Nationwide study of surgery for primary infected abdominal aortic and common iliac artery aneurysms.

BACKGROUND: Primary infected aneurysms of the abdominal aorta and iliac arteries are potentially life-threatening. However, because of the rarity of the disease, its pathogenesis and optimal treatment strategy remain poorly defined. METHODS: A nationwide retrospective cohort study investigated patients who underwent surgical treatment for a primary infected abdominal aortic and/or common iliac artery (CIA) aneurysm between 2011 and 2017 using a Japanese clinical registry. The study evaluated the relationships between preoperative factors and postoperative outcomes including 90-day and 3-year mortality, and persistent or recurrent aneurysm-related infection. Propensity score matching was used to compare survival between patients who underwent in situ prosthetic grafting and those who had endovascular aneurysm repair (EVAR). RESULTS: Some 862 patients were included in the analysis. Preceding infection was identified in 30.2 per cent of the patients. The median duration of postoperative follow-up was 639 days. Cumulative overall survival rates at 30 days, 90 days, 1 year, 3 years and 5 years were 94.0, 89.7, 82.6, 74.9 and 68.5 per cent respectively. Age, preoperative shock and hypoalbuminaemia were independently associated with short-term and late mortality. Compared with open repair, EVAR was more closely associated with persistent or recurrent aneurysm-related infection (odds ratio 2.76, 95 per cent c.i. 1.67 to 4.58; P < 0.001). Propensity score-matched analyses demonstrated no significant differences between EVAR and in situ graft replacement in terms of 3-year all-cause and aorta-related mortality rates (P = 0.093 and P =0.472 respectively). CONCLUSION: In patients undergoing surgical intervention for primary infected abdominal aortic and CIA aneursyms, postoperative survival rates were encouraging. Eradication of infection following EVAR appeared less likely than with open repair, but survival rates were similar in matched patients between EVAR and in situ graft replacement.

Long-term follow-up of the randomized trial of mesorectal excision with or without lateral lymph node dissection in rectal cancer (JCOG0212).

BACKGROUND: Japan Clinical Oncology Group (JCOG) 0212 (ClinicalTrials.gov NCT00190541) was a non-inferiority phase III trial of patients with clinical stage II-III rectal cancer without lateral pelvic lymph node enlargement. The trial compared mesorectal excision (ME) with ME and lateral lymph node dissection (LLND), with a primary endpoint of recurrence-free survival (RFS). The planned primary analysis at 5 years failed to confirm the non-inferiority of ME alone compared with ME and LLND. The present study aimed to compare ME alone and ME with LLND using long-term follow-up data from JCOG0212. METHODS: Patients with clinical stage II-III rectal cancer below the peritoneal reflection and no lateral pelvic lymph node enlargement were included in this study. After surgeons confirmed R0 resection by ME, patients were randomized to receive ME alone or ME with LLND. The primary endpoint was RFS. RESULTS: A total of 701 patients from 33 institutions were assigned to ME with LLND (351) or ME alone (350) between June 2003 and August 2010. The 7-year RFS rate was 71.1 per cent for ME with LLND and 70·7 per cent for ME alone (hazard ratio (HR) 1·09, 95 per cent c.i. 0·84 to 1·42; non-inferiority P = 0·064). Subgroup analysis showed improved RFS among patients with clinical stage III disease who underwent ME with LLND compared with ME alone (HR 1·49, 1·02 to 2·17). CONCLUSION: Long-term follow-up data did not support the non-inferiority of ME alone compared with ME and LLND. ME with LLND is recommended for patients with clinical stage III disease, whereas LLND could be omitted in those with clinical stage II tumours.

