Hepatic artery ligation for arterial rupture following liver transplantation: a reasonable option.

Hepatic artery (HA) rupture after liver transplantation is a rare complication with high mortality. This study aimed to review the different managements of HA rupture and their results. From 1997 to 2007, data from six transplant centers were reviewed. Of 2649 recipients, 17 (0.64%) presented with HA rupture 29 days (2-92) after transplantation. Initial management was HA ligation in 10 patients, reanastomosis in three, aorto-hepatic grafting in two and percutaneous arterial embolization in one. One patient died before any treatment could be initiated. Concomitant biliary leak was present in seven patients and could be subsequently treated by percutaneous and/or endoscopic approaches in four patients. Early mortality was not observed in patients with HA ligation and occurred in 83% of patients receiving any other treatment. After a median follow-up of 70 months, 10 patients died (4 after retransplantation), and 7 patients were alive without retransplantation (including 6 with HA ligation). HA ligation was associated with better 3-year survival (80% vs. 14%; p=0.002). Despite its potential consequences on the biliary tract, HA ligation should be considered as a reasonable option in the initial management for HA rupture after liver transplantation. Unexpectedly, retransplantation was not always necessary after HA ligation in this series.

Molecular basis for group-specific activation of the virulence regulator PlcR by PapR heptapeptides.

The transcriptional regulator PlcR and its cognate cell-cell signalling peptide PapR form a quorum-sensing system that controls the expression of extra-cellular virulence factors in various species of the Bacillus cereus group. PlcR and PapR alleles are clustered into four groups defining four pherotypes. However, the molecular basis for group specificity remains elusive, largely because the biologically relevant PapR form is not known. Here, we show that the in vivo active form of PapR is the C-terminal heptapeptide of the precursor, and not the pentapeptide, as previously suggested. Combining genetic complementation, anisotropy assays and structural analysis we provide a detailed functional and structural explanation for the group specificity of the PlcR-PapR quorum-sensing system. We further show that the C-terminal helix of the PlcR regulatory domain, specifically the 278 residue, in conjunction with the N-terminal residues of the PapR heptapeptide determines this system specificity. Variability in the specificity-encoding regions of plcR and papR genes suggests that selection and evolution of quorum-sensing systems play a major role in adaptation and ecology of Bacilli.

Mycophenolate mofetil monotherapy for severe side effects of calcineurin inhibitors following liver transplantation.

Withdrawal of calcineurin inhibitors (CNI) followed by mycophenolate mofetil (MMF) monotherapy after liver transplantation (LT) remains controversial due to the increased risk of acute rejection and graft loss. The aim of the present study, performed in a large cohort of liver-transplanted patients with severe CNI-induced side effects, was to assess renal function recovery, and safety in terms of liver function, of complete CNI withdrawal and replacement by MMF monotherapy. Fifty-two patients treated with MMF monotherapy for CNI-induced toxicity were analyzed. Mean estimated glomerular filtration rate (eGFR) increased significantly during the period of MMF monotherapy, from 37 +/- 10 to 44.7 +/- 15 mL/min/1.73 m(2) at 6 months (p = 0.001) corresponding to a benefit of +17.4% in renal function. eGFR stabilized or improved in 86.5%, 81% and 79% of cases, and chronic renal dysfunction worsened in 13.5%, 19% and 21% of cases, at 6, 12 and 24 months after CNI withdrawal, respectively. Only two patients experienced acute rejection. MMF monotherapy may be efficient at reversing/stabilizing CRD, and appears relatively safe in terms of liver graft function in long-term liver-transplanted patients. However, clinicians must bear in mind the potential risk of rejection and graft loss, and should be very cautious in the management of such 'difficult-to-treat patients'.

Fatal hemophagocytic syndrome related to human herpesvirus-6 reinfection following liver transplantation: a case report.

Human herpesvirus-6 (HHV-6) has been identified as the causal agent of exanthema subitum in early childhood (also called sixth disease or roseola), a mononucleosis-like disease in adults, and as an opportunistic pathogen in transplant recipients. In the latter setting, most infections are caused by reactivation of the latent virus in the recipient and generally have a paucisymptomatic course. Only limited data on HHV-6 infection are available for liver transplant recipients. Herein we have reported a case of fatal hemophagocytic syndrome related to HHV-6 reactivation 2 weeks after liver transplantation (LT). This case suggests that this virus may be a serious and potentially life-threatening pathogen following LT.

Overlapping reading frames at the LYS5 locus in the yeast Yarrowia lipolytica.

