[Pre- and post-prostatectomy rehabilitation by the certified nurse-urologist pair (REHAB): Feasibility study]. / Parcours de réhabilitation par le binôme IDE-urologue avant et après prostatectomie (REHAB) : étude de faisabilité.

INTRODUCTION: The functional results of radical prostatectomy are a crucial issue for patients to resume fulfilling sexuality. We assessed the feasibility of a care pathway dedicated to sexual rehabilitation in order to improve information, screening of risk situations and the implementation of therapeutic measures. METHODS: From January to May 2023, sexually active patients under 75 years of age undergoing prostatectomy for cancer were offered the opportunity to participate in two sexual rehabilitation sessions (REHAB) led by a nurse-urologist pair. The sessions took place in parallel with the care pathway already in place before and after surgery. The evaluations were carried out by carrying out questionnaires and a clinical examination. A satisfaction questionnaire was given to the patient after the two sessions to assess the format and relevance of the sessions. RESULTS: Fifteen patients were included in the REHAB program. All patients attended both sessions in person and the majority of them (91%) felt they had obtained new information for their rehabilitation. Post-operatively at 6 weeks, 82% of patients were dissatisfied with their sexuality (compared to 64% pre-operatively), Five patients (33%) had regained orgasmic abilities and 20% (n=3) had a penetrative ability. The average IIEF5 score was 19 (6-28) compared to 22.5 pre-operatively (14-30). All patients would recommend these sessions. CONCLUSION: The REHAB sexual rehabilitation program after prostatectomy could be implemented with significant patient adherence and satisfaction.

[Endoscopic papillary abnormalities and urinary stone recognition (EPSR): How and why?] / Reconnaissance endoscopique des anomalies papillaires et des calculs urinaires (REPC) : comment et quel intérêt ?

INTRODUCTION: The purpose of this article is to present the endoscopic papillary abnormalities and stone recognition (EPSR) to state-certified nurses (IDE and IBODE) working in the operating room. METHODS: This article is based on a literature review and the author's experience concerning the endoscopic papillary abnormalities and stone recognition. RESULTS: Since the advent of minimally invasive surgery and the laser, stones are no longer sent as one piece to laboratories, but fragmented. This has made it more difficult for biologists to fully analyze the stones, because they have less morphological data than before. Therefore, endoscopic papillary abnormalities and stone recognition have positioned themselves as tools that can compensate for this loss of information. They play a pivotal role in the identification of the lithogenesis cause, and thus allow a recurrence risk reduction of stones. CONCLUSION: Endoscopic papillary abnormalities and stone recognition are recent tools that require learning. However, the benefit of their uses is proven and is necessary for a complete management of urolithiasis.

[Double J stent removal's cost estimate using a re-sterilizable fibroscope in a French private structure]. / Évaluation du coût en structure privée d'une ablation de sonde double J en externe à l'aide d'un fibroscope re-stérilisable.

In front of the arrival of new devices intended to simplify the removal of double J stent, it poses the problem of the knowledge of the real cost of such an ablation under the current conditions of realization. MATERIAL AND METHOD: This is a monocentric economic evaluation of cost and remuneration needed data-gathering of quotation (CCAM, GHS/SE, …), estimate of the associated costs of wear and damping of the endoscopic equipments (endoscopes, cables, …), estimate of the cost of sterilization, estimate of the associated costs to the intervention of staff (Auxiliary nurse [AS] and Nurse [IDE]) with timing of the various tasks. RESULTS: Quotation CCAM JCGE004 (48€) gives access to fixed price SE1 (73.71€ for private clinic, and 75.89€ for public institution) without hospitalization nor anaesthesia. The costs were reported to an act of single double J removal. Concerning the equipments: 4.42€HT for the fibroscopes, graspers, cable and light. The costs of sterilization were: 17.95€HT. The timed workforce's costs were: 7.61-9.51€ for AS and 9.92-10.84€ for IDE. The cost of consumable was about 1.37 €HT, by excluding the common base from the extractions (1.876€HT). The total costs in France in 2016 were thus about 47.4 to 50.496€ including all taxes. CONCLUSIONS: This estimate will be used certainly for reflection on the investments and the future studies of the economic impact of the new devices of extraction, by correlating it of course with the various maintenance contracts from each institution. LEVEL OF PROOF: 4.

