[A case of fetal death resulting from a massive fetomaternal hemorrhage]. / LE CAS CLINIQUE DU MOIS. Mort foetale in utero due à une hémorragie foeto-maternelle massive.

We report the case of a late stillbirth which unexpectedly occurred in a patient without any medical history and after a meticulous obstetrical follow up. Stillbirth is unfortunately not unusual and implies a complete etiological work up. In the present observation, the Kleihauer test and anatomoclinical examination concluded that the death was due to an acute cerebral anoxy resulting from a massive fetomaternal hemorrhage (HFM). HFM is rarely considered as the cause of a late stillbirth, but its occurrence is certainly underestimated. Yet, if HFM is identified before fetal death, an .adequate management could considerably improve the fetal prognosis and, sometines, save the child's life.

Modulation of ethanol effect on hepatocyte proliferation by polyamines.

An occurrence and a magnitude of alcoholic liver diseases depend on the balance between ethanol-induced injury and liver regeneration. Like ethanol, polyamines including putrescine, spermidine, and spermine modulate cell proliferation. Thus, the purpose of this study was to evaluate the relationship between effect of ethanol on hepatocyte (HC) proliferation and polyamine metabolism using the HepaRG cell model. Results showed that ethanol effect in proliferating HepaRG cells was associated with a decrease in intracellular polyamine levels and ornithine decarboxylase (ODC) activity. Ethanol also induced disorders in expression of genes coding for polyamine-metabolizing enzymes. The α-difluoromethyl ornithine, an irreversible inhibitor of ODC, amplified ethanol toxicity on cell viability, protein level, and DNA synthesis through accentuation of polyamine depletion in proliferating HepaRG cells. Conversely, putrescine reversed ethanol effect on cell proliferation parameters. In conclusion, this study suggested that ethanol effect on HC proliferation was closely related to polyamine metabolism and that manipulation of this metabolism by putrescine could protect against the anti-proliferative activity of ethanol.

[Psychomotor disadaptation syndrome: a poorly known clinical entity]. / Le syndrome de désadaptation psychomotrice (SDPM): une entité clinique encore méconnue.

Psychomotor disadaptation syndrome is a typical geriatric clinical syndrome. It includes postural disorders such as body retropulsion, specific gait disorders, with axial akinesia and hypertonia, and psychobehavioral disorders akin to those found in depression. The diagnosis is essentially based on clinical observation rather than on iconographic data. This syndrome causes falls which induce a fear of falling. The old patient has a tendency to put himself down, to withdraw from society and to lose autonomy. This article briefly describes the physiopathology of the syndrome, recalls the diagnostic tools, and makes some suggestions regarding the care of patients suffering from this clinical entity.

Regulation of experimental peritonitis: a complex orchestration.

Experimental peritonitis is a frequently used inflammatory model to evaluate leukocyte recruitment. By the intrinsic characteristics of the peritoneal cavity, the various resident cell populations have a role to play in the initiation, the modulation and the resolution of peritoneal inflammation. Through various manipulations of these cell populations, we gained important knowledge on their respective roles in peritoneal inflammation. In this brief review, we will focus on the cellular regulation of leukocyte recruitment in experimental peritonitis.

Psychopathology and quality of life burden in chronic daily headache: influence of migraine symptoms.

The aim of this study is to compare the psychopathology and the quality of life of chronic daily headache patients between those with migraine headache and those with tension-type headache. We enrolled 106 adults with chronic daily headache (CDH) who consulted for the first time in specialised centres. The patients were classified according to the IHS 2004 criteria and the propositions of the Headache Classification Committee (2006) with a computed algorithm: 8 had chronic migraine (without medication overuse), 18 had chronic tension-type headache (without medication overuse), 80 had medication overuse headache and among them, 43 fulfilled the criteria for the sub-group of migraine (m) MOH, and 37 the subgroup for tension-type (tt) MOH. We tested five variables: MADRS global score, HAMA psychic and somatic sub-scales, SF-36 psychic, and somatic summary components. We compared patients with migraine symptoms (CM and mMOH) to those with tension-type symptoms (CTTH and ttMOH) and neutralised pain intensity with an ANCOVA which is a priori higher in the migraine group. We failed to find any difference between migraine and tension-type groups in the MADRS global score, the HAMA psychological sub-score and the SF36 physical component summary. The HAMA somatic anxiety subscale was higher in the migraine group than in the tension-type group (F(1,103) = 10.10, p = 0.001). The SF36 mental component summary was significantly worse in the migraine as compared with the tension-type subgroup (F(1,103) = 5.758, p = 0.018). In the four CDH subgroups, all the SF36 dimension scores except one (Physical Functioning) showed a more than 20 point difference from those seen in the adjusted historical controls. Furthermore, two sub-scores were significantly more affected in the migraine group as compared to the tension-type group, the physical health bodily pain (F(1,103) = 4.51, p = 0.036) and the mental health (F(1,103) = 8.17, p = 0.005). Considering that the statistic procedure neutralises the pain intensity factor, our data suggest a particular vulnerability to somatic symptoms and a special predisposition to develop negative pain affect in migraine patients in comparison to tension-type patients.

