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Thoracic aortic aneurysms evolving within a type IIIb chronic aortic dissection are mostly treated with the deployment of an endograft. However, several cases of dissecting aneurysms are associated with a significant dilatation of the aortic arch. These cases are usually managed in 2 steps: arch reconstruction or supra-aortic trunk debranching at first and a secondary graft deployment for the descending thoracic aorta. We present through this case series an alternative approach for this severe condition which consists in the replacement of the thoracic aorta from its hemi-arch to the distal thoracic or visceral aorta using a left thoracotomy. We deliberately neglected the remaining dissecting aorta if its diameter was below 45 mm, hypothesizing its nonevolution after repair. From 2012 to 2021, 9 patients have been treated for a thoracic aneurysm evolving after a IIIb chronic aortic dissection using a left thoracotomy and a 19°C circulatory arrest. Immediate postoperative results show no mortality or neurological disorders, and the 7 years follow-up for all of these 9 cases enlightened the absence of aneurysmal evolution especially for the distal anastomosis and the remaining dissected aorta. This work suggests that this direct approach strategy can definitively treat a thoracic dissecting aneurysm unsuitable for a simple endovascular treatment., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Endovascular aortic surgery is increasingly becoming the standard treatment. Percutaneous access preclosing systems appear to be effective and notably the Proglide (PG). We aimed to prospectively assess the clinical effectiveness of combining ultrasound-guided femoral puncture with ultrasound-guided PG deployment., Methods: Our single-center study consecutively included patients managed at a tertiary center from May to September 2023, undergoing endovascular aortic surgery. The placement of PG was performed under ultrasound guidance. Preoperative patient characteristics were evaluated using preoperative computed tomography scans. Clinical and technical success were defined, respectively, as the ability to achieve complete hemostasis confirmed by ultrasound 48 hr postprocedure and as the successful placement of a PG under ultrasound guidance contributing to final hemostasis., Results: Twenty patients were included over a 6-month period, totaling 34 common femoral arteries (CFAs). Fourteen were male, with an average age of 72.8 ± 8.2 years. Among the 34 CFA, CFA had diameter of 12.05 ± 2.4 mm and a depth of 38.0 ± 13.4 mm. The mean introducer sheath diameter was 6.2 ± 1.5 mm with a sheath to femoral artery ratio of 0.54 ± 0.18. Successful Proglide placement under ultrasound guidance was achieved in 100% of cases. No PG failure occurred. Clinical and technical success were, respectively, of 95% and 100%. One small pseudoaneurysm was observed at 48 hr treated medically. No CFA access reintervention was required., Conclusions: The technique of ultrasound-guided PG deployment in aortic surgery is a safe and effective method for achieving hemostasis. It effectively prevents PG failures at a lower cost., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The purpose of this article is to highlight an innovative technique in the surgical management of aortic aneurysms in the presence of a horseshoe kidney. The technique involves an anterograde aortic bypass from the distal thoracic aorta to the major renal artery with the primary advantage to a significant reduction in renal ischemia time., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Among bone cells, osteocytes are the most abundant, but also the most challenging to study because they are located inside a dense mineralized matrix. Due to their involvement in bone homeostasis, diverse tools are needed to understand their roles in bone physiology and pathology. This work was aimed at establishing a laser-assisted microdissection protocol to isolate osteocytes and analyze their gene expressions. The goal was to overcome the limitations of the technique currently most used: RNA extraction from the whole bone. To perform laser microdissection and subsequent gene expression analysis, the five main steps of the protocol have been adapted for the bone tissue. After testing many parameters, we found that the best options were (1) take unfixed snap-frozen tissue, (2) cryosection with a supported tape system to improve the tissue morphology if necessary, (3) microdissect regions of interest, and (4) recover the bone pieces by catapulting, if feasible, or by gravity. Finally, RNA extraction (5) was the most efficient with a precipitation method and allowed quantifying the expression of well described osteocyte genes ( Gja1/Cx43 , Phex , Pdpn , Dmp1 , Sost ). This work describes two protocols optimized for femur and calvaria and gives an overview of the many optimization options that one could try when facing difficulties with laser microdissection., Competing Interests: No competing interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of the American Society for Bone and Mineral Research.)