Compliance with clothing regulations and traffic flow in the operating room: a multi-centre study of staff discipline during surgical procedures.

This multi-centre study assessed operating room (OR) staff compliance with clothing regulations and traffic flow during surgical procedures. Of 1615 surgical attires audited, 56% respected the eight clothing measures. Lack of compliance was mainly due to inappropriate wearing of jewellery (26%) and head coverage (25%). In 212 procedures observed, a median of five people [interquartile range (IQR) 4-6] were present at the time of incision. The median frequency of entries to/exits from the OR was 10.6/h (IQR 6-29) (range 0-93). Reasons for entries to/exits from the OR were mainly to obtain materials required in the OR (N=364, 44.5%). ORs with low compliance with clothing regulations tended to have higher traffic flows, although the difference was not significant (P=0.12).

[Ureterolysis: technique, indications]. / Urétérolyse: technique, indications.

Ureteral obstruction due to idiopathic retroperitoneal fibrosis is a rare but severe clinical problem. The open approaches, as well as surgical techniques used to prevent stenosis recurrence, are described. Ureterolysis remains the procedure to relieve ureteral obstruction. The ureter is dissected and freed from the fibrotic process, and then separated to prevent the recurrence of the stenosis. Recently, the development of Laparoscopic urology has allowed for minimal invasive treatment of many urological problems. We present our technique of ureterolysis for extrinsic ureteral obstruction. Advantages and complications of each method are considered and indications are proposed.

Neurotensin induces mating in Saccharomyces cerevisiae cells that express human neurotensin receptor type 1 in place of the endogenous pheromone receptor.

Heterologous expression of the human neurotensin receptor type I (hNT1-R) has been achieved in the yeast Saccharomyces cerevisiae. Immunoanalysis of membranes prepared from cells expressing a c-myc-tagged version of hNT1-R revealed multiple c-myc cross-reacting polypeptides of high molecular mass, suggesting that hNT1-R was glycosylated in yeast. High-affinity binding sites for 125I-labeled-[monoiodo-Tyr3]neurotensin ([125I-Tyr3]NT) were detected on hNT1-R-expressing cells with Kd and Bmax values of 3.2 nM and of 500 receptors per cell, respectively. Competition binding studies of neurotensin with SR142948 and SR48692, two nonpeptidic antagonists of hNT1-R, indicated that the yeast-produced recombinant receptor displayed the same pharmacological properties as hNT1-R expressed in mammalian cells. Interestingly, neurotensin activated the pheromone pathway in hNT1-R-expressing cells in a dose-dependent fashion, as revealed by a beta-galactosidase activity assay with a pheromone-responsive Fus1:lacZ construct. Mutational inactivation of the SST2 and STE2 genes increased the level of beta-galactosidase activity in response to neurotensin by twofold. Recombinant hNT1-R-producing cells, which lacked the endogenous G-protein-coupled receptor for the alpha pheromone, mated with wild-type MATalpha haploid cells in response to neurotensin, leading to bona fide diploid zygote formation. This is the first report of a mammalian receptor that can replace the endogenous pheromone receptor when produced in yeast, by signaling a fully effective, agonist-induced, mating process.

Role of a new mammalian gene family in the biosynthesis of very long chain fatty acids and sphingolipids.

Whereas the physiological significance of microsomal fatty acid elongation is generally appreciated, its molecular nature is poorly understood. Here, we describe tissue-specific regulation of a novel mouse gene family encoding components implicated in the synthesis of very long chain fatty acids. The Ssc1 gene appears to be ubiquitously expressed, whereas Ssc2 and Cig30 show a restricted expression pattern. Their translation products are all integral membrane proteins with five putative transmembrane domains. By complementing the homologous yeast mutants, we found that Ssc1 could rescue normal sphingolipid synthesis in the sur4/elo3 mutant lacking the ability to synthesize cerotic acid (C(26:0)). Similarly, Cig30 reverted the phenotype of the fen1/elo2 mutant that has reduced levels of fatty acids in the C(20)-C(24) range. Further, we show that Ssc1 mRNA levels were markedly decreased in the brains of myelin-deficient mouse mutants known to have very low fatty acid chain elongation activity. Conversely, the dramatic induction of Cig30 expression during brown fat recruitment coincided with elevated elongation activity. Our results strongly implicate this new mammalian gene family in tissue-specific synthesis of very long chain fatty acids and sphingolipids.

