Risk of biliary complications in bariatric surgery.

Gallstones are commonly observed after rapid weight loss, particularly after bariatric surgery. Preventive measures of gallstone formation and potential related complications are still debated. This study aimed to propose a standardized strategy according to the results of the literature. Thus, preventive measures should be determined according to patient status (evaluated clinically and by routine ultrasound) and the type of bariatric surgery. Cholecystectomy should be performed in patients with symptomatic gallstones irrespective of the planned operation, or for asymptomatic gallstones during a gastric by-pass. In other settings, ursodesoxycholic acid should be given postoperatively for 6 months.

Retained appendicolith after laparoscopic appendectomy: the need for systematic double ligature of the appendiceal base.

Appendicoliths are considered to be strong indicators of appendicitis and the complications of appendicitis. We report the case of a 29-year-old woman who underwent a laparoscopic appendectomy for appendicitis with an appendicolith. The appendix was divided with a single ligature at the appendiceal base, and an appendicolith escaped into the pelvis. Thereafter, the patient suffered recurrent pelvic abscess. The diagnosis of retained appendicolith was made by repeated CT scans that revealed a mobile spontaneous calcification within the abscess. This postoperative complication could have been avoided if a systematic division of the appendix had been performed between double ligatures.

[Acute peritonitis: a rare presentation of chylous ascites]. / Péritonite aiguë: un mode de révélation rare d'ascite chyleuse.

Chylous ascites is a rare case of peritonitis. We report here a case arising in a 21-year-old lady.

Adenosylmethionine as a source of 5'-deoxyadenosyl radicals.

The combination of an iron-sulfur cluster and S-adenosylmethionine provides a novel mechanism for the initiation of radical catalysis in an unanticipated variety of metabolic processes. Molecular details of the cluster-mediated reductive cleavage of S-adenosylmethionine to methionine and, presumably, a 5'-deoxyadenosyl radical are the targets of recent studies.

Activation of class III ribonucleotide reductase from E. coli. The electron transfer from the iron-sulfur center to S-adenosylmethionine.

The anaerobic ribonucleotide reductase (ARR) from E. coli is the prototype for enzymes that use the combination of S-adenosylmethionine (AdoMet) and an iron-sulfur center for generating catalytically essential free radicals. ARR is a homodimeric alpha2 protein which acquires a glycyl radical during anaerobic incubation with a [4Fe-4S]-containing activating enzyme (beta) and AdoMet under reducing conditions. Here we show that the EPR-active S = 1/2 reduced [4Fe-4S]+ cluster is competent for AdoMet reductive cleavage, yielding 1 equiv of methionine and almost 1 equiv of glycyl radical. These data support the proposal that the glycyl radical results from a one-electron oxidation of the reduced cluster by AdoMet. Reduced protein beta alone is also able to reduce AdoMet but only in the presence of DTT. However, in that case, 2 equiv of methionine per reduced cluster was formed. This unusual stoichiometry and combined EPR and Mössbauer spectroscopic analysis are used to tentatively propose that AdoMet reductive cleavage proceeds by an alternative mechanism involving catalytically active [3Fe-4S] intermediate clusters.

Activation of class III ribonucleotide reductase by flavodoxin: a protein radical-driven electron transfer to the iron-sulfur center.

In its active form, Escherichia coli class III ribonucleotide reductase homodimer alpha(2) relies on a protein free radical located on the Gly(681) residue of the alpha polypeptide. The formation of the glycyl radical, namely, the activation of the enzyme, involves the concerted action of four components: S-adenosylmethionine (AdoMet), dithiothreitol (DTT), an Fe-S protein called beta or "activase", and a reducing system consisting of NADPH, NADPH:flavodoxin oxidoreductase, and flavodoxin (fldx). It has been proposed that a reductant serves to generate a reduced [4Fe-4S](+) cluster absolutely required for the reductive cleavage of AdoMet and the generation of the radical. Here, we suggest that the one-electron reduced form of flavodoxin (SQ), the only detectable product of the in vitro enzymatic reduction of flavodoxin, can support the formation of the glycyl radical. However, the redox potential of the Fe-S center of the enzyme is shown to be approximately 300 mV more negative than that of the SQ/fldx couple and not shifted to a more positive value by AdoMet binding. It is also more negative than that of the HQ/SQ couple, HQ being the fully reduced form of flavodoxin. Our interpretation is that activation of ribonucleotide reductase occurs through coupling of the reduction of the Fe-S center by flavodoxin to two thermodynamically favorable reactions, the oxidation of the cluster by AdoMet, yielding methionine and the 5'-deoxyadenosyl radical, and the oxidation of the glycine residue to the corresponding glycyl radical by the 5'-deoxyadenosyl radical. The second reaction plays the major role on the basis that a Gly-to-Ala mutation results in a greatly decreased production of methionine.