ANTECEDENTES: El JCOG0212 (ClinicalTrials.gov: NCT00190541) fue un ensayo fase III de no inferioridad en pacientes con cáncer de recto en estadio clínico II/III sin ganglios linfáticos aumentados de tamaño en la pared pélvica lateral. El ensayo comparó la escisión del mesorrecto (mesorectal excision, ME) con la ME con disección de los ganglios linfáticos laterales (lateral lymph node dissection, LLND), siendo el criterio de valoración principal la supervivencia libre de recidiva (recurrence free survival, RFS). El análisis primario planificado a los 5 años de seguimiento no pudo confirmar la no inferioridad de la ME frente a la ME con LLND. Este estudio tuvo como objetivo comparar la ME como procedimiento único y la ME con LLND utilizando datos de seguimiento a largo plazo del ensayo JCOG0212. MÉTODOS: En este estudio se incluyeron pacientes con cáncer de recto en estadio clínico II/III por debajo de la reflexión peritoneal sin ganglios linfáticos aumentados de tamaño en la pared pélvica lateral. Después de que los cirujanos confirmaran la resección R0 mediante la ME, los pacientes fueron asignados al azar al brazo de ME sola o al brazo de ME con LLND. El criterio de valoración principal fue la supervivencia libre de recidiva (RFS). RESULTADOS: Un total de 701 pacientes de 33 instituciones fueron asignados al azar para ser tratados mediante una ME con LLND (n = 351) o EM sola (n = 350) entre junio de 2003 y agosto de 2010. Las tasas de RFS a 7 años fueron del 71,1% para ME con LLND y 70,7 % para ME sola (cociente de riesgos instantáneos, hazard ratio, HR: 1,09 (i.c. del 95% 0,84-1,42), no inferioridad P = 0,064)). El análisis de subgrupos mostró una mejor RFS entre los pacientes en estadio clínico III que se sometieron a ME con LLND en comparación con ME sola (HR: 1,49 (i.c. del 95%: 1,02-2,17)). CONCLUSIÓN: Los datos de seguimiento a largo plazo no justificaron la no inferioridad de la ME en comparación con la ME con LLND. Se recomienda la ME con LLND para pacientes en estadio clínico III, mientras que LLND podría omitirse para pacientes en estadio clínico II.

Frequency-Dependent Squeezing for Advanced LIGO.

The first detection of gravitational waves by the Laser Interferometer Gravitational-Wave Observatory (LIGO) in 2015 launched the era of gravitational-wave astronomy. The quest for gravitational-wave signals from objects that are fainter or farther away impels technological advances to realize ever more sensitive detectors. Since 2019, one advanced technique, the injection of squeezed states of light, is being used to improve the shot-noise limit to the sensitivity of the Advanced LIGO detectors, at frequencies above ∼50 Hz. Below this frequency, quantum backaction, in the form of radiation pressure induced motion of the mirrors, degrades the sensitivity. To simultaneously reduce shot noise at high frequencies and quantum radiation pressure noise at low frequencies requires a quantum noise filter cavity with low optical losses to rotate the squeezed quadrature as a function of frequency. We report on the observation of frequency-dependent squeezed quadrature rotation with rotation frequency of 30 Hz, using a 16-m-long filter cavity. A novel control scheme is developed for this frequency-dependent squeezed vacuum source, and the results presented here demonstrate that a low-loss filter cavity can achieve the squeezed quadrature rotation necessary for the next planned upgrade to Advanced LIGO, known as "A+."

Randomized clinical trial of duct-to-mucosa versus invagination pancreaticojejunostomy after pancreatoduodenectomy.