The widespread use of computed tomography (CT) for diagnosing and screening abdominal conditions often reveals rare, asymptomatic anomalies. There is a wide range of documented congenital variations in the anatomy of the inferior vena cava (IVC) and hepatic veins. In this report, we detail an exceptionally unusual variant of the IVC that follows a frontward and intraliver course, terminating at the anterior section of the right atrium. To gain a deeper insight into this anomaly, we employed 3D reconstruction techniques using the software Slicer and Blender., (© 2024. The Author(s), under exclusive licence to Springer-Verlag France SAS, part of Springer Nature.)

Objective: Management of postdissection thoracoabdominal aneurysms with a fenestrated and/or branched endograft (F/BEVAR) is associated with favorable outcomes. Treatment should include both true lumen endografting and false lumen occlusion (FLO). Favorable results have recently been reported for FLO in the false lumen of the thoracic aorta. The purpose of this study is to analyze the results of FLO of the abdominal aorta in patients treated for post dissection thoracoabdominal aneurysm., Methods: A multicenter retrospective analysis of prospective data of consecutive patients managed for post dissection thoracoabdominal aortic aneurysm from April 2019 to December 2022 with F/BEVAR associated with FLO in the abdominal false lumen was conducted. The STrengthening the Reporting of OBservational studies in Epidemiology (STROBE) was followed. Baseline demographics, operative details, and early outcomes (mortality, length of stay) were recorded. Primary endpoints were technical and clinical success. FLO technical success was defined as complete occlusion of false lumen backflow above the FLO on completion angiogram., Results: During the 3-year study, 23 patients were treated for post dissection thoracoabdominal aneurysm with F/BEVAR and the use of abdominal FLO. Twenty-one patients (91.3%) had received prior endovascular treatment. The technical and clinical success was 95.7%. The abdominal FLO had a technical success rate of 78.3%. The median diameter of the FLO was 34 mm. No patient died during the perioperative period, and one patient had spinal cord ischemia (4.3%) with partial recovery. Six patients (26.1%) required early reintervention. The median duration of hospitalization in the intensive care unit and overall was 1 day (interquartile range, 0-3 days) and 7.5 days (interquartile range, 2-22 days), respectively. During the mean follow-up of 9.9 ± 9.0 months, no patient died. False lumen occlusion was complete or partial in nine (39.1%) and nine (39.1%) patients, respectively. No aortic rupture occurred during follow-up. Maximum aortic diameter decreased in 48% and remained stable in 39% of cases., Conclusions: Abdominal aorta FLO during endovascular treatment of post dissection thoracic abdominal aortic aneurysm is associated with favorable outcomes. It offers an additional staging therapeutic option before extensive aorto-bi-iliac coverage, associated with low spinal cord ischemia rates. FLO also provides high rates of false lumen occlusion and false lumen remodeling during follow-up. Longer follow-up and larger cohorts are required to confirm these very promising early findings., (Copyright © 2023 Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved.)

Introduction: Arteriovenous fistula (AVF) rarely occurs in the portal venous system. Aetiologies include iatrogenic, surgical, and penetrating trauma of the abdomen. Clinical manifestations of superior mesenteric portal arteriovenous fistula (SMPAVF) are right heart failure, mesenteric ischaemia, or signs of portal hypertension., Report: The case of a 42 year old man with a history of Crohn's disease who had a delayed symptomatic mesenteric portal AVF, occurring 20 years after ileocecal resection, which was subsequently managed by endovascular approach is reported. The patient presented with post-prandial abdominal pain for almost one year, and dyspnoea New York Heart Association stage II. There were no signs of portal hypertension. Pre-operative contrast enhanced computed tomography showed a high flow SMPAVF, with a short and wide neck (9 mm × 16 mm) at the level of the last collateral of the superior mesenteric artery. It was associated with a large aneurysm of the mesenteric vein. Vascular plug embolisation (Amplatzer 18 × 18 mm, Abbott, Chicago, IL, USA) by femoral access allowed exclusion of the SMPAVF and preserved arterial flow in the distal collaterals. During follow up, the patient developed portal vein thrombosis and required therapeutic anticoagulation for six months., Discussion: In most cases, endovascular approaches are preferred in the management of SMPAVF. Endovascular approaches are based on minimally invasive techniques including embolisation (coiling or plug) and covered stenting. Vascular plug embolisation of SMPAVF is feasible and seems to be an effective technique, with the advantage of saving collaterals. Therapeutic anticoagulation should be considered post-operatively in cases with venous dilatation and reduced flow linked to exclusion of the AVF., Competing Interests: None., (© 2023 Published by Elsevier Ltd on behalf of European Society for Vascular Surgery.)