Activation of class III ribonucleotide reductase by thioredoxin.

Anaerobic ribonucleotide reductase provides facultative and obligate anaerobic microorganisms with the deoxyribonucleoside triphosphates used for DNA chain elongation and repair. In Escherichia coli, the dimeric alpha2 enzyme contains, in its active form, a glycyl radical essential for the reduction of the substrate. The introduction of the glycyl radical results from the reductive cleavage of S-adenosylmethionine catalyzed by the reduced (4Fe-4S) center of a small activating protein called beta. This activation reaction has long been known to have an absolute requirement for dithiothreitol. Here, we report that thioredoxin, along with NADPH and NADPH:thioredoxin oxidoreductase, efficiently replaces dithiothreitol and reduces an unsuspected critical disulfide bond probably located on the C terminus of the alpha protein. Activation of reduced alpha protein does not require dithiothreitol or thioredoxin anymore, and activation rates are much faster than previously reported. Thus, in E. coli, thioredoxin has very different roles for class I ribonucleotide reductase where it is required for the substrate turnover and class III ribonucleotide reductase where it acts only for the activation of the enzyme.

[Normal values of plasma methoxyamines in the setting of renal insufficiency and peri-operative stress. Consequences for the etiological diagnosis of hypertension]. / Valeurs normales des méthoxyamines plasmatiques en situation d'insuffisance rénale et de stress péri-opératoire.

INTRODUCTION: HPLC plasma methoxyamines measurements are the updated technique for the diagnosis of adrenergic hypersecretion. Their reliability meets that of urinary measurements. Significance of increased values is not yet fully established for the etiological diagnosis of hypertension in some situations, especially in case of renal insufficiency and in the peri-operative period. The aim of this study is to define the "normal" range of the values of plasma methoxyamines in both of those conditions. PATIENTS AND METHODS: in a General and Endocrine Surgical Unit, 3 homogeneous group of 20 patients each have been studied: group 1, control (patients awaiting thyroidectomy); group 2, patients on maintenance hemodialysis submitted for hyperparathyroidism; group 3, patients submitted to digestive surgery. Measurements were done pre-operatively in group 1, pre and post-operatively in group 2, and post-operatively in group 3. RESULTS: in comparison to the control (11.8 nmol/l), we observed in group 2 a 18 fold increase preoperatively, and a 29 fold increase at post-operative day 1. In group 3, we observed a 2.3, 2.7 and 2 fold increase at post-operative days 1,2 and 3 respectively. All those results were statistically significant. CONCLUSION: Results of measurements of plama methoxyamines should always be matched to the serum creatinine levels. They are meaningful for the diagnosis of endocrine origin of hypertension only late after the early post-operative period.

Acute manifestation of LHON and coincidental finding of a pituitary adenoma: a case report.

A patient with Leber Hereditary Opticus Neuropathy (LHON) and a pituitary adenoma is presented. The different ophthalmological signs of LHON are described and illustrated. A definite diagnosis is made by detecting a mitochondrial mutation. A radiological examination revealed a pituitary adenoma. The pituitary adenoma may be a trigger factor in the penetrance of LHON.

The activating component of the anaerobic ribonucleotide reductase from Escherichia coli. An iron-sulfur center with only three cysteines.