Sterol metabolism and ERG2 gene regulation in the yeast Saccharomyces cerevisiae.

Certain exogenously-supplied sterols, like ergost-8-enol, are efficiently converted into ergosterol in yeast. We have taken advantage of this property to study the regulation of the Delta8-Delta7-sterol isomerase-encoding ERG2 gene in an ergosterol auxotrophic mutant devoid of squalene-synthase activity. Ergosterol starvation leads to an 8-16-fold increase in ERG2 gene expression. Such an increase was also observed in wild-type cells either grown anaerobically or treated with SR31747A a sterol isomerase inhibitor. Exogenously-supplied zymosterol is entirely transformed into ergosterol, which represses ERG2 transcription. By contrast, exogenously-supplied ergosterol has little or no effect on ERG2 transcription.

Colocalization of sterol isomerase and sigma(1) receptor at endoplasmic reticulum and nuclear envelope level.

SR31747A is a sigma ligand previously described as having original immunosuppressive properties. Two SR31747A targets were recently identified and termed sigma(1) or SR-BP-1 (SR31747A-binding protein-1) and hSI (human sterol isomerase). In order to characterize these proteins further, we examined their expression and localization at the subcellular level. Based on the amino acid sequence deduced from the cloned hSI, anti-hSI polyclonal antibody was raised against the N-terminal fragment of the protein. Using this antibody, we performed Western-blot experiments to demonstrate the presence of hSI in various B and T cell lines, and hSI expression was quantified in these cell lines by flow cytometry and estimated at 15 000-30 000 sites per cell. Subcellular localization studies by both confocal and electron microscopy, performed on THP1 cells with anti-hSI antibody and with the previously described anti-(SR-BP-1) monoclonal antibody, demonstrated that: (a) hSI was colocalized with SR-BP-1; (b) hSI and SR-BP-1 were associated with the endoplasmic reticulum and with the outer and inner membranes of the nuclear envelope; (c) both proteins were delocalized during the cell cycle at the mitosis step when the nuclear membranes disappeared. Taken together our results suggest that both SR31747A-binding proteins not only play a role in sterol metabolism but indirectly affect lipoprotein functions.

Antiproliferative effects of SR31747A in animal cell lines are mediated by inhibition of cholesterol biosynthesis at the sterol isomerase step.

SR31747A is a new sigma ligand exhibiting immunosuppressive properties and antiproliferative activity on lymphocyte cells. Only two subtypes of sigma receptor, namely the sigma1 receptor and emopamil-binding protein, have been characterised molecularly. Only the sigma1 receptor has been shown to bind (Z)N-cyclohexyl-N-ethyl-3-(3-chloro4-cyclohexylphenyl)pro pen-2-ylamine hydrochloride (SR31747A) with high affinity. It was demonstrated that the SR31747A effect on the inhibition of T-cell proliferation was consistent with a sigma1 receptor-mediated event. In this report, binding experiments and sterol isomerase assays, using recombinant yeast strains, indicate that the recently cloned emopamil-binding protein is a new SR31747A-binding protein whose activity is inhibited by SR31747A. Sterol analyses reveal the accumulation of a delta8-cholesterol isomer at the expense of cholesterol in SR31747A-treated cells, suggesting that cholesterol biosynthesis is inhibited by SR31747A at the delta8-delta7 sterol isomerase step in animal cells. This observation is consistent with a sterol isomerase role of the emopamil-binding protein in the cholesterol biosynthetic pathway in animal cells. In contrast, there is no evidence for such a role of the sigma1 receptor, in spite of the structural similarity shared by this protein and yeast sterol isomerase. We have found that SR31747A also exerts anti-proliferative effects at nanomolar concentrations on various established cell lines. The antiproliferative activity of SR31747A is reversed by cholesterol. Sterol-isomerase overproduction enhances resistance of CHO cells. This last observation strongly suggests that sterol isomerase is implicated in the antiproliferative effect of the drug in established cell lines.

Both the immunosuppressant SR31747 and the antiestrogen tamoxifen bind to an emopamil-insensitive site of mammalian Delta8-Delta7 sterol isomerase.