Chronic transplant vasculopathy microenvironment present in the renal allograft reprograms macrophage phenotype.

Macrophages and monocytes are important in chronic allograft dysfunction. Chronic transplant vasculopathy is an important cause of chronic renal allograft dysfunction. It is characterized by the presence of apoptotic endothelial cells, which generate an apocrine loop that leads to typical myointimal proliferation. However, the exact nature of the reprogramming induced by this microenvironment on recruited monocyte and macrophage phenotypes is ill defined. Human umbilical vein endothelial cells with a serum-starved condition (SSC) for 4 hours were used as a model of apoptotic endothelial cells. This conditioned medium was centrifuged to isolate the apoptotic bodies. Monocytes were harvested from healthy donors using Ficoll gradient and immunomagnetic selection. Blood monocyte-derived macrophages (5-7 days of maturation) were exposed to centrifuged apoptotic cell-conditioned media for 24 hours. Cells were harvested for immunoblotting, and supernates were retained for enzyme-linked immunosorbent assay to determine cytokine and chemokine production. Macrophages generated less IL-6 and IL-8 when cultured in SSC compared with control. Immunoblotting for STAT3 (signal transducer and activator of transcription 3) in macrophages revealed that SSC increased STAT3 levels, which persisted after lipopolysaccharide stimulation. These results suggest that SSC attenuates the pro-inflammatory phenotype in macrophages, through a STAT3-dependent mechanism.

Lymphocytes modulate peritoneal leukocyte recruitment in peritonitis.

OBJECTIVE AND DESIGN: To investigate the modulating role of lymphocytes in leukocyte recruitment in a murine model of peritonitis. MATERIALS OR SUBJECTS: RAG-1 knockout (KO) mice, NUDE mice and microMT KO mice were compared to their wild-type controls. TREATMENT: Mice were administered with 1 ml of Brewer's thioglycollate (BTG) and terminal peritoneal lavages were performed at 8, 24, 72 and 120 h after treatment. METHODS: Leukocyte numbers recruited at the different time points following a BTG administration were determined. Chemokine and cytokine levels were assessed by either ELISAs or cytometric bead array. RESULTS: RAG-1 KO mice (absent B and T cells) exhibited increased early neutrophil infiltration and blunted late monocyte/macrophage infiltration. NUDE mice (absent T cells) exhibited both increased neutrophil and monocyte/macrophage infiltration. In contrast, microMT KO mice (absent B cells) demonstrated reduced influx of both neutrophils and monocyte/macrophages. Chemokine analysis revealed various differences in important chemokines. CONCLUSIONS: These data suggest that T cells act to suppress leukocyte recruitment while B cells promote leukocyte recruitment.

Automatic analysis of pacemaker diagnostic data in the identification of atrial tachyarrhythmias in patients with no prior history of them.

AIMS: Modern pacemakers provide a large amount of diagnostic data. Given the limited time available during a pacemaker follow-up visit essential information may be overlooked. This registry was conducted to assess the utility of an expert system that analyses the diagnostic data collected by an implanted pacing device and notifies and advises the physician about suspected technical issues and arrhythmias that need further attention. METHODS: Patients with various standard indications for pacing were included in this registry and received single or dual chamber pacemakers. Data were collected and analysed by the expert system during at least two subsequent follow-up visits. The evaluation of this system focused on data obtained from patients with a dual chamber pacing device without prior history of atrial arrhythmias. RESULTS: A total number of 239 patients without prior history of atrial tachyarrhythmias were included in this analysis. Atrial tachyarrhythmias were detected in 73 (31%) of these patients. The highest incidence of newly detected arrhythmias occurred in the group of patients with high-degree AV block and VDD pacemakers. Furthermore, newly detected arrhythmias predominantly occurred in the period shortly after implantation. Device programming suggestions by the expert system were adopted in 30% of the cases. Following detection of atrial tachyarrhythmias by the expert system, pharmacological management was adjusted at 71% of the first follow-up visits and at 27% of later follow-up visits. CONCLUSION: Results of this registry show that this expert system provides a valuable tool for the detection of atrial tachyarrhythmias during pacemaker follow-up visits.