Class III anaerobic ribonucleotide reductase small component, named protein beta, contains a (4Fe-4S) center. Its function is to mediate electron transfer from reduced flavodoxin to S-adenosylmethionine, required for the introduction of a glycyl radical in the large component, named protein alpha, which then becomes active for the reduction of ribonucleotides. By site-directed mutagenesis we demonstrate that the three cysteines of the conserved CXXXCXXC sequence are involved in iron chelation. Such a sequence is also present in the activase of the pyruvate formate-lyase and in the biotin synthase, both carrying an iron-sulfur center involved in reductive activation of S-adenosylmethionine. Even though they are able to bind iron in the (4Fe-4S) form, as shown by Mössbauer spectroscopy, the corresponding Cys to Ala mutants are catalytically inactive. Mutation of the two other cysteines of the protein did not result in inactivation. We thus conclude that the (4Fe-4S) cluster has, in the wild type protein, only three cysteine ligands and a fourth still unidentified ligand.

Iron-sulfur interconversions in the anaerobic ribonucleotide reductase from Escherichia coli.

The anaerobic ribonucleotide reductase from Escherichia coli contains an iron-sulfur cluster which, in the reduced [4Fe-4S](+) form, serves to reduce S-adenosylmethionine and to generate a catalytically essential glycyl radical. The reaction of the reduced cluster with oxygen was studied by UV-visible, EPR, NMR, and Mössbauer spectroscopies. The [4Fe-4S](+) form is shown to be extremely sensitive to oxygen and converted to [4Fe-4S](2+), [3Fe-4S](+/0), and to the stable [2Fe-2S](2+) form. It is remarkable that the oxidized protein retains full activity. This is probably due to the fact that during reduction, required for activity, the iron atoms, from 2Fe and 3Fe clusters, readily reassemble to generate an active [4Fe-4S] center. This property is discussed as a possible protective mechanism of the enzyme during transient exposure to air. Furthermore, the [2Fe-2S] form of the protein can be converted into a [3Fe-4S] form during chromatography on dATP-Sepharose, explaining why previous preparations of the enzyme were shown to contain large amounts of such a 3Fe cluster. This is the first report of a 2Fe to 3Fe cluster conversion.

The anaerobic ribonucleotide reductase from Escherichia coli. The small protein is an activating enzyme containing a [4fe-4s](2+) center.

For deoxyribonucleotide synthesis during anaerobic growth, Escherichia coli cells depend on an oxygen-sensitive class III ribonucleotide reductase. The enzyme system consists of two proteins: protein alpha, on which ribonucleotides bind and are reduced, and protein beta, of which the function is to introduce a catalytically essential glycyl radical on protein alpha. Protein beta can assemble one [4Fe-4S] center per polypeptide enjoying both the [4Fe-4S](2+) and [4Fe-4S](1+) redox state, as shown by iron and sulfide analysis, Mössbauer spectroscopy (delta = 0.43 mm.s(-1), DeltaE(Q) = 1.0 mm.s(-1), [4Fe-4S](2+)), and EPR spectroscopy (g = 2. 03 and 1.93, [4Fe-4S](1+)). This iron center is sensitive to oxygen and can decompose into stable [2Fe-2S](2+) centers during exposure to air. This degraded form is nevertheless active, albeit to a lesser extent because of the conversion of the cluster into [4Fe-4S] forms during the strongly reductive conditions of the assay. Furthermore, protein beta has the potential to activate several molecules of protein alpha, suggesting that protein beta is an activating enzyme rather than a component of an alpha(2)beta(2) complex as previously claimed.

Essential hypertension: first reason for persistent hypertension after unilateral adrenalectomy for primary aldosteronism?