SR31747 is a novel agent that elicits immunosuppressive and anti-inflammatory effects. This drug was shown to inhibit Delta8-Delta7 sterol isomerase in yeast. To test whether this enzyme could also be an SR31747 target in mammals, the binding, antiproliferative and sterol biosynthesis inhibitory properties of various drugs were studied in recombinant sterol isomerase-producing yeast cells. Our results clearly show that SR31747 is a high affinity ligand of recombinant mammalian sterol isomerase (Kd = 1 nM). Tridemorph, a sterol biosynthesis inhibitor that is widely used in agriculture as an antifungal agent, is also a powerful inhibitor of murine and human sterol isomerases (IC50 value in the nanomolar range). Some drugs, like cis-flupentixol, trifluoperazine, 7-ketocholestanol and tamoxifen, inhibit SR31747 binding only with the mammalian enzymes, whereas other drugs, like haloperidol and fenpropimorph, are much more effective with the yeast enzyme than with the mammalian ones. Emopamil, a high affinity ligand of human sterol isomerase, is inefficient in inhibiting SR31747 binding to its mammalian target, suggesting that the SR31747 and emopamil binding sites on mammalian sterol isomerase do not overlap. In contrast, SR31747 binding inhibition by tamoxifen is very efficient and competitive (IC50 value in the nanomolar range), indicating that mammalian sterol isomerase contains a so-called antiestrogen binding site. Tamoxifen is found to selectively inhibit sterol biosynthesis at the sterol isomerase step in the cells that are producing the mammalian enzyme in place of their own sterol isomerase. Finally, we also show that tridemorph, a sterol biosynthesis inhibitor widely used in agriculture as an antifungal agent, is not selective of yeast Delta8-Delta7 sterol isomerase but is also highly efficient against murine Delta8-Delta7 sterol isomerase or human Delta8-Delta7 sterol isomerase. This observation contrasts with our already published results showing that fenpropimorph, another sterol isomerase inhibitor used in agriculture, is only poorly efficient against the mammalian enzymes.

Human lamin B receptor exhibits sterol C14-reductase activity in Saccharomyces cerevisiae.

Lamin B receptor (LBR), a nuclear protein of avian and mammalian cells, contains an hydrophobic domain that shares extensive structural similarities with the members of the sterol reductase family. To test if the sterol-reductase-like domain of LBR could be enzymatically competent, several sterol reductase-defective strains of Saccharomyces cerevisiae were transformed with a human-LBR expressing vector. LBR production did not change the ergosterol biosynthesis defect in an erg4 mutant impaired in sterol C24(28) reductase. In contrast, the sterol C14 reduction step and ergosterol prototrophy were restored in LBR-producing erg24 transformants which lack endogenous sterol C14 reductase. To test the effects of C14 reductase inhibitors on LBR activity, we constructed EMY54, an ergosterol-requiring strain that is devoid of both sterol C8-C7 isomerase and sterol C14 reductase activities. EMY54 cells recovered the capability of synthesizing ergost-8-en-3beta-ol upon transformation with a vector that expressed either yeast sterol C14 reductase or hLBR. In addition, growth in sterol-free medium was restored in these transformants. Sterol biosynthesis and proliferation of LBR-producing cells were found to be highly susceptible to fenpropimorph and tridemorph, but only moderately susceptible to SR 31747. Our results strongly suggest that hLBR is a sterol C14 reductase.

The Mr 18,000 subunit of the peripheral-type benzodiazepine receptor exhibits both benzodiazepine and isoquinoline carboxamide binding sites in the absence of the voltage-dependent anion channel or of the adenine nucleotide carrier.

The peripheral type benzodiazepine receptor (PBR) binds benzodiazepines such as RO5-4864 and isoquinoline carboxamide derivatives such as PK11195. This receptor includes an Mr 18,000 isoquinoline-binding subunit predominantly located in mitochondrial mem- branes. This protein has been found to copurify with two other mitochondrial proteins, namely the outer membrane voltage-dependent anion channel (VDAC), also known as mitochondrial porin, and the inner membrane adenine nucleotide carrier. In vitro reconstitution experiments suggested that the PBR was a multimeric complex in which the isoquinoline binding site was on the Mr 18,000 subunit, denoted pk18, whereas the benzodiazepine binding site required the association of this subunit with VDAC to be expressed. Untransformed cells of the yeast Saccharomyces cerevisiae are devoid of specific binding sites for isoquinolines and benzodiazepines, whereas yeast cells transformed with a pk18-expressing vector exhibit RO5-4864 and PK11195 binding sites that are pharmacologically identical to those of the PBR. To clarify the role of VDAC and of the adenine nucleotide carrier, if any, in the constitution of the benzodiazepine binding site, yeast host strains were constructed in which the corresponding genes had been knocked out. Mitochondria prepared from pk18-producing cells devoid of either VDAC or adenine nucleotide carrier exhibit both benzodiazepine and isoquinoline carboxamide binding sites with little or no change in the Kd values as compared with the wild-type background. These results rule out the contention that VDAC is indispensable for establishing the benzodiazepine binding site and are in agreement with the hypothesis that the Mr 18,000 subunit carries both the isoquinoline carboxamide and benzodiazepine binding domains.