Mutants affected at the LYS5 locus of Yarrowia lipolytica lack detectable dehydrogenase (SDH) activity. The LYS5 gene has previously been cloned, and we present here the sequence of the 2.5-kilobase-pair (kb) DNA fragment complementing the lys5 mutation. Two large antiparallel open reading frames (ORF1 and ORF2) were observed, flanked by potential transcription signals. Both ORFs appear to be transcribed, but several lines of evidence suggest that only ORF2 is translated and encodes SDH. (i) The global amino acid compositions of Saccharomyces cerevisiae SDH and of the putative ORF2 product are similar and that of ORF1 is dissimilar. (ii) An in-frame translational fusion of ORF2 with the Escherichia coli lacZ gene was introduced into yeast cells and resulted in a beta-galactosidase activity regulated similarly to SDH; no beta-galactosidase activity was obtained with an in-frame fusion of ORF1 with lacZ. (iii) The introduction of a stop codon at the beginning of ORF2 prevented SDH expression in yeast cells, whereas no phenotypic effect was observed when ORF1 translation was blocked.

Structural insights into the regulation of bacterial signalling proteins containing PRDs.

PRD-containing proteins are bacterial transcriptional antiterminators and activators characterised by a duplicated phosphorylation domain involved in the regulation of catabolic operons. Recent genetic and biochemical studies have suggested how the activity of these regulators is positively or negatively controlled through the multiple phosphorylation of conserved histidyl residues. The regulation mode of these proteins has been examined in light of the recently determined first crystal structure of the phosphorylatable domain of the LicT antiterminator.

Activation of Bacillus licheniformis alpha-amylase through a disorder-->order transition of the substrate-binding site mediated by a calcium-sodium-calcium metal triad.

BACKGROUND: The structural basis as to how metals regulate the functional state of a protein by altering or stabilizing its conformation has been characterized in relatively few cases because the metal-free form of the protein is often partially disordered and unsuitable for crystallographic analysis. This is not the case, however, for Bacillus licheniformis alpha-amylase (BLA) for which the structure of the metal-free form is available. BLA is a hyperthermostable enzyme which is widely used in biotechnology, for example in the breakdown of starch or as a component of detergents. The determination of the structure of BLA in the metal-containing form, together with comparisons to the apo enzyme, will help us to understand the way in which metal ions can regulate enzyme activity. RESULTS: We report here the crystal structure of native, metal-containing BLA. The structure shows that the calcium-binding site which is conserved in all alpha-amylases forms part of an unprecedented linear triadic metal array, with two calcium ions flanking a central sodium ion. A region around the metal triad comprising 21 residues exhibits a conformational change involving a helix unwinding and a disorder-->order transition compared to the structure of metal-free BLA. Another calcium ion, not previously observed in alpha-amylases, is located at the interface between domains A and C. CONCLUSIONS: We present a structural description of a major conformational rearrangement mediated by metal ions. The metal induced disorder-->order transition observed in BLA leads to the formation of the extended substrate-binding site and explains on a structural level the calcium dependency of alpha-amylases. Sequence comparisons indicate that the unique Ca-Na-Ca metal triad and the additional calcium ion located between domains A and C might be found exclusively in bacterial alpha-amylases which show increased thermostability. The information presented here may help in the rational design of mutants with enhanced performance in biotechnological applications.

Probing structural determinants specifying high thermostability in Bacillus licheniformis alpha-amylase.

Bacillus licheniformis alpha-amylase (BLA) is a starch-degrading enzyme that is highly thermostable although it is produced by a rather mesophilic organism. Over the last decade, the origin of BLA thermal properties has been extensively investigated in both academic and industrial laboratories, yet it is poorly understood. Here, we have used structure-based mutagenesis in order to probe the role of amino acid residues previously proposed as being important for BLA thermostability. Residues involved in salt-bridges, calcium binding or potential deamidation processes have been selected and replaced with various amino acids using a site-directed mutagenesis method, based on informational suppression. A total of 175 amylase variants were created and analysed in vitro. Active amylase variants were tested for thermostability by measuring residual activities after incubation at high temperature. Out of the 15 target residues, seven (Asp121, Asn126, Asp164, Asn192, Asp200, Asp204 and Ala269) were found to be particularly intolerant to any amino acid substitutions, some of which lead to very unstable mutant enzymes. By contrast, three asparagine residues (Asn172, Asn188 and Asn190) could be replaced with amino acid residues that significantly increase the thermostability compared to the wild-type enzyme. The highest stabilization event resulted from the substitution of phenylalanine in place of asparagine at position 190, leading to a sixfold increase of the enzyme's half-life at 80 degrees C (pH 5.6, 0.1 mM CaCl(2)). These results, combined with those of previous mutational analyses, show that the structural determinants contributing to the overall thermostability of BLA concentrate in domain B and at its interface with the central A domain. This region contains a triadic Ca-Na-Ca metal-binding site that appears extremely sensitive to any modification that may alter or reinforce the network of electrostatic interactions entrapping the metal ions. In particular, a loop spanning from residue 178 to 199, which undergoes pronounced conformational changes upon removal of calcium, appears to be the key feature for maintaining the enzyme structural integrity. Outside this region, most salt-bridges that were destroyed by mutations were found to be dispensable, except for an Asp121-Arg127 salt-bridge that contributes to the enhanced thermostability of BLA compared to other homologous bacterial alpha-amylases. Finally, our studies demonstrate that the natural resistance of BLA against high temperature is not optimized and can be enhanced further through various means, including the removal of possibly deamidating residues.

