, but they remain empirical, lacking sufficient accuracy to be used in clinical practice. The aims of this study were to propose a novel echocardiographic measurement of PVR based on a theoretical formula and investigate the feasibilty and accuracy of this method in patients with heart failure., Methods and results: Echocardiography was performed in 27 patients before right heart catheterization. Peak tricuspid regurgitation pressure gradient (TRPG), pulmonary regurgitation pressure gradient in end-diastole (PRPGed), and cardiac output derived from the time-velocity integral and the diameter in the left ventricular outflow tract (COLVOT) were measured. PVR based on a theoretical formula (PVRtheo) was calculated as (TRPG-PRPGed)/3COLVOTin Wood units (WU). The results were compared with PVR obtained by right heart catheterization (PVRcath) using linear regression and Bland-Altman analysis. Mean PVRcathwas 2.4±1.4 WU. PVRtheocorrelated well with PVRcath(r=0.83, P<0.001). On Bland-Altman analysis the mean difference was 0.1±0.7 WU. The limits of agreements were smaller than for other non-invasive estimations previously reported., Conclusions: The new echocardiographic approach based on a theoretical formula provides a non-invasive and accurate assessment of PVR in patients with heart failure.

A 76-year-old woman was referred to our hospital with a non-healing ulcer and intractable rest pain. Diagnostic angiography revealed total occlusion of long iliac (from right common iliac artery to common femoral artery) and femoropopliteal [from superficial femoral artery (SFA) to popliteal artery] TASC D lesions. The iliac lesion was successfully treated with implantation of two nitinol stents. However, because of severe calcification, the SFA lesion could not be crossed with an antegrade approach alone and a retrograde approach from the high anterior tibial artery was, therefore, added. After wire crossing, the SFA lesion was stented directly followed by balloon dilation. Five months after endovascular treatment, the ulcer had healed completely thereby avoiding major amputation.

A 4-year field trial for the salt tolerant Eucalyptus globulus Labill. harboring the choline oxidase (codA) gene derived from the halobacterium Arthrobacter globiformis was conducted to assess the impact of transgenic versus non-transgenic trees on biomass production, the adjacent soil microbial communities and vegetation by monitoring growth parameters, seasonal changes in soil microbes and the allelopathic activity of leaves. Three independently-derived lines of transgenic E. globulus were compared with three independent non-transgenic lines including two elite clones. No significant differences in biomass production were detected between transgenic lines and non-transgenic controls derived from same seed bulk, while differences were seen compared to two elite clones. Significant differences in the number of soil microbes present were also detected at different sampling times but not between transgenic and non-transgenic lines. The allelopathic activity of leaves from both transgenic and non-transgenic lines also varied significantly with sampling time, but the allelopathic activity of leaves from transgenic lines did not differ significantly from those from non-transgenic lines. These results indicate that, for the observed variables, the impact on the environment of codA-transgenic E. globulus did not differ significantly from that of the non-transformed controls on this field trial.

Although endovascular therapy (EVT) has been widely used for iliac lesions due to acceptable patency with stenting, EVT has not been established as primary treatment for aortic occlusion, partly because of uncertainty regarding long-term results. The purpose of this study was to investigate outcomes following EVT with stenting for chronic aortic occlusion. This study was a single-center retrospective analysis of a prospectively maintained database. Between September 2005 and May 2012, twenty-four lesions from 25 patients with a clinical diagnosis of chronic aortic occlusion (mean age, 71 years; 80 % male) were treated with EVT with stenting. Kaplan-Meier estimators were used to determine the patency rates according to Society for Vascular Surgery criteria. In results, lesion type was Trans Atlantic Inter-Society Consensus D in all patients. Mean lesion length was 145 mm. Initial success rate and procedural complication rate were 96 % (24/25) and 8 % (2/25), respectively. At 36 months, primary and secondary patency rates were 76 and 94 %, respectively. Restenosis was observed in 5 patients, all of whom underwent reintervention (four successful, one failure and intensity of medical treatment). EVT can be safely done in patients with chronic aortic occlusion. Procedural morbidity and mid-term durability were comparable to those of bypass surgery up to 3 years.

The performance of tree species is influenced by environmental factors and growth stages. To evaluate the practical performance of transgenic tree species, it is insufficient to grow small, young trees under controlled conditions, such as in a growth chamber. Three transgenic Eucalyptus globulus lines, carrying the choline oxidase gene, were investigated for their salt tolerance and expression of the transgene at the young plantlet stage in a special netted-house. To clarify the characteristics at the young as well during the later stages, salt tolerance and the properties of the transgenic lines at large juvenile and adult stages were evaluated in the special netted-house. All transgenic lines showed high glycinebetaine content, particularly in young leaves. Trees of the transgenic line 107-1 showed low damage because of salinity stress based on the results from the chlorophyll analysis and malondialdehyde content, and they survived the high-salt-shock treatment at the large juvenile and adult stages. Only this line showed salt tolerance at all stages in the special netted-house. In this evaluation in the special netted-house, the tolerant line among young plantlets might perform better at all stages. Since evaluation in these special netted-house mimics field evaluation, line 107-1 is a potential tolerant line.