BACKGROUND: The postoperative pancreatic fistula (POPF) rate for duct-to-mucosa and invagination anastomosis after pancreatoduodenectomy is still debated. The aim of this RCT was to investigate the POPF rate for duct-to-mucosa versus invagination pancreaticojejunostomy. METHODS: Patients were stratified by pancreatic texture and diameter of the main pancreatic duct and randomized to the duct-to-mucosa or invagination group. The primary endpoint was the rate of clinically relevant POPF (defined as grade B or C). Secondary endpoints were suture material cost for pancreaticojejunostomy, drain insertion duration and duration of postoperative hospital stay. RESULTS: Some 120 patients undergoing pancreatoduodenectomy were included following consent. Clinically relevant POPF developed in six of 59 patients (10 per cent) in the invagination group and in 14 of 61 patients (23 per cent) in the duct-to-mucosa group (P = 0·077). Duration of drain insertion (6 versus 7 days respectively; P = 0·027) and postoperative hospital stay (19 versus 24 days; P = 0·015) were shorter in the invagination group. Subgroup analysis for 61 patients with a soft pancreas revealed a lower rate of clinically relevant POPF in the invagination group (10 per cent versus 42 per cent in the duct-to-mucosa group; P = 0·010). Among 20 patients with a clinically relevant POPF, the six patients in the invagination group had a shorter duration of drain insertion (38·5 days versus 49 days for 14 patients in the duct-to-mucosa group; P = 0·028) and postoperative hospital stay (42 versus 54·5 days respectively; P = 0·028). CONCLUSION: This study did not demonstrate a superiority of invagination over duct-to-mucosa pancreaticojejunostomy in the risk of POPF. However, in high-risk patients with a soft pancreas, invagination may reduce the risk of clinically relevant POPF compared with duct-to-mucosa. Registration number: UMIN000005890 (http://www.umin.ac.jp).

Efficacy of pegfilgrastim administration in patients with esophageal cancer treated with docetaxel, cisplatin, and 5-fluorouracil.

Chemotherapy is among the standard treatments for esophageal cancer. The docetaxel, 5-fluorouracil, and cisplatin (DCF) protocol yields a better response rate than 5-fluorouracil plus cisplatin. However, the incidence of side effects, such as febrile neutropenia and hematologic toxicity, is also significantly high with the DCF protocol. The granulocyte colony-stimulating factor and pegfilgrastim are prophylactically administered to prevent febrile neutropenia. This retrospective study evaluated the efficacy and safety of pegfilgrastim in patients receiving DCF therapy. Of the 65 patients who were administered DCF therapy in our hospital from 2011 through 2016, 21 received pegfilgrastim 24 hours or more after the end of chemotherapy. The protocol comprised 70 mg/m2 each of docetaxel and cisplatin on day 1 and 700 mg/m2 5-fluorouracil on days 1 to 5 via intravenous injection in a 3-week cycle. The primary endpoint was the rate of grade 3-4 neutropenia and febrile neutropenia. The mean patient age was 66.4 years. The incidence of grade 3 and 4 neutropenia was 14.2 % and 11.4 %, respectively, in the pegfilgrastim group and 31.9 % and 37.8 %, respectively, in the non-pegfilgrastim group. The incidence of febrile neutropenia in the pegfilgrastim group and non-pegfilgrastim group was 11.4 % and 40.3 %, respectively. Statistical analysis showed that the incidence of neutropenia and febrile neutropenia was significantly different (p<0.05) between the two groups. Pegfilgrastim prevents severe neutropenia and febrile neutropenia in patients with esophageal cancer who are treated according to the DCF protocol.

Controlled direct electron transfer kinetics of fructose dehydrogenase at cup-stacked carbon nanofibers.

Graphene edge sites not only facilitate heterogeneous electron transfer reactions of redox species because of localization of electrons, but also allow sensitivities and selectivities to be tuned by controlling the atomic oxygen/carbon (O/C) ratio. Here, we immobilized fructose dehydrogenase (FDH) onto the surface of cup-stacked carbon nanofibers (CSCNFs), which provide highly ordered graphene edges with a controlled O/C ratio, and investigated the direct electron communication with FDH. As the O/C ratio decreased at the CSCNF surface, the negative zeta potential was mitigated and the electrochemical communication with FDH was facilitated. This is likely due to improved orientation of FDH molecules on the CSCNF surface. CSCNFs with a controlled O/C ratio could be applied to FDH-based d-fructose biosensors with tunable dynamic range and fructose biofuel cells with a controlled maximum current.

Feeding catheter gastrostomy with the round ligament of the liver prevents mechanical bowel obstruction after esophagectomy.