RNA recognition by transcriptional antiterminators of the BglG/SacY family: functional and structural comparison of the CAT domain from SacY and LicT.

Transcriptional antiterminators of the BglG/SacY family are regulatory proteins that mediate the induction of sugar metabolizing operons in Gram-positive and Gram-negative bacteria. Upon activation, these proteins bind to specific targets in nascent mRNAs, thereby preventing abortive dissociation of the RNA polymerase from the DNA template. We have previously characterized the RNA-binding domain of SacY from Bacillus subtilis and determined its three-dimensional structure by both NMR and crystallography. In the present study, we have characterized the paralogous domain from LicT and we present the first structural comparison between two BglG/SacY family members. Similar to SacY, the RNA-binding activity of LicT is contained within the 56 N-terminal amino acid residue fragment corresponding to the so-called co-antiterminator (CAT) domain. Surface plasmon resonance affinity measurements show that, compared to SacY-CAT, LicT-CAT binds more tightly and more specifically to its cognate RNA target, with a KD value of about 10(-8) M. The crystal structure of LicT-CAT has been determined at 1.8 A resolution and compared to that of SacY-CAT. Both molecules fold as symmetrical dimers, each monomer comprising a four-stranded antiparallel beta-sheet that stacks against the beta-sheet of the other monomer in a very conserved manner. Comparison of the proposed RNA-binding surfaces shows that many of the conserved atoms concentrate in a central region across one face of the CAT dimer, whereas variable elements are mostly found at the edges. Interestingly, the electrostatic potential maps calculated for the two molecules are quite different, except for the core of the RNA-binding site, which appears essentially neutral in both structures.

Dimer stabilization upon activation of the transcriptional antiterminator LicT.

LicT belongs to the BglG/SacY family of transcriptional antiterminators that induce the expression of sugar metabolizing operons in Gram positive and Gram negative bacteria. These proteins contain a N-terminal RNA-binding domain and a regulatory domain called PRD which is phosphorylated on conserved histidine residues by components of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). Although it is now well established that phosphorylation of PRD-containing transcriptional regulators tunes their functional response, the molecular and structural basis of the regulation mechanism remain largely unknown.A constitutively active LicT variant has been obtained by introducing aspartic acid in replacement of His207 and His269, the two phosphorylatable residues of the PRD2 regulatory sub-domain. Here, the functional and structural consequences of these activating mutations have been evaluated in vitro using various techniques including surface plasmon resonance, limited proteolysis, analytical centrifugation and X-ray scattering. Comparison with the native, unphosphorylated form shows that the activating mutations enhance the RNA-binding activity and induce tertiary and quaternary structural changes. Both mutant and native LicT form dimers in solution but the native dimer exhibits a less stable and more open conformation than the activated mutant form. Examination of the recently determined crystal structure of mutant LicT regulatory domain suggests that dimer stabilization is accomplished through salt-bridge formation at the PRD2:PRD2 interface, resulting in domain motion and dimer closure propagating the stabilizing effect from the protein C-terminal end to the N-terminal effector domain. These results suggest that LicT activation arises from a conformational switch inducing long range rearrangement of the dimer interaction surface, rather than from an oligomerization switch converting an inactive monomer into an active dimer.

Impact of the lysine-188 and aspartic acid-189 inversion on activity of trypsin.

The impact of the charge rearrangement on the specificity of trypsin was tested by an inversion of sequence K188D/D189K maintaining the integrity of the charges of the substrate binding pocket when switching their polarity. In native trypsin, aspartate 189 situated at the bottom of the primary substrate binding pocket interacts with arginine and lysine side chains of the substrate. The kinetic parameters of the wild-type trypsin and K188D/D189K mutant were determined with synthetic tetrapeptide substrates. Compared with trypsin, the mutant K188D/D189K exhibits a 1.5- to 6-fold increase in the Km for the substrates containing arginine and lysine, respectively. This mutant shows a approximately 30-fold decrease of its k(cat) and its second-order rate constant k(cat)/Km decreases approximately 40- and 150-fold for substrates containing arginine and lysine, respectively. Hence, trypsin K188D/D189K displays a large increase in preference for arginine over lysine.