Purification and characterization of the human SR 31747A-binding protein. A nuclear membrane protein related to yeast sterol isomerase.

SR 31747A, defined as a sigma ligand, is a novel immunosuppressive agent that blocks proliferation of human and mouse lymphocytes. Using a radiolabeled chemical probe, we here purified a target of SR 31747A and called it SR 31747A-binding protein (SR-BP). Purified SR-BP retained its binding properties and migrated on SDS-polyacrylamide gel as a Mr 28,000 protein. Cloning of the cDNA encoding human SR-BP shows an open reading frame for a 223-amino acid protein, which is homologous to the recently cloned sigma 1 receptor. Interestingly, the deduced amino acid sequence was found to be related to fungal C8-C7 sterol isomerase, encoded by the ERG2 gene. The ERG2 gene product has been identified recently as the molecular target of SR 31747A that mediates antiproliferative effects of the drug in yeast. Northern blot analysis of SR-BP gene expression revealed a single transcript of 2 kilobases which was widely expressed among organs, with the highest abundance in liver and the lowest abundance in brain. Subcellular localization analysis in various cells, using a specific monoclonal antibody raised against SR-BP, demonstrated that this protein was associated with the nuclear envelope. When studying the binding of SR 31747A on membranes from yeast expressing SR-BP, we found a pharmacological profile of sigma 1 receptors; binding was displaced by (+)-pentazocine, haloperidol, and (+)-SKF 10,047, with (+)-SKF 10, 047 being a more potent competitor than (-)-SKF 10,047. Scatchard plot analysis revealed Kd values of 7.1 nM and 0.15 nM for (+)-pentazocine and SR 31747A, respectively, indicating an affinity of SR-BP 50-fold higher for SR 31747A than for pentazocine. Additionally, we showed that pentazocine, a competitive inhibitor of SR 31747A binding, also prevents the immunosuppressive effect of SR 31747A. Taken together, these findings strongly suggest that SR-BP represents the molecular target for SR 31747A in mammalian tissues, which could be critical for T cell proliferation.

N-acetyl-heparosan lyase of Escherichia coli K5: gene cloning and expression.

The structure of the capsular polysaccharide of Escherichia coli K5 is identical to that of N-acetyl-heparosan, a nonsulfated precursor of heparin, which makes this E. coli antigen an attractive starting point for the chemical synthesis of analogs of low-molecular-weight heparin. This polysaccharide is synthesized as a high-molecular-weight molecule that can be depolymerized by an enzyme displaying endo-beta-eliminase activity. The eliminase-encoding gene, designated elmA, has been cloned from E. coli K5 by expression in E. coli K-12. The K-12 genome is devoid of the elmA sequence. The elmA gene product is 820 amino acids long. Active recombinant eliminase is produced by K-12 cells in both cell-bound and secreted forms. Deletion analyses have shown that the C terminus and the N terminus are required for activity and secretion, respectively.

Emopamil-binding protein, a mammalian protein that binds a series of structurally diverse neuroprotective agents, exhibits delta8-delta7 sterol isomerase activity in yeast.

