Delivery after previous cesarean: a risk evaluation. Swiss Working Group of Obstetric and Gynecologic Institutions.

OBJECTIVE: To examine the risks of vaginal delivery after previous cesarean and to find criteria to help decide whether a trial of labor or an elective repeat cesarean should be preferred. METHODS: We evaluated 29,046 deliveries after previous cesarean registered in a pooled database of 457,825 deliveries used to assess quality control in gynecology and obstetrics departments in Switzerland. RESULTS: Among the 17,613 trial-of-labor cases logged (attempt rate 60.64%), the success rate was 73.73% (65.56% after inducing labor and 75.06% after the spontaneous onset of labor). The following complications were significantly more frequent in the previous-cesarean group: maternal febrile episodes (relative risk [RR] 2.77; 95% confidence interval [CI] 2.52, 3.05), thromboembolic events (RR 2.81; CI 2.23, 3.55), bleeding due to placenta previa during pregnancy (RR 2.06; CI 1.70, 2.49), uterine rupture (92 cases; RR 42.18; CI 31.09, 57.24), and perinatal mortality (118 cases, including six associated with uterine rupture; RR 1.33; CI 1.10, 1.62). The postcesarean group also showed a 0.28% rate of peripartum hysterectomy (81 cases; RR 6.07; CI 4.71, 7.83). There was one maternal death in the group, compared with 14 maternal deaths in the group without previous cesarean (no statistical significance). The risk of uterine rupture for patients with previous cesareans was elevated in the trial-of-labor group compared with the group without trial of labor (RR 2.07; CI 1.29, 3.30), but all other maternal risks, including peripartum hysterectomy (RR 0.36; CI 0.23, 0.56), were lower. When comparing the women having a trial of labor, the 70 with uterine rupture more often had induced labor (24.29% compared with 13.92% in the nonrupture group; P = .013), had epidural anesthesia (24.29% compared with 8.44%; P < .001), had an abnormal fetal heart rate tracing (32.86% compared with 8.53%; P < .001), and had failure to progress (21.43% compared with 7.98%; P = .001). CONCLUSION: A history of cesarean delivery significantly elevates the risks for mother and child in future deliveries. Nonetheless, a trial of labor after previous cesarean is safe. Induction of labor, epidural anesthesia, failure to progress, and abnormal fetal heart rate pattern are all associated with failure of a trial of labor and uterine rupture.

Characterization of a beta-Asp33 isoform of recombinant hirudin sequence variant 1 by low-energy collision-induced dissociation.

A new low-concentration congener (Ib) of recombinant hirudin sequence variation 1 was structurally characterized as a beta-Asp33 isoform of the parent protein (Ia). alpha-beta Isomerization at the 33-position was expected in view of the previous isolation of a potential precursor (Asp33-Gly34-anhydro-hirudin (Ic)), i.e., a succinimide-type dehydration product liable to undergo facile hydrolysis with ring opening, yielding beta- (along with alpha-) aspartates. In order to identify and locate the modified site in Ib, a sufficiently small peptide ([28-35]-octapeptide IIIb) was prepared by disulfide bond reduction, S-alkylation (pyridylethylation) and twofold enzymatic degradation (Glu-C protease followed by trypsin). When [M + H] + ions of IIIb were analyzed by electrospray ionization tandem mass spectrometry (ESIMS/MS) and low-energy collision-induced dissociation (CID), a singular [bn + H2O]+ ion indicative of beta-Asp in the neighboring 'n + 1' position was observed for n = 5. This located the beta-Asp residue unambiguously in the 6-position of IIIb and thus, as expected, in the 33-position of Ib. The formation of this highly diagnostic [bn + H2O]+ ion, for which precedents had only been reported for CID under high-energy conditions, requires net OH migration from one to another amino acid position. Confirmatory results from 18O-labeling of the suspected migratory oxygen atom (beta-Asp33-CO18OH) together with the low-energy genesis suggest a specific charge-triggered rather than charge-remote mechanism for the formation of the ion. The analogy of this process to the ejection of the C-terminal amino acid similarly involving net OH rearrangement is discussed.

NMR solution structure of the recombinant tick anticoagulant protein (rTAP), a factor Xa inhibitor from the tick Ornithodoros moubata.