BACKGROUND: Despite cure of primary aldosteronism by surgical resection, hypertension persists postoperatively in 30% to 50% of patients. The aim of this study was to determine factors influencing long-term outcome of blood pressure after unilateral adrenalectomy for primary aldosteronism. METHODS: Records of 100 patients who underwent unilateral adrenalectomy for primary aldosteronism from 1970 through 1997 were reviewed. Patients were distributed in 2 groups according to whether blood pressure was normal (criteria of World Health Organization). Clinical, biochemical, and pathologic data were compared. RESULTS: All patients were biochemically cured. Blood pressure was normal in 56 patients and improved in 44 (mean follow-up, 69 and 59 months). Persistent hypertension correlated with age, known duration and seriousness of preoperative hypertension, family history of hypertension, no preoperative response to spironolactone, and contralateral adrenal hypertrophy. Gender, surgical approach, and pathologic findings were not predictive factors of blood pressure outcome. The prevalence of hypertension was almost the same in these postoperative patients as the prevalence of essential hypertension in a random population of the same age. CONCLUSIONS: Early unilateral adrenalectomy allows cure or improvement of hypertension in all patients with primary aldosteronism induced by unilateral excessive source of aldosterone secretion regardless of the pathologic findings. Persistent hypertension suggests that coexisting essential hypertension is present.

Activation of the anaerobic ribonucleotide reductase from Escherichia coli. The essential role of the iron-sulfur center for S-adenosylmethionine reduction.

The anaerobic ribonucleotide reductase of Escherichia coli catalyzes the synthesis of the deoxyribonucleotides required for anaerobic DNA synthesis. The enzyme is an alpha2beta2 heterotetramer. In its active form, the large alpha2 subunit contains an oxygen-sensitive glycyl radical, whereas the beta2 small protein harbors a [4Fe-4S] cluster that joins its two polypeptide chains. Formation of the glycyl radical in the inactive enzyme requires S-adenosylmethionine (AdoMet), dithiothreitol, K+, and either an enzymatic (reduced flavodoxin) or chemical (dithionite or 5-deazaflavin plus light) reducing system. Here, we demonstrate that AdoMet is directly reduced by the Fe-S center of beta2 during the activation of the enzyme, resulting in methionine and glycyl radical formation. Direct binding experiments showed that AdoMet binds to beta2 with a Kd of 10 microM and a 1:1 stoichiometry. Binding was confirmed by EPR spectroscopy that demonstrated the formation of a complex between AdoMet and the [4Fe-4S] center of beta2. Dithiothreitol triggered the cleavage of AdoMet, leading to an EPR-silent form of beta2 and, in the case of alpha2beta2, to glycyl radical formation. In both instances, 3 methionines were formed per mol of protein. Our results indicate that the Fe-S center of beta2 is directly involved in the reductive cleavage of AdoMet and suggest a new biological function for an iron-sulfur center, i.e redox catalysis, as recently proposed by others (Staples, R. C., Ameyibor, E., Fu, W., Gardet-Salvi, L., Stritt-Etter, A. L., Schürmann, P., Knaff, D. B., and Johnson, M. K. (1996) Biochemistry 35, 11425-11434).

The anaerobic Escherichia coli ribonucleotide reductase. Subunit structure and iron sulfur center.

During anaerobic growth Escherichia coli uses a specific ribonucleoside triphosphate reductase for the production of deoxyribonucleoside triphosphates. The active species of this enzyme was previously found to be a large homodimer of 160 kDa (alpha 2) with a stable, oxygen-sensitive radical located at Gly-681 of the 80-kDa polypeptide chain. The radical is formed in an enzymatic reaction involving S-adenosylmethionine, NADPH, a reducing flavodoxin system and an additional 17.5-kDa polypeptide, previously called activase. Here, we demonstrate by EPR spectroscopy that this small protein contains a 4Fe-4S cluster that joins two peptides in a 35-kDa small homodimer (beta 2). A degraded form of this cluster may have been responsible for an EPR signal observed earlier in preparations of the large 160-kDa subunit that suggested the presence of a 3Fe-4S cluster in the reductase. These preparations were contaminated with a small amount of the small protein. The large and the small proteins form a tight complex. From sucrose gradient centrifugation, we determined a 1:1 stoichiometry of the two proteins in the complex. The anaerobic reductase thus has an alpha 2 beta 2 structure. We speculate that the small protein interacts with S-adenosylmethionine and forms a transient radical involved in the generation of the stable glycyl radical in the large protein that participates in the catalytic process.

The free radical of the anaerobic ribonucleotide reductase from Escherichia coli is at glycine 681.