Background: We hypothesized that supraceliac aortic cross clamping could induce lung injury mediated by an inflammatory ischemia-reperfusion (IR) trigger. We aimed to characterize glycocalyx (GCX), a component of endothelial membrane, participating to remote lung injury., Methods: Rats underwent supraceliac aortic cross clamping for 40 min and were sacrificed at 0, 3, 6, and 24 hr of reperfusion (n = 10/group). Each group was compared to sham (n = 6/group). GCX products (syndecan-1 [Sdc-1] and heparan sulfate [HS]), tumor necrosis factor-alpha (TNF-α), and interleukin-1β (IL-1β) were measured in plasma (enzyme-linked immunosorbent assay[ELISA]). Lungs were harvested for measurements of TNF-α, IL-1β (polymerase chain reaction) and Sdc-1 (western blotting [WB]). Histologic lung injury scoring and pulmonary gravimetry were analyzed in a blinded manner., Results: Plasmatic Sdc-1, HS, TNF-α, and IL-1β reached peak levels at 3 hr. Levels were significantly higher in clamping groups than sham at 6 hr for Sdc-1, at 0 and 3 hr for HS, at 3 and 6 hr for TNF-α, and at 3 hr for IL-1β. Lung TNF-α and Interleukin-1β reached peak levels at 6 hr. Levels were significantly higher than sham at 6 and 24 hr for TNF-α and at 6 hr for IL-1β. Lung Sdc-1 was lowest at 3 hr. Sdc-1 was not significantly different compared to sham at the different reperfusion times. At 3 hr, it was 0.27 ± 0.03 vs. 0.33 ± 0.02 (sham) (P = 0.09). Histopathologic scores at 6 and 24 hr were higher in clamping groups than sham. At 6 and 24 hr, it was higher for hemorrhage, polynuclear neutrophil (PNN) infiltration and intravascular leukocytes. Pulmonary edema was higher by gravimetry at 0 and 6 hr., Conclusions: Supra celiac aortic clamping causes early lung injury in relation with a systemic inflammatory response associated with altered GCX structure., (Copyright © 2022. Published by Elsevier Inc.)

Tissue engineering strategies aim at characterizing and at optimizing the cellular component that is combined with biomaterials, for improved tissue regeneration. Here, we present the immunoMap of apical papilla, the native tissue from which SCAPs are derived. We characterized stem cell niches that correspond to a minority population of cells expressing Mesenchymal stromal/Stem Cell (CD90, CD105, CD146) and stemness (SSEA4 and CD49f) markers as well as endothelial cell markers (VWF, CD31). Based on the colocalization of TKS5 and cortactin markers, we detected migration-associated organelles, podosomes-like structures, in specific regions and, for the first time, in association with stem cell niches in normal tissue. From six healthy teenager volunteers, each with two teeth, we derived twelve cell banks, isolated and amplified under 21 or 3% O 2 . We confirmed a proliferative advantage of all banks when cultured under 3% versus 21% O 2 . Interestingly, telomerase activity was similar to that of the highly proliferative hiPSC cell line, but unrelated to O 2 concentration. Finally, SCAPs embedded in a thixotropic hydrogel and implanted subcutaneously in immunodeficient mice were protected from cell death with a slightly greater advantage for cells preconditioned at 3% O 2 .