The solution structure of the recombinant tick anticoagulant protein (rTAP) was determined by 1H nuclear magnetic resonance (NMR) spectroscopy in aqueous solution at pH 3.6 and 36 degrees C. rTAP is a 60-residue protein functioning as a highly specific inhibitor of the coagulation protease factor Xa, which was originally isolated from the tick Ornithodoros moubata. Its regular secondary structure consists of a two-stranded antiparallel beta-sheet with residues 22-28 and 32-38, and an alpha-helix with residues 51-60. The relative orientation of these regular secondary structure elements has nearly identical counterparts in the bovine pancreatic trypsin inhibitor (BPTI). In contrast, the loop between the beta-sheet and the C-terminal alpha-helix as well as the N-terminal 20-residue segment preceding the beta-sheet adopt different three-dimensional folds in the two proteins. These observations are discussed with regard to the implication of different mechanisms of protease inhibition by rTAP and by Kunitz-type protein proteinase inhibitors.

Isolation and characterization of recombinant annexin V expressed in Saccharomyces cerevisiae.

The cloning, purification and characterization of full-length annexin V, expressed intracellularly in Saccharomyces cerevisiae is detailed. Following homogenization in a glass bead mill, clarification by ultracentrifugation and fractional ammonium sulfate precipitation, the 319 amino acid protein was purified by column chromatography on phenyl-Sepharose and heparin-Sepharose. Annexin V elutes on reverse phase C4 silica as a single peak with greater than 97% homogeneity and is further characterized by a molecular mass of 34 kDa from electrophoresis under reducing conditions on SDS gels. Dynamic light scattering experiments reveal annexin V exists as a monomer in solution. Amino terminal Edman degradation afforded no sequence, therefore the carbamidomethylated protein was chemically cleaved with cyanogen bromide. Separation of the resulting peptide fragments on reverse phase HPLC followed by N-terminal sequencing and electrospray mass spectrometry supported the correct sequence as well as the existence of an acetyl blocking group on the N-terminus. The protein exhibits an isoelectric point of 4.73 by column chromatofocusing. Secondary structure predictions from CD spectroscopy indicate that the molecule is correctly folded. In anticoagulant assays, the purified protein exhibits dose-response effects in activated partial thromboplastin time (APTT) prolongation and doubles the clotting time of control human plasma at 70 micrograms ml-1. More specifically, in a factor Xa inhibition assay in which the activation of factor X via the tissue factor-factor VIIa complex is monitored by the cleavage of a factor Xa chromogenic substrate, recombinant annexin V exhibits a 50% inhibitory concentration (IC50) in the low nanomolar range.

Characterization of succinimide-type dehydration products of recombinant hirudin variant 1 by electrospray tandem mass spectrometry.

Two congeners Q4 and Q5 inferred in earlier analyses to be cyclic succinimide-type dehydration products of recombinant hirudin (variant 1) were structurally fully characterized. After isotopic labeling by ring-opening with H2(18)O, the suspected anhydro-positions Asp53 and Asp33 were confirmed by tandem mass spectrometry (MS/MS) sequencing of relevant smaller peptides directly in the enzymatic hydrolysates (V8 protease) using electrospray MS/MS. The chosen strategy proved highly efficient and sensitive.

Production of disulfide-linked hirudin dimer by in vitro folding.

A simple process of in vitro folding has been developed for the preparation of hirudin dimer. A variant of recombinant hirudin with Asp33 replaced by Cys was expressed in yeast and isolated by HPLC. Crude Cys33-hirudin contains heterogeneous products that are made of one species of primary sequence. They were together reduced/denatured, and allowed to re-fold in the sodium bicarbonate buffer (pH 8.3) alone. Active, homogeneous Cys33-hirudin monomer folded spontaneously with a first order rate constant of 0.05 +/- 0.01 min-1, followed by the oxidation of two Cys33 to produce the pure dimer. The folding yield was 90%. On an equal weight basis, both Cys33-hirudin monomer and the dimer exhibit thrombin inhibitory activity comparable to that of wild-type hirudin. Due to the presence of an extra cysteine, the folding of active hirudin monomer (formation of three native disulfides) was accelerated by at least 12-fold.

Crystal structure of the thrombin-hirudin complex: a novel mode of serine protease inhibition.