Initial studies of the molecular basis of peptide mimicry of group B streptococcal type III capsular polysaccharide.

We have previously shown that the peptide FDTGAFDPDWPA is a mimetic of the group B streptococcal type III capsular polysaccharide (CPS(III)). It binds to anti-CPS(III) antibodies and can be used to immunize mice and produce anti-CPS(III). In this paper we investigate the molecular mechanisms underlying this peptide-carbohydrate mimicry in two ways. First, we have examined the conformation of the peptide by NMR spectroscopy in water. Next, we have produced monoclonal anti-peptide and anti-CPS(III) antibodies, compared their fine specificity, and have sequenced them. The results indicate that the peptide assumes a conformation that may be similar to that of the CPS(III) in solution and that the peptide and CPS(III) elicit an overlapping repertoire of antibodies.

Blood oxidative stress in amyotrophic lateral sclerosis.

It has been suggested that amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder resulting in motor neuron death, is associated with oxidative damage induced by free radicals. Our study aimed to get an assessment of the blood oxidative stress status in a population of 167 ALS patients (aged 59+/-13 years), treated or not with riluzole, compared with 62 age-matched healthy control subjects (aged 60+/-11 years) simultaneously included in the study. We determined the level of plasma lipid peroxidation (thiobarbituric acid-reactive substances, TBARS); the status of the major lipophilic plasma antioxidant defenses (vitamin E, vitamin A and beta-carotene); the activities of erythrocyte Cu,Zn-superoxide dismutase (Cu,Zn-SOD) and of plasma and erythrocyte glutathione peroxidase (GSH-Px). Plasma selenium was also determined as a trace element essential to the activity of the GSH-Px. In comparison with controls, we observed in ALS patients (mean+/-S.D.) significantly higher TBARS values (ALS=1.34+/-0.28 micromol/l; controls=1.11+/-0. 20 micromol/l) and a significant enhancement of the erythrocyte SOD activity (ALS=710+/-114 U/g Hb; controls=667+/-93 U/g Hb). No differences were observed for selenium level, GSH-Px activity, plasma vitamin E, beta-carotene and vitamin A concentrations. These data confirm the presence of an oxidative stress in blood of ALS patients. The elevated plasma TBARS, without any deficiency in plasma lipophilic antioxidants such as vitamin E, vitamin A and beta-carotene, suggest an enhancement in the production of free radicals. No correlation was found in our study between the level of any of the blood oxidative stress markers and the disease duration. Comparison between patients treated or not with riluzole did not display any modification of the plasma TBARS concentration, but we observed a slight decrease of erythrocyte SOD activity in treated patients (treated=705+/-113 U/g Hb; not treated=725+/-118 U/g Hb), suggesting a possible activity of riluzole on the oxygenated free radical production.

[Effects of the preparation on anxiety before bronchoscopy]. / Effets de la préparation sur l'anxiété avant la fibroscopie bronchique.

In their daily practice, the nurses note the patients' anxiety when they are in hospital for diagnosis exams. Considering this observation, we wanted to assess the potential benefits provided by the behavioural and relational techniques, such as sophrology, maintenance of the help relation, visualization-relaxation. In order to carry out this survey, we adopted the model of Betty NEUMAN, who relies on the concept of homeostasis and on the stress theory of Hans Seyle. The measurement of anxiety by the STAI (State Trait Anxiety Inventory), a scale worked out by SPIELBERGER, enabled us to prove that these relational tools, used by the nurses, made it possible for the patients to better mobilize their adjustment or coping strategies. Recommendations concerning the management of anxiety were set out as not to trigger an attitude of vigilant coping.

Elevation of tumor necrosis factor-alpha and increased risk of recurrent coronary events after myocardial infarction.