Delta8-delta7 sterol isomerase is an essential enzyme on the sterol biosynthesis pathway in eukaryotes. This endoplasmic reticulum-resident membrane protein catalyzes the conversion of delta8-sterols to their corresponding delta7-isomers. No sequence data for high eukaryote sterol isomerase being available so far, we have cloned a murine sterol isomerase-encoding cDNA by functional complementation of the corresponding deficiency in the yeast Saccharomyces cerevisiae. The amino acid sequence deduced from the cDNA open reading frame is highly similar to human emopamil-binding protein (EBP), a protein of unknown function that constitutes a molecular target for neuroprotective drugs. A yeast strain in which the sterol isomerase coding sequence has been replaced by that of human EBP or its murine homologue recovers the ability to convert delta8-sterol into delta7-sterol, both in vivo and in vitro. In these recombinant strains, both cell proliferation and the sterol isomerization reaction are inhibited by the high affinity EBP ligand trifluoperazine, as is the case in mammalian cells but not in wild type yeast cell. In contrast, the recombinant strains are much less susceptible to the sterol inhibition effect of haloperidol and fenpropimorph, as compared with wild type yeast strains. Our results strongly suggest that EBP and delta8-delta7 sterol isomerase are identical proteins in mammals.

The immunosuppressant SR 31747 blocks cell proliferation by inhibiting a steroid isomerase in Saccharomyces cerevisiae.

SR 31747 is a novel immunosuppressant agent that arrests cell proliferation in the yeast Saccharomyces cerevisiae, SR 31747-treated cells accumulate the same aberrant sterols as those found in a mutant impaired in delta 8- delta 7-sterol isomerase. Sterol isomerase activity is also inhibited by SR 31747 in in vitro assays. Overexpression of the sterol isomerase-encoding gene, ERG2, confers enhanced SR resistance. Cells growing anaerobically on ergosterol-containing medium are not sensitive to SR. Disruption of the sterol isomerase-encoding gene is lethal in cells growing in the absence of exogenous ergosterol, except in SR-resistant mutants lacking either the SUR4 or the FEN1 gene product. The results suggest that sterol isomerase is the target of SR 31747 and that both the SUR4 and FEN1 gene products are required to mediate the proliferation arrest induced by ergosterol depletion.

Cloning and characterization of the eapB and eapC genes of Cryphonectria parasitica encoding two new acid proteinases, and disruption of eapC.

Two new proteinases secreted by Cryphonectria parasitica, namely EapB and EapC, have been purified. The corresponding structural genes were isolated by screening a cosmid library, and sequenced. Comparison of genomic and cDNA sequences revealed that the eapB and eapC genes contain three and two introns, respectively. The products of the eapB and eapC genes as deduced from the nucleotide sequences, are 268 and 269 residues long, respectively. N-terminal amino acid sequencing data indicates that EapC is synthesized as a zymogen, which yields a mature 206-amino acid enzyme after cleavage of the prepro sequence. Similarly, sequence alignment studies suggest that EapB is secreted as a 203-residue form which shares extensive similarities not only with EapC but also with two other acid fungal proteinases. However, they display distinct structural features; for example, no cysteine residue is found in EapC. The eapC gene was mutated using a two-step gene replacement strategy which allowed the specific introduction of several stop codons at the beginning of the eapC coding sequence in an endothiapepsin-deficient (EapA-) C. parasitica strain. Although the resulting strain did not secrete EapC, it still exhibited residual extracellular proteolytic activity, which could be due to EapB.

Secretion and maturation study of endothiapepsin in Saccharomyces cerevisiae. A first step toward improving its substrate specificity.

The gene encoding endothiapepsin (EAP), an extracellular aspartic proteinase from the filamentous ascomycete Cryphonectria parasitica, was expressed into Saccharomyces cerevisiae. Efficient secretion of an active and correctly processed enzyme was achieved when expressing the entire cDNA encoding prepro-EAP under the control of the galactose-inducible GRAP1 yeast promoter. Since three independent, site-directed mutations of EAP, including the substitution of an aspartyl catalytic residue, resulted in the intracellular accumulation of zymogen forms, we assumed that the EAP propeptide was autocatalytically processed. As a prerequisite to further improve the specificity of EAP, we therefore attempted to bypass this self-processing step in three different ways: 1) introduction of a Kex2-like recognition site between the pro and the mature part, 2) deletion of the prosequence (pre-EAP), and 3) co-expression in trans of the pre-EAP with its preprosequence. No improvement in the secretion of mutant enzymes was obtained in any of these experiments. As an alternative, we finally replaced the EAP processing site by the chymosin cleavage sequence of kappa-casein. Such a modification remained efficient in directing the secretion of active EAP only when a putative alpha-helix structural motif was conserved at the C terminus of the pro region.