Jejunostomy, which requires the fixation of the jejunum to the abdominal wall, is commonly used as an enteral feeding access after esophagectomy. However, this procedure sometimes causes severe complications, such as mechanical bowel obstruction. In 2009, we developed a modified approach to insert an enteral feeding tube through the reconstructed gastric tube using the round ligament of the liver. The aim of this study is to investigate the usefulness of this approach as compared to the approach through jejunostomy. Between January 2005 and March 2015, 420 patients with thoracic esophageal cancer underwent esophagectomy via thoracotomy and laparotomy. Of these, 214 underwent feeding jejunostomy (FJ group) and 206 patients underwent feeding via gastric tube with round ligament of the liver (FG group). Catheter-related complications, other postoperative complications, and mortality were compared between the two groups. The incidence of catheter site infection during catheterization in the FG group was significantly lower (n = 1/206, 0.5%) compared to the FJ group (n = 11/214, 5.1%) (P < 0.01). The postoperative bowel obstruction did not occur in the FG group, while it occurred in eight patients (3.7%) in the FJ group (P < 0.01). The incidences of other catheter-related and postoperative complications were similar between the two groups. Feeding catheter gastrostomy with the round ligament of the liver can be a useful enteral feeding access after esophagectomy, because the incidence rate of severe catheter-related complications, such as surgical site infection and mechanical obstruction tend to be lower with this technique compare to jejunostomy.

Clinical outcomes after infrainguinal bypass grafting for critical limb ischaemia in patients with dialysis-dependent end-stage renal failure.

OBJECTIVE: To assess and risk-stratify the medium-term clinical outcomes after infrainguinal bypass grafting (IBG) to treat critical limb ischaemia (CLI) in patients with end-stage renal disease. METHODS: This was a retrospective single-centre study. Between April 2007 and March 2011, 112 limbs from 89 patients were studied. In particular, amputation-free survival (AFS), 30 day mortality, freedom from major adverse limb events (MALE), limb salvage, and overall survival were examined. The aim was to identify outcome predictors. RESULTS: Eight patients (9%) died within 30 days of IBG. The only positive predictor of 30-day mortality was an ejection fraction (EF) < 40% (hazard ratio [HR] 5.57, 95% confidence interval [CI] 1.16-26.83; p = .03). The mean follow-up duration was 14 months. The 1- and 2-year AFS rates were 64% and 43%, respectively, and the rates of freedom from MALE were 81% and 77%, respectively. In addition, the 1- and 2-year limb salvage rates were 89% and 85%, and the survival rates were 68% and 50%, respectively. Non-ambulatory status was negatively associated with AFS (HR 3.04, 95% CI 1.59-5.82; p < .01), freedom from MALE (HR 4.98, 95% CI 1.91-12.96; p < .01), and limb salvage (HR 5.18, 95% CI 1.47-18.30; p = .01). The other negative predictors of overall survival were a serum albumin level <3.0 g/dL (HR 2.26, 95% CI 1.12-4.58; p = .02) and an EF <40% (HR 2.24, 95% CI 1.05-4.79; p = .04). CONCLUSION: Patients with CLI on dialysis enjoyed satisfactory freedom from MALE and limb salvage, but survival and AFS were significantly less than reported for IBG in patients with CLI who did not receive dialysis. In addition, patients with an EF <40%, lower serum albumin (<3.0 g/dL), or non-ambulatory status experienced particularly poor clinical outcomes after IBG.

Occurrence of selected pharmaceuticals in river water in Japan and assessment of their environmental risk.

The existence of pharmaceuticals in the water environment is thought to be a potential problem for aquatic organisms. In this study, we conducted a nationwide survey to clarify the occurrence of 24 selected pharmaceuticals in major Japanese rivers and evaluated their environmental risk to aquatic organisms. We found a total of 22 substances in river waters at concentrations from several nanograms per liter to several micrograms per liter. We found the highest, which was 2.4 µg/L of caffeine, followed by 1.5 µg/L of crotamiton and 1.4 µg/L of sulpiride. We conducted an environmental risk assessment of the 22 pharmaceuticals detected in river water, for which predicted no-effect concentration (PNEC) values for crustacea and algae had been obtained. The measured environmental concentration/PNEC values of four substances, caffeine, carbamazepine, clarithromycin, and ketoprofen, exceeded 0.1 with the maximum value of 9.0 for clarithromycin. As clarithromycin exhibits a high environmental risk to aquatic organisms, particular attention is required.