Consecutive successful pregnancies in a combined liver and kidney transplant recipient with type 1 primary hyperoxaluria.

Pregnancy is now a common, but high-risk event, in young women who have received transplants. Consequences to the fetus are known, but pregnancy may also interfere with graft function. We report the outcome of two successive and successful pregnancies in a 29-year-old woman with type 1 hyperoxaluria, who received a combined liver and kidney transplant. Two healthy children were born at 35 and 37 weeks of gestation, with low birth weight. Liver function remained normal before, during, and after pregnancies up to 52 months after transplantation. Renal function was impaired before the first conception, worsened during both pregnancies, and returned to the previous level in both immediate postpartum periods. However, renal function has declined 17 months after the last delivery. This report shows the feasibility of successive pregnancies in multiple organ transplant recipients, but raises the question of long-term maternal kidney graft survival.

Selection of diabetic patients for islet transplantation. A single-center experience.

OBJECTIVE: Since the Edmonton protocol, islet transplantation (IT) offers the prospect of adequate glycemic control with no major surgical risk. In our single-center experience of IT, we studied the recruitment of eligible diabetic patients. METHODS: Between 1998 and 2002, we screened 79 diabetic patients that were divided into 2 groups according to their renal status: 41 were not receiving dialysis (ND) while 38 were receiving ongoing dialysis (D). RESULTS: In the ND group, 20 patients initiated the contact with our team, 8 patients were recruited during hospitalization for very poor glycemic imbalance, and 13 were referred by their diabetologist. 14/41 (34%) patients were ineligible for IT either because of very good glycemic balance, detectable C-peptide (C-p), kidney or liver problems, or plans for future pregnancy. 16/41 (39%) did not wish to proceed, 7 of whom were more interested by a pump. 11/41 (27%) were eligible, among which 8 are currently being assessed, 1 is on the waiting list and 2 have been transplanted. In the D group, 17/38 (45%) had a detectable C-p and received a kidney graft alone. Among the remaining 21 C-p negative diabetic patients, 3 were not eligible for kidney transplantation mainly for psychological reasons, and 4 were enlisted for kidney+pancreas transplantation. The remaining 14 C-p negative patients were kidney-transplanted. Among them, 6 were not eligible for IT, mainly for lack of motivation, slightly positive C-p stimulation tests, obesity, cancer, or increased creatininemia. The remaining 8/14 C-p negative kidney-engrafted patients were enlisted for IT. 3 had secondary failure with the pre-Edmonton immunosuppressive (IS) protocol. Five have been transplanted with the Edmonton-like IS regimen. CONCLUSION: Twenty-five per cent of the 79 patients for whom islet transplantation was considered underwent pregraft assessment and 12% (10 patients, 8 kidney-transplanted and 2 islet alone) of the 79 have been transplanted. The main eligibility criteria were undetectable Cpeptide, normal kidney function, average weight, glycemic imbalance, hypoglycemia unawareness, and glycemic brittleness.

[Late portal vein thrombosis caused by hematoma after liver transplantation]. / Thrombose porte tardive par hématome après transplantation hépatique.

Late portal vein thrombosis of liver grafts is uncommon and is usually revealed by variceal bleeding. We herein report a case of thrombosis discovered in an adult 10 months after liver transplantation, due to stenosis of the portal vein by extrinsic compression, probably secondary to a biopsy-induced hematoma. Development of venous collateral circulation allowed for normal function of the liver graft.

[Pregnancy after hepatic transplantation: what is the maternal risk?]. / Grossesse après transplantation hépatique: quel est le risque maternel?

OBJECTIVES AND METHODS: We report 7 pregnancies which occurred from 1988 to 1995 in 5 women who underwent liver transplantation. The immunosuppression regimen associated cyclosporine, azathioprine and prednisone. RESULTS: Mean age at conception was 25. During pregnancy, cholestasis occurred in 2 women. None of the patients experienced rejection. An increase in serum creatinine was observed in 3 cases. Serum uric acid increased in the third trimester of pregnancy in 6 cases, associated with arterial hypertension in 3 cases. In 4 cases, toxemia led to premature delivery. Seven childbirths occurred between the 34th and 38th week of gestation, by vaginal delivery (n = 3) or caesarean section (n = 4). Newborn weights ranged from 1,350 g to 3,100 g. A favorable outcome was observed in all mothers, with a follow-up ranging from 2 months to 7 years after delivery. CONCLUSION: These results suggest that a successful pregnancy is possible after liver transplantation in young women with normal hepatic function and treated with cyclosporine. The risk of toxemia is mainly related to renal function before pregnancy.