BACKGROUND: Levels of tumor necrosis factor-alpha (TNF-alpha) increase with acute ischemia. However, whether elevations of TNF-alpha in the stable phase after myocardial ischemia (MI) are associated with increased risk of recurrent coronary events is unknown. METHODS AND RESULTS: A nested case-control design was used to compare TNF-alpha levels obtained an average of 8.9 months after initial MI among 272 participants in the Cholesterol And Recurrent Events (CARE) trial who subsequently developed recurrent nonfatal MI or a fatal cardiovascular event (cases) and from an equal number of age- and sex-matched participants who remained free of these events during follow-up (controls). Overall, TNF-alpha levels were significantly higher among cases than controls (2.84 versus 2.57 pg/mL, P=0.02). The excess risk of recurrent coronary events after MI was predominantly seen among those with the highest levels of TNF-alpha, such that those with levels in excess of 4.17 pg/mL (the 95th percentile of the control distribution) had an approximately 3-fold increase in risk (RR=2.7, 95% CI 1.4 to 5.2, P=0.004). Risk estimates were independent of other risk factors and were similar in subgroup analyses limited to cardiovascular death (RR=2.1) or to recurrent nonfatal MI (RR=3.2). CONCLUSIONS: Plasma concentrations of TNF-alpha are persistently elevated among post-MI patients at increased risk for recurrent coronary events. These data support the hypothesis that a persistent inflammatory instability is present among stable patients at increased vascular risk. Novel therapies designed to attenuate inflammation may thus represent a new direction in the treatment of MI.

Oxidizability of atherogenic low-density lipoprotein subspecies in severe familial hypercholesterolemia: impact of long-term low-density lipoprotein apheresis.

BACKGROUND: Oxidative modification of low-density lipoprotein (LDL) plays a key role in the pathophysiology of atherosclerosis. LDL-apheresis, which involves direct removal of plasma LDL from circulating blood, is an efficient treatment of homozygous familial hypercholesterolemia (FH). METHODS: We evaluated impact of long-term LDL apheresis treatment on the atherogenicity of the major LDL subclasses (light, LDL1, and LDL2, density [d] 1.018-1.030 g/mL; intermediate, LDL3, d 1.030-1.040 g/mL, and dense LDL, LDL4 and LDL5, d 1.040-1.065 g/mL) separated by density gradient ultracentrifugation in severe FH patients. Therefore, we compared the oxidative resistance as well as the chemical and physical properties of each LDL subpopulation in the FH group with those in the corresponding LDL subfractions from normocholesterolemic control subjects. RESULTS: Both intermediate and dense LDL subfractions were significantly more resistant to copper-mediated oxidation in FH patients treated regularly by LDL-apheresis than their counterpart controls. The lag phases for LDL3, LDL4, and LDL5: 63.9+/-11.6, 55.8+/-1.2, and 47.2+/-6.5 min. in FH patients were significantly longer than those of the corresponding subfractions in normocholesterolemic controls (P <.01 for LDL3 and LDL5, P<.005 for LDL4). This protective effect was reflected in the delayed formation of biologically active lipid oxidation products such as oxysterols, lipid hydroperoxides, dienes, and dienals in the intermediate and dense LDL subfractions of FH patients. These findings may result from lower "seed" contents of lipid hydroperoxide (LOOH) detected as dienes in plasma LDL from apheresis-treated FH patients; indeed, baseline LOOH/diene contents in all 5 LDL subclasses from FH patients were significantly lower than those of the corresponding subclasses in normolipidemic subjects (P<.0005). On the other hand, the enhanced oxidative resistance of both intermediate (LDL3) and dense (LDL4 and LDL5) LDL subpopulations in FH patients could not be accounted for by any consistent modification in chemical composition or in lipophilic antioxidant content, although minor differences were observed between patients and controls in unsaturated fatty acid profile. In contrast, sphingomyelin content was enriched in FH LDL subclasses, potentially resulting in reduced penetration of the hydrophilic surface layer of LDL by oxygen radicals. CONCLUSION: We conclude that low concentrations of preformed lipid hydroperoxides and dienes, together with surface sphingomyelin enrichment, can account for the enhanced oxidative resistance of intermediate (LDL3) and atherogenic dense LDL (LDL4, LDL5) induced by long-term LDL apheresis in severe FH patients.

Cholesteryl ester hydroperoxide lability is a key feature of the oxidative susceptibility of small, dense LDL.