Cilostazol stimulates revascularisation in response to ischaemia via an eNOS-dependent mechanism.

OBJECTIVES: Cilostazol is known to be a selective inhibitor of phosphodiesterase 3 and is generally used to treat intermittent claudication caused by peripheral arterial disease. However, there is little information concerning the effect of cilostazol on angiogenesis. Here, we investigated whether cilostazol modulates the angiogenic process in vivo employing a hindlimb model of ischaemia-induced angiogenesis. DESIGN: This was an experimental study. MATERIALS AND METHODS: Wild-type (WT) mice were randomly divided into two groups and were treated with or without cilostazol. One week later, the mice were subjected to unilateral hindlimb ischaemia. Angiogenesis was determined by laser Doppler analysis and capillary density stained with CD31. The expression of endothelial nitric oxide synthase (eNOS) was assessed by immunoblotting. RESULTS: WT mice treated with cilostazol showed accelerated neo-vascularisation following hindlimb ischaemic surgery on post-operative day 14 based upon laser Doppler measurements of blood flow (cilostazol-treated group, 0.54 ± 0.13 vs. control group, 0.38 ± 0.11; P-<-0.05). The capillary density in the ischaemic hindlimb was also significantly greater in WT mice treated with cilostazol than in non-treated WT mice (cilostazol-treated group, 1.63 ± 0.10 vs. control group, 1.15 ± 0.12; P-<-0.01). Cilostazol stimulated an ischaemia-induced increase in the phosphorylation of eNOS in the ischaemic limbs. Administration of NOS inhibitor N-nitro-l-arginine methyl ester (l-NAME) abolished cilostazol-induced increase in limb perfusion. CONCLUSIONS: Our observations indicate that cilostazol can promote neo-vascularisation in response to tissue ischaemia via an eNOS-dependent mechanism. Cilostazol could be useful for treatment of ischaemic limb diseases.

Mass balance analysis of triclosan, diethyltoluamide, crotamiton and carbamazepine in sewage treatment plants.

The behavior of antibacterial triclosan, insect-repellent diethyltoluamide (DEET), anticonvulsant carbamazepine, and antipruritic crotamiton was investigated at two sewage treatment plants (STPs) to clarify their complete mass balance. Twenty-four-hour flow-proportional composite samples were collected from the influent and effluent of primary and final sedimentation tanks, a biofiltration tank and disinfection tanks. Sludge samples (i.e., activated and excess sludge) and samples of the return flow from the sludge treatment process were collected in the same manner. The analytes in both the dissolved and particulate phases were individually determined by a gas chromatograph equipped with mass spectrometer. Triclosan was dominantly detected in the particulate phase especially in the early stage of treatment (up to 83%) and was efficiently removed (over 90%) in STPs, mainly by sorption to sewage sludge. Limited removal was observed for DEET (55+/-24%), while no significant removal was demonstrated for crotamiton or carbamazepine. The solid-water distribution coefficients (K(d), n=4) for triclosan (log K(d): 3.7-5.1), DEET (1.3-1.9) and crotamiton (1.1-1.6) in the sludge samples are also determined in this study. These findings indicate the limitations of current sewage treatment techniques for the removal of these water-soluble drugs (i.e. DEET, carbamazepine, and crotamiton).

Nomograms predicting survival and recurrence in colonic cancer in the era of complete mesocolic excision.

