The antioxidants of legume nodule mitochondria.

The mitochondria of legume root nodules are critical to sustain the energy-intensive process of nitrogen fixation. They also generate reactive oxygen species at high rates and thus require the protection of antioxidant enzymes and metabolites. We show here that highly purified mitochondria from bean nodules (Phaseolus vulgaris L. cv. Contender x Rhizobium leguminosarum bv. phaseoli strain 3622) contain ascorbate peroxidase primarily in the inner membrane (with lesser amounts detected occasionally in the matrix), guaiacol peroxidases in the outer membrane and matrix, and manganese superoxide dismutase (MnSOD) and an ascorbate-regenerating system in the matrix. This regenerating system relies on homoglutathione (instead of glutathione) and pyridine nucleotides as electron donors and involves the enzymes monodehydroascorbate reductase, dehydroascorbate reductase, and homoglutathione reductase. Homoglutathione is synthesized in the cytosol and taken up by the mitochondria and bacteroids. Although bacteroids synthesize glutathione, it is not exported to the plant in significant amounts. We propose a model for the detoxification of peroxides in nodule mitochondria in which membrane-bound ascorbate peroxidase scavenges the peroxide formed by the electron transport chain using ascorbate provided by L-galactono-1,4-lactone dehydrogenase in the inner membrane. The resulting monodehydroascorbate and dehydroascorbate can be recycled in the matrix or cytosol. In the matrix, the peroxides formed by oxidative reactions and by MnSOD may be scavenged by specific isozymes of guaiacol peroxidase, ascorbate peroxidase, and catalase.

Biliary anastomotic complications in 400 living related liver transplantations.

The purpose of this study was to evaluate the cause and outcome of biliary anastomotic complications occurring after living related liver transplantation (LRLT). A database of 391 patients undergoing 400 LRLT from June 1990 to August 1998 was reviewed. The overall incidence of biliary anastomotic complications was 18.2% (71 patients). There were 45 bile leaks, 35 anastomotic strictures, and the bile duct was ligated inadvertently in 3 cases. Univariative analysis revealed that the manner of stent usage, intrapulmonary shunting, and gender of recipients were significant risk factors for leakage. Anastomotic leaks, cytomegalovirus infection, hepatic artery complications, and gender of recipients were significant risk factors for stricture. In pediatric patients older than 2 years old, ABO blood type compatibility was another risk factor for leakage and stricture. Choice of stent usage and earlier transplantation for patients with intrapulmonary shunting should reduce the rate of biliary leaks, and prophylaxis of leaks for patients with intrapulmonary shunting, and minimizing hepatic artery complications should reduce the rate of biliary stricture after LRLT. Avoidance of ABO-incompatible donors or innovative immunosuppression in ABO-incompatible transplantation should be considered in children.

Glutathione and homoglutathione synthetases of legume nodules. Cloning, expression, and subcellular localization.

The thiol tripeptides glutathione (GSH) and homoglutathione (hGSH) are very abundant in legume root nodules and their synthesis is catalyzed by the enzymes gamma-glutamylcysteine synthetase (gammaECS), GSH synthetase (GSHS), and hGSH synthetase (hGSHS). As an essential step to elucidate the role of thiols in N(2) fixation we have isolated cDNAs encoding the three enzymes and have quantified the transcripts in nodules. Assay of enzyme activities in highly purified nodule organelles revealed that gammaECS is localized in the plastids, hGSHS in the cytosol, and GSHS in the cytosol and mitochondria. These results are consistent with sequence analyses. Subcellular fractionation of nodules also showed that bacteroids contain high thiol concentrations and high specific gammaECS and GSHS activities. Results emphasize the role of nodule plastids in antioxidant protection and in control of thiol synthesis, and suggest that plastids may be important in the stress response of nodules. Overall, our results provide further evidence that thiol synthesis is critical for nodule functioning.

Glutathione and homoglutathione synthesis in legume root nodules.