Purpose: The incidence of type II endoleaks (ELII) after endovascular aneurysm repair (EVAR) ranges from 10-44%. Aneurysm thrombus density after EVAR could predict successful aneurysm exclusion., Materials and Methods: Twenty-seven patients with an abdominal aortic aneurysm (AAA) who had a CT scan within the first 45 days (early group) post-surgery or after 7 months (late group) were included. Thrombus density was analyzed on non-contrast enhanced CT scans., Results: A total of 5/13 (38%) patients in the early group had an ELII and 9/14 (64.3%) in the late group had a persistent ELII since surgery. In the early group, thrombus density was similar in patients with or without an ELII (mean: 39.9 ± 4.8 vs. 41.9 ± 3.4, p = 0.7; median: 38.7 ± 4.8 vs. 39.7 ± 3.1, p = 0.8). In patients with an ELII, there was no difference in thrombus density at 45 days and after 7 months (mean: 39.9 ± 4.8 vs. 40.2 ± 2.1, p = 0.9; median: 38.7 ± 4.8 vs. 38 ± 2.6, p = 0.9). In patients without an ELII, thrombus density was significantly higher at 45 days than after 7 months (mean: 41.9 ± 3.44 vs. 25.7 ± 2.0, p = 0.005; median: 39.7 ± 3.11 vs. 24.4 ± 1.5, p = 0.004). In patients with an ELII, thrombus density was significantly higher after 7 months than in patients without an ELII (mean: 40.2 ± 2.1 vs. 25.7 ± 2.0. p = 0.001; median: 38 ± 2.6 vs. 24.4 ± 1.5, p = 0.003)., Conclusion: Low thrombus density after EVAR on late unenhanced CT scans predicts aneurysm exclusion.

We present an uncommon case of a 16-year-old woman with type 1 neurofibromatosis and renovascular hypertension due to bilateral renal stenosis associated with asymptomatic digestive artery stenosis. Our patient was treated by several autologous bypasses to the superior mesenteric artery and the left and right renal arteries. She had no postoperative complications and good clinical and imaging outcomes at 10 years. Furthermore, there are few data on the vascular impairment of this rare disease, thus justifying its presentation., (Copyright © 2020 Elsevier Inc. All rights reserved.)

We present a case of abdominal aortic aneurysm open repair planned with temporary left ventricular support (Impella®) in a patient suffering from severe chronic heart failure. Only 1 single similar case was reported in the literature from 1974., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Open surgical repair (OSR) for thoracoabdominal aortic aneurysms (TAA) is associated with a high pulmonary and renal morbidity rate. Ischemic preconditioning (IPC) is a mechanism of protection against the deleterious effects of ischemia-reperfusion. To our knowledge IPC has never been tested during OSR for TAA., Methods: The primary objective of the study is to evaluate the efficacy of IPC during OSR for TAA with respect to acute kidney injury (AKI) according to KDIGO and pneumonia/prolonged ventilation-time during the first 8 postoperative days. The secondary objectives are to compare both arms with respect to cardiac complications within 48 h, renal and pulmonary complications within 21 days and mortality at 60 days. To assess the efficacy of IPC with respect to pulmonary and renal morbidity, a cox model for competing risks will be used. Assuming that the event occurs among 36% of the patients when no IPC is performed, the allocation of 55 patients to each arm should allow detecting a hazard ratio of at least 2.75 with a power of 80% when admitting 5% for an error of first kind. This means that 110 patients, enrolled in this multicenter study, may be randomised within 36 months of the first randomization. Randomization will be performed to allocate patients either to surgery with preconditioning before aortic cross clamping (Arm 1) or to surgery without preconditioning before aortic cross clamping (Arm 2). Randomization takes place during the intervention after intravenous injection of heparin, or after the start of femoral assistance. The procedure for IPC will be a supra-visceral thoracic aortic cross clamping for 5 min followed by an unclamping period of 5 min. This procedure will be repeated twice before starting thoracic aortic cross clamping needed to perform surgery., Conclusions: Our hypothesis is that ischemic preconditioning could reduce clinical morbidity and the incidence of lung damage associated with supra-visceral aortic clamping., Trial Registration: EPICATAStudy registered in ClinicalTrial.gov / number: NCT03718312 on Oct.24.2018 URL number.