Background: More extensive lymphadenectomy may improve survival after resection of colonic cancer. Nomograms were created predicting overall survival and recurrence for patients who undergo D2-D3 lymph node dissection, and their validity determined. Methods: This was a multicentre study of patients with colonic cancer who underwent resection with D2-D3 lymph node dissection in Japan. Inclusion criteria included R0 resection. A training cohort of patients operated on from 2007 to 2008 was analysed to construct prognostic models predicting survival and recurrence. Discrimination and calibration were performed using an external validation cohort from the Japanese colorectal cancer registry (procedures in 2005-2006). Results: The training cohort consisted of 2746 patients. Predictors of survival were: age (hazard ratio (HR) 1·04), female sex (HR 0·71), depth of tumour invasion (HR 1·15, 1·22, 2·96 and 3·14 for T2, T3, T4a and T4b respectively versus T1), lymphatic invasion (HR 1·11, 1·15 and 2·95 for ly1, ly2 and ly3 versus ly0), preoperative carcinoembryonic antigen (CEA) level (HR 1·21, 1·59 and 1·99 for 5·1-10·0, 10·1-20·0 and 20·1 and over versus 0-5·0 ng/ml), number of metastatic lymph nodes (HR 1·07), number of lymph nodes examined (HR 0·98) and extent of lymphadenectomy (HR 0·23, 0·13 and 0·11 for D1, D2 and D3 versus D0). Predictors of recurrence were: female sex (HR 0·82), macroscopic type (HR 3·82, 4·56, 6·66, 7·74 and 3·22 for types I, II, III, IV and V versus type 0), depth of invasion (HR 1·25, 2·66, 5·32 and 6·43 for T2, T3, T4a and T4b versus T1), venous invasion (HR 1·43, 3·05 and 4·79 for v1, v2 and v3 versus v0), preoperative CEA level (HR 1·39, 1·43, 1·56 and 1·85 for 5·1-10·0, 10·1-20·0, 20·1-40·0 and 40·1 or more versus 0-5 ng/ml), number of metastatic lymph nodes (HR 1·07) and number of lymph nodes examined (HR 0·98). The validation cohort comprised 4446 patients. The internal and external validated Harrell's C-index values for the nomogram predicting survival were 0·75 and 0·74 respectively. Corresponding values for recurrence were 0·78 and 0·75. Conclusion: These nomograms could predict survival and recurrence after curative resection of colonic cancer.

Serum procalcitonin (PCT) as a negative screening test for colonic ischemia after open abdominal aortic surgery.

BACKGROUND AND AIM: We assessed serum procalcitonin (PCT) as a screening test for early detection of ischemic colitis. PATIENTS AND METHODS: Ninety-three patients (81 men and 12 women) undergoing elective aortic surgery were enrolled in this study. Their mean age was 70.3+/-8.1 years old. Serum procalcitonin was measured postoperatively. RESULTS: Four patients suffered from colon ischemia. Based on a cut off value of serum PCT>/=2.0 ng/ml, fourteen patients had a false positive but none had a false negative result. Sensitivity was 100%, and specificity was 83.9% in detecting ischemic colitis. Negative predictive vale was 100%. CONCLUSION: Serum PCT is a non-invasive test that has a high negative predictive vale in ruling out colon ischemia after aortic surgery.

Archaeal primase: bridging the gap between RNA and DNA polymerases.

In the evolution of life, DNA replication is a fundamental process, by which species transfer their genetic information to their offspring. DNA polymerases, including bacterial and eukaryotic replicases, are incapable of de novo DNA synthesis. DNA primases are required for this function, which is sine qua non to DNA replication. In Escherichia coli, the DNA primase (DnaG) exists as a monomer and synthesizes a short RNA primer. In Eukarya, however, the primase activity resides within the DNA polymerase alpha-primase complex (Pol alpha-pri) on the p48 subunit, which synthesizes the short RNA segment of a hybrid RNA-DNA primer. To date, very little information is available regarding the priming of DNA replication in organisms in Archaea. Available sequenced genomes indicate that the archaeal DNA primase is a homolog of the eukaryotic p48 subunit. Here, we report investigations of a p48-like DNA primase from Pyrococcus furiosus, a hyperthermophilic euryarchaeote. P. furiosus p48-like protein (Pfup41), unlike hitherto-reported primases, does not catalyze by itself the synthesis of short RNA primers but preferentially utilizes deoxynucleotides to synthesize DNA fragments up to several kilobases in length. Pfup41 is the first DNA polymerase that does not require primers for the synthesis of long DNA strands.