[Postoperative acute adrenal insufficiency]. / Insuffisance surrénalienne aiguë postopératoire.

Acute adrenal insufficiency is an uncommon complication of lung cancer and adrenal metastasis resection. Diagnosis is difficult to establish but an early recognition and treatment may be life-saving. A 55-year-old man underwent right upper lobectomy and adrenalectomy for lung carcinoma with right adrenal metastasis. Anaesthesia was obtained with propofol, alfentanil, atracurium and isoflurane. Blood pressure remained stable throughout surgical procedure and blood loss was about 3,000 ml. Several hours after the end of the procedure which was uneventful the circulator status worsened. The blood pressure was initially controlled with 500 ml of gelatin. External blood loss was about 200 ml. Clinical examination, chest X-ray and ECG were normal. Postoperative laboratory data showed a serum sodium at 134 mmol-1.l-1 and a serum potassium 5.1 mmol.l-1; haemoglobin concentration was 93 g.l-1. Arterial blood gas analysis, with a 5.1.min-1 nasal O2 flow showed a PaO2 at 108 mmHg, a PaCO2 at 30 mmHg and a pH at 7.44. Twelve hours later, a transient cardiac arrest occurred which responded to fluid load, dopamine and dobutamine. Six hours later, the patient went in ventricular fibrillation responding to an external electric countershock. No change in clinical status was noticed, except hyperthermia at 39.5% degrees C. Serum potassium concentration before cardiac arrest was 4.7 mmol-l-1. Main considered diagnoses were septic shock and acute adrenal insufficiency. Antibiotics (imipenem, amikacin and vancomycin) and hormonal treatment (hydrocortisone 200 mg.day-1), after blood samples had been obtained for bacteriological and hormonal examinations. The patient's condition improved dramatically within 48 hours. Shock was under control, dopamine and dobutamine were rapidly discontinued.(ABSTRACT TRUNCATED AT 250 WORDS)

[Transplantation for primary hyperoxaluria. Role of oxalate crystal deposits in the occurrence of kidney failure]. / Transplantation pour hyperoxalurie primaire. Rôle des dépôts cristallins d'oxalate dans la survenue de l'insuffisance rénale.

We analyzed the records of 3 patients transplanted for end-stage renal failure due to primary hyperoxaluria and evaluated on repeat biopsies the role played by oxalate deposits in the constitution of renal failure after isolated kidney graft, or combined liver and kidney transplantation. Early failure of the renal graft is frequent and often interpreted as the consequence of recurrence because of the presence of oxalate deposits on the graft biopsy. In fact, the decrease in oxalate deposits observed in our 2 cases of combined liver and kidney transplantation despite the progressive renal failure, indicates that crystal deposition is not responsible for the renal lesions. However, we cannot exclude that the oxalate molecule toxicity plays a role in the constitution of the diffuse sclerosis which occurred in these two cases after a primary renal non function, aggravating a hemodynamic process by using cyclosporin. On the other hand, as observed in our isolated kidney graft, renal crystal deposition occurring before the onset of renal failure suggests the true mechanism explaining the slow recurrence of renal oxalosis.

Hyperthermostable variants of a highly thermostable alpha-amylase.

Genetic screening at temperatures between 70-80 degrees C far exceeds the range of growth of most bacteria, and is not applicable to isolate easily thermostable protein variants. We describe a temperature shift protocol and an in vivo screening method which allowed us to identify a hyperthermostable variant of the thermostable alpha-amylase from Bacillus licheniformis. Our strategy was to select, after hydroxylamine mutagenesis, an intragenic suppressor mutation which overcomes a mutation leading to a thermolabile enzyme. Sequence analysis of the mutated gene revealed only one change in the amino acid sequence, substituting a valine for alanine at position 209. This single amino acid replacement increased the half-life of the protein at 90 degrees C by a factor of two to three relative to the wild-type enzyme. When this substitution was combined with another stabilizing substitution (H133Y) we described previously, the stabilizing effects were additive. The half-life of the new protein was about 12 hours at 90 degrees C, corresponding to a nine to ten-fold increase over the wild-type enzyme and the industrial Bacillus licheniformis alpha-amylase Termamyl. These mutations are located in a predicted folding domain of the protein which appears crucial in determining thermal stability.