Background: Although durable clinical outcomes have been reported, arterial repair after paclitaxel-coated nitinol drug-eluting stent (Zilver PTX) implantation in the superficial femoral artery (SFA) remains unclear., Methods and Results: Angioscopic evaluation was performed in SFA intra-stent surfaces 80±29 (range, 49-135) days or 84±18 (range, 52-112) days following Zilver PTX (20 stents in 10 patients; mean age, 72±8 years; 40% men) or bare metal stent (BMS; 14 stents in 9 patients; mean age, 70±7 years; 67% men) implantation, respectively. Neointimal coverage (NIC) was graded as 0, stent struts exposed; grade 1, struts bulging into the lumen, but still transparently visible although covered; grade 2, struts embedded in the neointima, but translucent; grade 3, struts fully embedded and invisible. NIC was defined as heterogeneous when the NIC grade variation was ≥1. Presence of yellow plaque and thrombus were investigated. Dominant NIC was significantly different between Zilver PTX (grade 0, 35%; grade 1, 20%; grade 2, 25%; grade 3, 20%) and BMS (grade 0, 7%; grade 1, 0%; grade 2, 14%; grade 3, 79%; P=0.001). NIC heterogeneity was less frequently observed in Zilver PTX (40% vs. 86%, P=0.009). Prevalence of yellow plaque or thrombus (75% vs. 79%, P=0.57) or thrombus (75% vs. 79%, P=0.57) were similar between Zilver PTX and BMS., Conclusions: Early phase arterial repair was different between Zilver PTX and BMS.

Several studies showed durable long-term clinical benefit of endovascular therapy with stenting in aorto-iliac occlusive disease. Although in-stent restenosis is easily treated in routine practice, we experienced an uncommon case of failed reconstitution of in-stent total occlusion at the common iliac artery (CIA). The case was treated with nitinol stent implantation outside of the in-stent occlusion site, and good vessel patency was observed at 14 months after the procedure. Nitinol stent implantation outside of an in-stent occlusion in the CIA is a novel reconstitution strategy when the guide wire cannot pass the occlusion site within a previously implanted stent.

Repeated gene targeting by recombinase-mediated cassette exchange (RMCE) is an efficient tool for the study of gene function and regulation because of the high predictability and repeatability of gene expression. We have developed the site-directed integration (SDI) vector system for Agrobacterium-mediated transformation to precisely integrate a single copy of a desired gene into a predefined chromosomal locus in the absence of any coexpressed selection marker gene (Nanto et al. Plant Biotechnol J 3:203-214, 2005; Nanto and Ebinuma Transgenic Res 17:337-344, 2008; Nanto et al. Plant Cell Rep 28:777-785, 2009; Ebinuma and Nanto (2009) Marker-free targeted transformation, in Molecular techniques in crop improvement (2nd Edition). (Jain, S. M. and Brar, D. S. eds.), Springer Netherlands, pp. 527-543; Ebinuma and Nanto in preparation). The SDI vector system consists of a target vector to introduce the target cassette and an exchange vector to reintroduce the exchange cassette for gene replacement. We describe the molecular design and experimental protocol that can efficiently enrich RMCE events through the removal of randomly integrated copies and select clean marker-free targeted transgenic plants by using a negative marker.

Eucalyptus globulus is one of the most economically important plantation hardwoods for paper making. However, its low transformation frequency has prevented genetic engineering of this species with useful genes. We found the hypocotyl section with a shoot apex has the highest regeneration ability among another hypocotyl sections, and have developed an efficient Agrobacterium-mediated transformation method using these materials. We then introduced a salt tolerance gene, namely a bacterial choline oxidase gene (codA) with a GUS reporter gene, into E. globulus. The highest frequency of transgenic shoot regeneration from hypocotyls with shoot apex was 7.4% and the average frequency in four experiments was 4.0%, 12-fold higher than that from hypocotyls without shoot apex. Using about 10,000 explants, over 250 regenerated buds were confirmed as transformants by GUS analysis. Southern blot analysis of 100 elongated shoots confirmed successful generation of stable transformants. Accumulation of glycinebetaine was investigated in 44 selected transgenic lines, which showed 1- to 12-fold higher glycinebetaine levels than non-transgenic controls. Rooting of 16 transgenic lines was successful using a photoautotrophic method under enrichment with 1,000 ppm CO(2). The transgenic whole plantlets were transplanted into potting soil and grown normally in a growth room. They showed salt tolerance to 300 mM NaCl. The points of our system are using explants with shoot apex as materials, inhibiting the elongation of the apex on the selection medium, and regenerating transgenic buds from the side opposite to the apex. This approach may also solve transformation problems in other important plants.