The anaerobic ribonucleoside triphosphate reductase of Escherichia coli is an iron-sulfur protein carrying an oxygen-sensitive organic radical, which is essential for catalysis. The radical was tentatively proposed to be on glycine 681, based on a comparison with the glycyl radical-containing enzyme pyruvate formate-lyase. By EPR spectroscopy of selectively 2H- and 13C-labeled anaerobic ribonucleotide reductase, the radical was now unambiguously assigned to carbon-2 of a glycine residue. The large 1H hyperfine splitting (1.4 millitesla) was assigned to the alpha-proton. Site-directed mutagenesis was used to change glycine 681 into an alanine residue. In separate experiments, the two adjacent residues, cysteine 680 and tyrosine 682, were changed into serine and phenylalanine, respectively. All mutated proteins were retained on dATP-Sepharose, indicating that the mutant proteins had intact allosteric sites. They also contained amounts of iron comparable with the wild type reductase and showed the same iron-sulfur-related spectrum, suggesting that the mutant proteins were properly folded. Of the three mutant proteins only the G681A protein completely lacked the detectable glycyl radical as well as enzyme activity. Our results identify glycine 681 as the stable free radical site in E. coli anaerobic ribonucleotide reductase.

Formate is the hydrogen donor for the anaerobic ribonucleotide reductase from Escherichia coli.

During anaerobic growth Escherichia coli uses a specific ribonucleoside-triphosphate reductase (class III enzyme) for the production of deoxyribonucleoside triphosphates. In its active form, the enzyme contains an iron-sulfur center and an oxygen-sensitive glycyl radical (Gly-681). The radical is generated in the inactive protein from S-adenosylmethionine by an auxiliary enzyme system present in E. coli. By modification of the previous purification procedure, we now prepared a glycyl radical-containing reductase, active in the absence of the auxiliary reducing enzyme system. This reductase uses formate as hydrogen donor in the reaction. During catalysis, formate is stoichiometrically oxidized to CO2, and isotope from [3H]formate appears in water. Thus E. coli uses completely different hydrogen donors for the reduction of ribonucleotides during anaerobic and aerobic growth. The aerobic class I reductase employs redox-active thiols from thioredoxin or glutaredoxin to this purpose. The present results strengthen speculations that class III enzymes arose early during the evolution of DNA.

Flavodoxin is required for the activation of the anaerobic ribonucleotide reductase.

The inactive anaerobic ribonucleotide reductase from Escherichia coli is transformed by a multienzyme system and S-adenosylmethionine + NADPH into a radical protein that is enzymatically active. One of the activating enzyme components was earlier shown to be ferredoxin (flavodoxin):NADP+ reductase. Here we present evidence that flavodoxin, but not ferredoxin, also is a component of the system. Light reduced deazaflavin can substitute for the flavodoxin system. An additional unidentified low-molecular weight component further stimulates the reaction.

An iron-sulfur center and a free radical in the active anaerobic ribonucleotide reductase of Escherichia coli.

Anaerobically grown Escherichia coli contain an oxygen-sensitive ribonucleotide reductase. The enzyme requires anaerobic activation by two E. coli fractions with S-adenosylmethionine, NADPH, dithiothreitol, and KCl. We now find that photochemically reduced deazaflavin can substitute for these two fractions and NADPH. The reductase contained roughly equimolar amounts of iron and sulfide, suggesting the presence of an Fe-S complex. The cluster is characterized by a charge transfer band at 420 nm and a low temperature EPR signal centered at g = 2.01 that is difficult to saturate at 14 K, suggested to be a (3Fe-4S)+ cluster. In five different preparations of essentially protein-pure reductase containing widely different amounts of iron, the catalytic activity correlated well with the iron content. The iron signal disappeared during reductive anaerobic activation, with the appearance of a new EPR signal at g = 2.0033 showing a temperature behavior and microwave power saturability consistent with an organic free radical. The signal disappeared after exposure of the activated enzyme to air. We suggest that activation involves generation of a specific amino acid free radical that is dependent on the reduced Fe-S cluster and S-adenosylmethionine. From other work it appears likely that the free radical is localized on glycine 681 of the polypeptide chain.