Occurrence of 70 pharmaceutical and personal care products in Tone River basin in Japan.

The occurrence of 70 pharmaceutical and personal care products (PPCPs) was investigated in the Tone River. The river has the largest basin in Japan, and the water is utilized not only for farming, but also as a source of water supply. One day in both January and October 2006, surface waters in the river and its tributaries and effluents from sewage treatment plants (STPs) directly discharging into the Tone River were collected, the location of which ranged over 150 km along the river. The 70 PPCPs in the samples were concentrated by solid phase cartridge and were measured by LC-MS/MS using three analytical methods. Fifty-seven PPCPs were detected in one or more samples. Bezafibrate, caffeine, carbamazepine, clarithromycin, crotamiton and sulpiride were frequently detected. Mass flow profiles of some PPCPs (e.g., crotamiton) were comparable to cumulative inhabitants in the basin, suggesting that these PPCPs could be markers of population. Total load of each PPCP into the basin from upstream, the tributaries, and the STPs were calculated. The contribution of selected PPCPs from the tributaries with lower sewerage system coverage was dominant compared to those from upstream and the STPs, suggesting the installation of sewerage systems is necessary to reduce the load of PPCPs in the Tone River basin.

PI-PfuI and PI-PfuII, intein-coded homing endonucleases from Pyrococcus furiosus. II. Characterization Of the binding and cleavage abilities by site-directed mutagenesis.

PI- Pfu I and PI- Pfu II from Pyrococcus furiosus are homing endonucleases, as shown in the accompanying paper. These two endonucleases are produced by protein splicing from the precursor protein including ribonucleotide reductase (RNR). We show here that both enzymes specifically interact with their substrate DNA and distort the DNA strands by 73 degrees and 67 degrees, respectively. They have two copies of the amino acid sequence motif LAGLIDADG, which is present in the majority of homing endonucleases and provides some of the catalytic residues necessary for DNA cleavage activity. Site-specific mutagenesis studies showed that two acidic residues in the motifs, Asp149 and Glu250 in PI- Pfu I, and Asp156 and Asp249 in PI- Pfu II, were critical for catalysis. The third residues of the active site triads, as predicted from the structure of PI- Sce I, were Asn225 in PI- Pfu I and Lys224 in PI- Pfu II. Substitution of Asn225 in PI- Pfu I by Ala did not affect catalysis. The cleavage activity of PI- Pfu II was 50-fold decreased by the substitution of Ala for Lys224. The binding affinity of the mutant protein for the substrate DNA also decreased 6-fold. The Lys in PI- Pfu II may play a direct or indirect role in catalysis of the endonuclease activity.

Hjc resolvase is a distantly related member of the type II restriction endonuclease family.

Hjc resolvase is an archaeal enzyme involved in homologous DNA recombination at the Holliday junction intermediate. However, the structure and the catalytic mechanism of the enzyme have not yet been identified. We performed database searching using the amino acid sequence of the enzyme from Pyrococcus furiosus as a query. We detected 59 amino acid sequences showing weak but significant sequence similarity to the Hjc resolvase. The detected sequences included DPN:II, HAE:II and Vsr endonuclease, which belong to the type II restriction endonuclease family. In addition, a highly conserved region was identified from a multiple alignment of the detected sequences, which was similar to an active site of the type II restriction endonucleases. We substituted three conserved amino acid residues in the highly conserved region of the Hjc resolvase with Ala residues. The amino acid replacements inactivated the enzyme. The experimental study, together with the results of the database searching, suggests that the Hjc resolvase is a distantly related member of the type II restriction endonuclease family. In addition, the results of our database searches suggested that the members of the RecB domain superfamily are evolutionarily related to the type II restriction endonuclease family.