Herpesviruses encode a serine protease that specifically cleaves assembly protein. This protease is critical for replication, and represents a new target for antiviral drug design. Here we report the three-dimensional structure of the protease from human cytomegalovirus (hCMV) at 2.27 angstroms resolution. The structure reveals a unique fold and new catalytic strategy for cleavage. The monomer fold of the enzyme, a seven-stranded beta-barrel encircled by a chain of helices that form the carboxy terminus of the molecule, is unrelated to those observed in classic serine proteases such as chymotrypsin and subtilisin. The serine nucleophile at position 132 is activated by two juxtaposed histidine residues at positions 63 and 157. Dimerization, which seems to be necessary for activity, is observed in the crystals. Correlations of the structure with the sequences of herpesvirus proteases suggest that dimerization may confer specificity and recognition in substrate binding.

Excessive coagulation is a typical response to the vascular injury occurring in gram negative sepsis. This study evaluated the pharmacological effects of the use of a recombinant Escherichia coli derived form of tissue factor pathway inhibitor (ala-TFPI) in a baboon model of septic shock. Several doses of ala-TFPI were administered either 30 or 120 min after the initiation of a lethal intravenous infusion of E. coli into baboons. Treatment at 30 min with either 2.7 or 7.4 mg/kg of ala-TFPI resulted in the same survival rates and attenuation of both the coagulation response and cellular injury, as measured by clinical chemistry. When administration of ala-TFPI was delayed for 120 min, a dose of ala-TFPI protein continued to provide a benefit to survival. Ala-TFPI reduced the drop in mean systemic arterial pressure compared to control baboons in addition to partially attenuating the coagulopathic response. Baboons given ala-TFPI also maintained lower levels of plasma interleukin-6 (IL-6) and thrombin-antithrombin. These results suggest that the site of action of the protein may involve the later stage components of the coagulation and inflammatory pathways.

Human tissue factor pathway inhibitor (TFPI) was expressed in E. coli as a non-glycosylated protein with an additional alanine attached to the aminoterminus of the wild type molecule. High-level expression was obtained with pMON6875, a plasmid containing a tac promoter, Gene 10 leader from bacteriophage T7, methionine-alanine-TFPI coding sequence, and the p22 transcriptional terminator. In this system, TFPI accounted for about 5-10% of the total cell protein. The inclusion bodies containing. TFPI were sulfitolyzed, purified by anion-exchange chromatography, refolded through a disulfide interchange reaction, and further fractionated by Mono S cation exchange chromatography. The Mono S resin resolved a peak of highly active TFPI from relatively inactive and possibly misfolded molecules. The E. coli TFPI was shown to be about two-fold more active, on a molar basis, than full-length human SK hepatoma TFPI in a tissue factor-induced clotting assay in human plasma.

The pharmacokinetics of recombinant tissue factor pathway inhibitor (TFPI) after an intravenous bolus injection was studied in rabbits. Clearance of TFPI was followed by measurement of the radioactivity of the 125I-labelled compound in the whole plasma or the trichloroacetic acid precipitate and by quantitation of the functional TFPI activity of the unlabelled compound using a tissue factor-induced coagulation assay. When iodinated TFPI was used, the ratios of the trichloroacetic acid precipitable counts vs. that of the whole plasma was about 1 in the first 10 min after TFPI injection, but this ratio gradually decreased to less than 0.5 after 2 h. This result suggested that the iodinated TFPI in the plasma was partially degraded after prolonged circulation in the animal. When unlabelled TFPI was used, the clearance of TFPI activity from the plasma exhibited bi-exponential elimination kinetics with a rapid alpha phase half-life (t1/2 alpha) of 2.3 min, and a terminal beta phase half-life (t1/2 beta) of 79 min. The plasma clearance was 4.2 ml kg-1 min-1. The tissue distribution of intravenously administered 125I-TFPI in the rabbit was studied using whole-body autoradiography. At 3 min after dosing, significant levels of TFPI were apparent in the liver, kidney, and other highly blood perfused tissues. Significant levels of 125I-TFPI-derived radioactivity were also apparent in the liver and kidney at 30 min after intravenous administration. The localization within the liver demonstrated a mottled appearance, suggesting regions of higher uptake within the liver. In the kidney, the outer cortex consistently revealed the highest activity.