Abundant evidence has been provided to substantiate the elevated cardiovascular risk associated with small, dense, low density lipoprotein (LDL) particles. The diminished resistance of dense LDL to oxidative stress in both normolipidemic and dyslipidemic subjects is established; nonetheless, the molecular basis of this phenomenon remains indeterminate. We have defined the primary molecular targets of lipid hydroperoxide formation in light, intermediate, and dense subclasses of LDL after copper-mediated oxidation and have compared the relative stabilities of the hydroperoxide derivatives of phospholipids and cholesteryl esters (CEs) as a function of the time course of oxidation. LDL subclasses (LDL1 through LDL5) were isolated from normolipidemic plasma by isopycnic density gradient ultracentrifugation, and their content of polyunsaturated molecular species of phosphatidylcholine (PC) and CE and of lipophilic antioxidants was quantified by reverse-phase high-performance liquid chromatography. The molar ratio of the particle content of polyunsaturated CE and PC species containing linoleate or arachidonate relative to alpha-tocopherol or beta-carotene did not differ significantly between LDL subspecies. Nonetheless, dense LDL contained significantly less polyunsaturated CE species (400 mol per particle) compared with LDL1 through LDL4 (range, approximately 680 to 490 mol per particle). Although the formation of PC-derived hydroperoxides did not vary significantly between LDL subspecies as a function of the time course of copper-mediated oxidation, the abundance of the C18:2 and C20:4 CE hydroperoxides was uniquely deficient in dense LDL (23 and 0.6 mol per particle, respectively, in LDL5; 47 to 58 and 1.9 to 2.3 mol per particle, respectively, in other LDL subclasses) at propagation half-time. When expressed as a lability ratio (mol hydroperoxides formed relative to each 100 mol of substrate consumed) at half-time, the oxidative lability of CE hydroperoxides in dense LDL was significantly elevated (lability ratio <25:100) relative to that in lighter, larger LDL particle subclasses (lability ratio >40:100) throughout the oxidative time course. We conclude that the elevated lability of CE hydroperoxides in dense LDL underlies the diminished oxidative resistance of these particles. Moreover, this phenomenon appears to result not only from the significantly elevated PC to free cholesterol ratio (1.54:1) in dense LDL particles (1.15:1 to 1.25:1 for other LDL subclasses) but also from their unique structural features, including a distinct apoB100 conformation, which may facilitate covalent bond formation between oxidized CE and apoB100.

When drug inactivation renders the target irrelevant to antibiotic resistance: a case story with beta-lactams.

By challenging the efficiency of some of our most useful antimicrobial weapons, bacterial antibiotic resistance is becoming an increasingly worrying clinical problem. A good antibiotic is expected to exhibit a high affinity for its target and to reach it rapidly, while escaping chemical modification by inactivating enzymes and elimination by efflux mechanisms. A study of the behaviour of a beta-lactamase-overproducing mutant of Enterobacter cloacae in the presence of several penicillins and cephalosporins showed that the minimum inhibitory concentration (MIC) values for several compounds were practically independent of the sensitivity of the target penicillin binding protein (PBP), even for poor beta-lactamase substrates. This apparent paradox was explained by analysing the equation that relates the antibiotic concentration in the periplasm to that in the external medium. Indeed, under conditions that are encountered frequently in clinical isolates, the factor characterizing the PBP sensitivity became negligible. The conclusions can be extended to all antibiotics that are sensitive to enzymatic inactivation and efflux mechanisms and must overcome permeability barriers. It would be a grave mistake to neglect these considerations in the design of future antibacterial chemotherapeutic agents.

Modifications of alpha-tocopherol and fatty acid concentrations in blood and adipose tissue of obese patients during a weight loss programme.