High-performance liquid chromatography (HPLC) with fluorescence detection was used to study thiol metabolism in legume nodules. Glutathione (GSH) was the major non-protein thiol in all indeterminate nodules examined, as well as in the determinate nodules of cowpea (Vigna unguiculata), whereas homoglutathione (hGSH) predominated in soybean (Glycine max), bean (Phaseolus vulgaris), and mungbean (Vigna radiata) nodules. All nodules had greater thiol concentrations than the leaves and roots of the same plants because of active thiol synthesis in nodule tissue. The correlation between thiol tripeptides and the activities of glutathione synthetase (GSHS) and homoglutathione synthetase (hGSHS) in the nodules of eight legumes, and the contrasting thiol contents and activities in alfalfa (Medicago sativa) leaves (98% hGSH, 100% hGSHS) and nodules (72% GSH, 80% GSHS) indicated that the distribution of GSH and hGSH is determined by specific synthetases. Thiol contents and synthesis decreased with both natural and induced nodule senescence, and were also reduced in the senescent zone of indeterminate nodules. Thiols and GSHS were especially abundant in the meristematic and infected zones of pea (Pisum sativum) nodules. Thiols and gamma-glutamylcysteinyl synthetase were also more abundant in the infected zone of bean nodules, but hGSHS was predominant in the cortex. Isolation of full-length cDNA sequences coding for gamma-glutamylcysteinyl synthetase from legume nodules revealed that they are highly homologous to those from other higher plants.

Stress-induced legume root nodule senescence. Physiological, biochemical, and structural alterations.

Nitrate-fed and dark-stressed bean (Phaseolus vulgaris) and pea (Pisum sativum) plants were used to study nodule senescence. In bean, 1 d of nitrate treatment caused a partially reversible decline in nitrogenase activity and an increase in O(2) diffusion resistance, but minimal changes in carbon metabolites, antioxidants, and other biochemical parameters, indicating that the initial decrease in nitrogenase activity was due to O(2) limitation. In pea, 1 d of dark treatment led to a 96% decline in nitrogenase activity and sucrose, indicating sugar deprivation as the primary cause of activity loss. In later stages of senescence (4 d of nitrate or 2-4 d of dark treatment), nodules showed accumulation of oxidized proteins and general ultrastructural deterioration. The major thiol tripeptides of untreated nodules were homoglutathione (72%) in bean and glutathione (89%) in pea. These predominant thiols declined by approximately 93% after 4 d of nitrate or dark treatment, but the loss of thiol content can be only ascribed in part to limited synthesis by gamma-glutamylcysteinyl, homoglutathione, and glutathione synthetases. Ascorbate peroxidase was immunolocalized primarily in the infected and parenchyma (inner cortex) nodule cells, with large decreases in senescent tissue. Ferritin was almost undetectable in untreated bean nodules, but accumulated in the plastids and amyloplasts of uninfected interstitial and parenchyma cells following 2 or 4 d of nitrate treatment, probably as a response to oxidative stress.

Slide tracheoplasty: a case report of successful concomitant reconstruction of extensive congenital tracheal stenosis and pulmonary artery sling.

An 8-month-old infant presented with an extensive congenital tracheal stenosis with an aberrant left pulmonary artery. The patient was treated successfully by relocation of the left pulmonary artery and tracheal reconstruction with slide tracheoplasty. This patient is the first survivor cited in the literature after concomitant repair using slide tracheoplasty.

Postoperative intussusception after surgery for malrotation and appendicectomy in a newborn.

Postoperative intussusception in the newborn is an infrequent condition. A 17-day-old female with duodenal stenosis and malrotation underwent excision of the membrane in the duodenum and incidental appendicectomy. Postoperatively, a ceco-colic type of intussusception occurred, necessitating a right hemicolectomy. We speculate that the causative factors are twofolds: the embedded appendiceal stump, a polyp-like protrusion that became a lead point, and the non-fixation of the ileocecal mesentery, which facilitated a ceco-colic type of invagination.