Recombinant tissue factor pathway inhibitor (rTFPI) has been expressed in four mammalian expression systems using human SK hepatoma, mouse C127, baby hamster kidney (BHK), and Chinese hamster ovary (CHO) cells as hosts. On sodium dodecyl sulfate polyacrylamide gel electrophoresis, the immunoaffinity purified rTFPIs all show broad bands and the mean molecular weight of SK hepatoma and C127 rTFPIs (M(r) approximately 38,000) appear larger than those of BHK and CHO rTFPIs (M(r) approximately 35,000). All these proteins inhibit factor Xa and appear to bind factor Xa with 1:1 stoichiometry. The ability of these proteins to inhibit tissue factor-induced coagulation in plasma was examined using a prothrombin time assay. The relative activities of SK rTFPI:C127 rTFPI:BHK rTFPI:CHO rTFPI were found to be 28:15:2.1:1. By Western blot using specific antisera against the amino- and carboxy-termini of TFPI as probes, it is found that all the immunoaffinity purified rTFPIs possess approximately equal amounts of the amino terminus, but the C127 and BHK rTFPIs are deficient in carboxy terminus and the CHO rTFPI is essentially devoid of this region of the protein. Mono S chromatography allowed separation of the full-length and the truncated molecules with high and low anticoagulant activities, respectively. The above results suggest that proteolysis of the carboxy terminus of TFPI occurs to different extent when TFPI is expressed in different cells and that the carboxy terminal region of the TFPI molecule is important for the inhibition of tissue factor-induced coagulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Lipoprotein-associated coagulation inhibitor produces feed-back inhibition of tissue factor (tissue thromboplastin)-induced coagulation in the presence of factor Xa Recombinant lipoprotein-associated coagulation inhibitor (rLACI) was tested for its ability to modify thromboplastin-induced intravascular coagulation in a rabbit model that allows monitoring of iodine-125 fibrin accumulation/disappearance in the lung and sampling of blood for the measurement of coagulation parameters. Infusion of thromboplastin into the rabbit caused a rapid increase of radioactivity over the lungs, possibly due to the accumulation of 125I fibrin in the lungs, followed by a rapid decline of radioactivity, suggestive of removal of fibrin from the lungs. Thromboplastin also caused a rapid decrease of systemic fibrinogen that was accompanied by a lengthening of the activated partial thromboplastin time and prothrombin time. The effect of coinfusion of rLACI with thromboplastin or bolus injection of rLACI before thromboplastin infusion was studied. At a high dose of rLACI (800 micrograms/kg body weight), the thromboplastin-induced radioactivity increase in the lungs and the systemic fibrinogen decrease were completely suppressed. The activated partial thromboplastin time and prothrombin time of the plasma samples lengthened, possibly due to the presence of thromboplastin in circulation. The thromboplastin-induced radioactivity increase over the lungs was not completely suppressed by lower doses of rLACI (135 to 270 micrograms/kg body weight), but these doses of rLACI prevented systemic fibrinogen decrease. At a bolus dose of 23 micrograms/kg body weight, rLACI provided 50% protection of the fibrinogen consumption (fibrinogen decreased to 82% compared with 65% in rabbits treated with thromboplastin alone). These results show that rLACI is effective in the inhibition of thromboplastin-induced coagulation in vivo.

Crystals of a chymotrypsin inhibitor from Erythrina caffra seeds have been grown out of lithium sulfate, by the hanging drop method of vapor diffusion. The crystals belong to the rhombohedral space group R32, with a = 67.2 A and alpha = 99.4 degrees, and diffract to 3 A resolution.