BACKGROUND AND AIM: The aim of the study was to describe qualitative and/or quantitative modifications of lipoproteins, including their fatty acid composition, in obese patients during a hypocaloric diet, and determine whether the variations observed paralleled modifications of alpha-tocopherol concentration in adipose tissue and blood. METHODS AND RESULTS: 15 healthy, obese volunteers (5 males, 10 females; age: 32-69 yr; BMI: 28.4-60.5 kg/m2) were given a 3-week hypocaloric diet (3.9 MJ (941 kcal)). Adipose tissue and blood samples were taken at the beginning and at the end of this period. At baseline and after 3 weeks measurements were made for alpha-tocopherol and fatty acids in total serum, lipoproteins and adipose tissue; thiobarbituric acid-reactive substances (TBARS) in serum. A significant drop in cholesterol-rich particles (LDL and HDL) was observed, in parallel to a significant enrichment of n-6 polyunsaturated fatty acids (PUFA) at the expense of both saturated and monounsaturated fatty acids in serum. A drop in alpha-tocopherol concentration (expressed as mumol alpha-tocopherol/g lipid) in serum and lipoprotein fractions paralleled the decrease in cholesterol-rich lipoproteins. CONCLUSIONS: Our results suggest that a hypocaloric diet not only decreases cholesterol-rich particle levels in serum, but also leads to a significant modification of fatty acid composition which may reflect improvement of insulin sensitivity. We did not observe any modification in adipose tissue after diet with regard to both alpha-tocopherol and fatty acid concentrations. Despite a drop in alpha-tocopherol concentration and an increase in n-6 PUFA content in serum, we did not find any enhancement of serum lipid peroxidation level evaluated by the thiobarbituric acid-reactive substance (TBARS) assay. If we assume that dietary intakes of alpha-tocopherol were not modified during this diet, it can be supposed that adipose tissue released alpha-tocopherol without any specific regulation, in parallel to the release of fatty acids.

[The plasma antioxidant status and trace elements in patients with familial hypercholesterolemia treated with LDL-apheresis]. / Statut plasmatique en antioxydants et en oligoéléments de patients atteints d'hypercholestérolémie familiale traités par LDL-aphérèse.

Oxidation of low density lipoprotein is involved in the pathogenesis of atherosclerosis. Epidemiological studies suggest a negative correlation between the occurrence of cardiovascular diseases and blood concentrations of lipophilic antioxidants such as vitamin A and E and beta-carotene. Trace elements such as selenium, zinc and copper are involved in the activity of antioxidant enzymes: glutathione peroxidase and superoxide dismutase. The aim of this work was to determine the antioxidant and trace elements status of patients with very severe hypercholesterolemia and who were treated by dextran sulphate low density lipoprotein apheresis, in comparison with two control populations: one constituted by normocholesterolemic subjects and the other by hypercholesterolemic patients before treatment. Our results showed that, as compared with normocholesterolemic subjects, patients treated by LDL-apheresis were not deficient in vitamin E, beta-carotene and copper but had low plasma levels of selenium, zinc and vitamin A. The low selenium and vitamin A levels were due to the treatment by LDL-apheresis by itself, while the hypercholesterolemia of these patients might have provoked the low plasma levels of zinc. This study pointed out the interest of a supplement of selenium, zinc and vitamin A in patients treated by LDL-apheresis.

Site-directed mutagenesis of the Actinomadura R39 DD-peptidase.

The role of various residues in the conserved structural elements of the Actinomadura R39 penicillin-sensitive dd-peptidase has been studied by site-directed mutagenesis. Replacement of Ser-298 of the 'SDN loop' by Ala or Gly significantly decreased the kcat/Km value for the peptide substrate, but only by a factor of 15 and had little effect on the other catalytic properties. Mutations of Asn-300 of the same loop and of Lys-410 of the KTG triad yielded very unstable proteins. However, the N300S mutant could be purified as a fusion protein with thioredoxin that exhibited decreased rates of acylation by the peptide substrate and various cephalosporins. Similar fusion proteins obtained with the N300A, K410H and K410N mutants were unstable and their catalytic and penicillin-binding properties were very strongly affected. In transpeptidation reactions, the presence of the acceptor influenced the kcat/Km values, which suggested a catalytic pathway more complex than a simple partition of the acyl-enzyme between hydrolysis and aminolysis. These results are compared with those obtained with two other penicillin-sensitive enzymes, the Streptomyces R61 dd-peptidase and Escherichia coli penicillin-binding protein (PBP) 5.

Dual multimodular class A penicillin-binding proteins in Mycobacterium leprae.

The ponA gene of cosmid L222 of the Mycobacterium leprae genome library encodes a multimodular class A penicillin-binding protein (PBP), PBP1. The PBP, labelled with a polyhistidine sequence, has been produced in Escherichia coli, extracted from the membranes with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane-sulfonate (CHAPS) and purified by Ni2(+)-nitrilotriacetic acid-agarose chromatography. In contrast to the pon1-encoded class A PBP1, PBP1 undergoes denaturation at temperatures higher than 25 degrees C, it catalyzes acyl transfer reactions on properly structured thiolesters, and it binds penicillin with high affinity.