Metabolic engineering aimed at monoterpene production has become an intensive research topic in recent years, although most studies have been limited to herbal plants including model plants such as Arabidopsis. The genus Eucalyptus includes commercially important woody plants in terms of essential oil production and the pulp industry. This study attempted to modify the production of monoterpenes, which are major components of Eucalyptus essential oil, by introducing two expression constructs containing Perilla frutescens limonene synthase (PFLS) cDNA, whose gene products were designed to be localized in either the plastid or cytosol, into Eucalyptus camaldulensis. The expression of the plastid-type and cytosol-type PFLS cDNA in transgenic E. camaldulensis was confirmed by real-time polymerase chain reaction (PCR). Gas chromatography with a flame ionization detector analyses of leaf extracts revealed that the plastidic and cytosolic expression of PFLS yielded 2.6- and 4.5-times more limonene than that accumulated in wild-type E. camaldulensis, respectively, while the ectopic expression of PFLS had only a small effect on the emission of limonene from the leaves of E. camaldulensis. Surprisingly, the high level of PFLS in Eucalyptus was accompanied by a synergistic increase in the production of 1,8-cineole and alpha-pinene, two major components of Eucalyptus monoterpenes. This genetic engineering of monoterpenes demonstrated a new potential for molecular breeding in woody plants.

We developed a site-directed integration (SDI) system for Agrobacterium-mediated transformation to precisely integrate a single copy of a desired gene into a predefined target locus by recombinase-mediated cassette exchange (RMCE). We produced site-specific transgenic tobacco plants from four target lines and examined expression of the transgene in T1 site-specific transgenic tobacco plants, which were obtained by backcrossing. We found that site-specific transgenic plants from the same target lines showed approximately the same level of expression of the transgene. Moreover, we demonstrated that site-specific transgenic plants showed much less variability of transgene expression than random-integration transgenic plants. Interestingly, transgenes in the same direction at the same target locus showed the same level of activity, but transgenes in different directions showed different levels of activity. The expression levels of transgene did not correlate with those of the target gene. Our results showed that the SDI system could benefit the precise comparisons between different gene constructs, the characterization of different chromosomal regions and the cost-effective screening of reliable transgenic plants.

Recently, site-specific recombination methods in plants have been developed to delete selection markers to produce marker-free transgenic plants or to integrate the transgene into a pre-determined genomic location to produce site-specific transgenic plants. However, these methods have been developed independently, and although the strategies of producing marker-free site-specific integration plants have been discussed, the concept has not been demonstrated. In the present study, we combined two approaches to site-specific recombination and demonstrated the concepts for removing the marker after site-specific integration for producing marker-free site-specific transgenic plants.

We describe the site-directed integration (SDI) system for Agrobacterium-mediated transformation to precisely integrate a single copy of a desired gene into a predefined target locus by recombinase-mediated cassette exchange (RMCE). The system requires the selection of a transformed line with an integrated copy of a target cassette, and subsequent introduction of an exchange vector. The target cassette contains the npt and cod genes between oppositely orientated recognition sites (RS). The exchange vector T-DNA possesses an exchange cassette containing the gene of interest and a selectable marker gene, such as hpt, between oppositely orientated (inner) RS. Adjacent to the exchange cassette are ipt and recombinase (R) genes and an additional (outer) RS. The recombinase catalyses double-crossover between target RS and exchange inner RS to replace the integrated target cassette with the introduced exchange cassette. Transgenic plants that contain randomly integrated copies of the exchange vector T-DNA show an abnormal phenotype as a result of the overproduction of cytokinin from ipt gene expression. The recombinase can also act on the directly orientated outer RS to remove such randomly integrated copies. The system resulted in single-copy exchange into the target site only in regenerated tobacco at a frequency of 1%-3% per treated explant, or 4%-9% per regenerated line of normal phenotype. Thus, transgenic plants with only an exchanged copy can be efficiently accumulated and selected. Here, we show that the SDI system can efficiently replace the target cassettes with the exchange cassettes in a heterozygous or homozygous condition. The SDI system may be useful for precise comparisons of different gene constructs, the characterization of different chromosomal regions and the cost-effective screening of reliable transgenic plants.

The polypeptide encoded by the partial fragment of cDNA of phenylalanine ammonia-lyase (PAL; EC 4.3.1.5), PALcDNA1 (Osakabe et al., 1995, Plant Sci. 105: 217-226), isolated from Populus kitakamiensis (P. sieboldii x P. grandidentata), was expressed in Escherichia coli cells. The polypeptide was purified and an antiserum raised against it. The antiserum recognized a protein of 77 kDa on nitrocellulose blots after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total protein and the partially purified PAL protein from P. kitakamiensis. Moreover, the antiserum recognized a protein on the blot after non-denaturing polyacrylamide gel electrophoresis of P. kitakamiensis proteins and this protein had PAL activity. Furthermore, the antibody inhibited PAL activity of extracts from stem tissues. These results showed that the antiserum against the partial PAL peptide recognized only the PAL subunits in extracts of P. kitakamiensis. Immunolocalization studies of P. kitakamiensis tissues revealed that the PAL protein was specifically localized in the xylem and the phloem fibers and no immunogold signal was found in the epidermis, the cortex, the pith, or the cambium of either stems or leaves.