N-[4-(5-Nitro-2-furyl)-2-thiazolyl]acetamide (NFTA) metabolism was examined in vitro using microsomes prepared from rat liver and renal cortex and from rabbit liver and renal cortex and outer and inner medulla. NFTA nitroreduction was observed with each tissue. Three mol of NADPH were used per mol of NFTA reduced. Substrate and inhibitor specificity suggested that the microsomal nitroreduction was due to NADPH:cytochrome c reductase. Metabolite(s) formed bound to protein, RNA, DNA, and synthetic polyribonucleotides. Maximum covalent binding was seen with polyguanylic acid. A guanosine-NFTA adduct was isolated. Binding was inhibited by sulfhydryl compounds and vitamin E. The [14C]NFTA:glutathione or [3H]glutathione:NFTA conjugates obtained from microsomal incubations showed identical chromatographic properties as the product obtained by the reaction of synthetic N-hydroxy-NFTA with [3H]glutathione. Structures of synthetic N-hydroxy-NFTA and the microsomal reduction product 1-[4-(2-acetylaminothiazolyl)]-3-cyano-1-propanone were established by mass spectrometry. The latter reduction product did not bind macromolecules. These results suggest that renal NADPH:cytochrome c reductase reduces NFTA to an N-hydroxy-NFTA intermediate that binds nucleophilic sites on macromolecules.

To probe the effects of N-glycosylation on the fibrin-dependent plasminogenolytic activity of tissue-type plasminogen activator (t-PA), we have expressed a human recombinant t-PA (rt-PA) gene in Chinese hamster ovary (CHO) cells and in a murine C127 cell line. The resulting rt-PA glycoproteins were isolated and their associated N-linked oligosaccharide structures determined by using a combination of high-resolution Bio-Gel P-4 gel filtration chromatography, sequential exoglycosidase digestion, and methylation analysis. The results show that CHO rt-PA is N-glycosylated differently from murine C127 derived rt-PA. Further, both rt-PA's are N-glycosylated differently from t-PA derived from a human colon fibroblast and the Bowes melanoma cell line (Parekh et al., 1989), confirming that N-glycosylation of the human t-PA polypeptide is cell-type-specific. Both CHO and murine rt-PA were fractionated on lysine-Sepharose chromatography. The N-glycosylation of the major forms was analyzed and their fibrin-dependent plasminogenolytic activity determined by using an indirect amidolytic assay with Glu-plasminogen and a chromogenic plasmin substrate. The results suggest that the various forms of rt-PA differ from one another with respect to the kinetics of their fibrin-dependent activation of plasminogen. Together, these data support the notion (Wittwer et al., 1989) that N-glycosylation influences the fibrin-dependent catalytic activity of t-PA and that t-PA when expressed in different cell lines may consist of kinetically and structurally distinct glycoforms.

It has been demonstrated that N-[4-(5-nitro-2-furyl)-2-thiazolyl]acetamide (NFTA), when fed with the diet, causes transitional carcinomas in rats. An important step in the mechanism of NFTA-induced carcinogenesis is endogenous metabolic activation to an ultimate carcinogen. We have proposed that the enzyme complex prostaglandin H synthase (PHS) is involved in the activation of certain renal and urinary tract carcinogens. This proposal was assessed by examining the activation of the 5-nitrofuran renal carcinogen NFTA and its deacetylated analogue 2-amino-4-(5-nitro-2-furyl)thiazole (ANFT) by PHS. Ram seminal vesicular and rabbit renal inner medullary microsomes were used as a source of PHS. Both NFTA and ANFT were activated by PHS to bind microsomal protein. Both microsomal preparations activated ANFT to bind DNA. However, only ram seminal vesicular microsomes activated NFTA to bind DNA. The rate of ANFT binding to macromolecules was considerably greater than NFTA with both microsomal preparations. Although activated ANFT was shown to bind several different homopolynucleotides, a preference for binding polyguanylic acid was demonstrated. Glutathione inhibition of carcinogen binding to macromolecules was shown to be due to the formation of a thioether conjugate. Deacetylation of NFTA was demonstrated in both tissues with deacetylation significantly exceeding acetylation of ANFT to NFTA in the kidney. Thus, renal PHS activation of both NFTA and ANFT was demonstrated with the rate of ANFT activation being considerably greater than NFTA. The conversion of NFTA to ANFT by intact tissue suggests that ANFT may contribute to NFTA renal carcinogenesis.