Thrombin is a serine protease that plays a central role in blood coagulation. It is inhibited by hirudin, a polypeptide of 65 amino acids, through the formation of a tight, noncovalent complex. Tetragonal crystals of the complex formed between human alpha-thrombin and recombinant hirudin (variant 1) have been grown and the crystal structure of this complex has been determined to a resolution of 2.95 A. This structure shows that hirudin inhibits thrombin by a previously unobserved mechanism. In contrast to other inhibitors of serine proteases, the specificity of hirudin is not due to interaction with the primary specificity pocket of thrombin, but rather through binding at sites both close to and distant from the active site. The carboxyl tail of hirudin (residues 48-65) wraps around thrombin along the putative fibrinogen secondary binding site. This long groove extends from the active site cleft and is flanked by the thrombin loops 35-39 and 70-80. Hirudin makes a number of ionic and hydrophobic interactions with thrombin in this area. Furthermore hirudin binds with its N-terminal three residues Val, Val, Tyr to the thrombin active site cleft. Val1 occupies the position P2 and Tyr3 approximately the position P3 of the synthetic inhibitor D-Phe-Pro-ArgCH2Cl. Thus the hirudin polypeptide chain runs in a direction opposite to that expected for fibrinogen and that observed for the substrate-like inhibitor D-Phe-Pro-ArgCH2Cl.

Physico-chemical properties of recombinant desulphatohirudin.

Physico-chemical properties of recombinant desulphatohirudin expressed in yeast (CIBA GEIGY code No. CGP 39393) were reinvestigated. As previously reported for natural hirudin, the recombinant molecule exhibited abnormal behaviour by gel filtration with an apparent molecular weight greater than that based on the primary structure. However, molecular weight estimation by SDS gel electrophoresis, FAB-mass spectrometry and Photon Correlation Spectroscopy were in agreement with the theoretical molecular weight, with little suggestion of dimer or aggregate formation. Circular dichroism studies of the recombinant molecule show similar spectra at different pH values but are markedly different from that reported by Konno et al. for a natural hirudin-variant. Our CD studies indicate the presence of about 60% beta sheet and the absence of alpha helix in the secondary structure of recombinant hirudin, in agreement with the conformation determined by NMR studies.

Benzodiazepine abuse in patients of doctors in domiciliary practice in the Basle area.

In the city of Basle with a catchment area of 300,000 inhabitants a significant number of physicians were asked for observations concerning benzodiazepine abuse by their patients. In 1985, 31 patients abusing benzodiazepine only were observed, which represents an incidence of 1:10,000 or 0.01%. In addition, concrete information on 88 patients with multiple abuse was obtained. Demographic data, diagnosis and main reason of abuse, data on most frequently abused drugs, consequences of abuse, as well as measures against the abused are described in relation to the two main groups of abusers.

Biotreatment of s-triazine-containing wastewater in a fluidized bed reactor.

Mixed cultures of microorganisms immobilized on sand were used to degrade s-triazine-containing industrial wastewater in a fluidized bed reactor. Immobilized cell concentrations of up to 18 g/L volatile suspended solids could be achieved with the s-triazines as sole nitrogen source for growth and carbon sources added at a C--N ratio of about 12. Maximal removal efficiencies of 80% of the s-triazines could be maintained only if (a) the bio-film thickness was limited to avoid oxygen deficiency and (b) the carbon source and complete wastewater (/=20-25 h.

Ring cleavage and degradative pathway of cyanuric acid in bacteria.

The degradative pathway of cyanuric acid [1,3,5-triazine-2,4,6(1H,3H,5H)-trione] was examined in Pseudomonas sp. strain D. The bacterium grew with cyanuric acid, biuret, urea or NH4+ as sole source of nitrogen, and each substrate was entirely metabolized concomitantly with growth. Enzymes from strain D were separated by chromatography on DEAE-cellulose and three reactions were examined. Cyanuric acid (1 mol) was converted stoichiometrically into 1.0 mol of CO2 and 1.1 mol of biuret, which was conclusively identified. Biuret (1 mol) was converted stoichiometrically into 1.1 mol of NH4+, about 1 mol of CO2 and 1.0 mol of urea, which was conclusively identified. Urea (1 mol) was converted into 1.9 mol of NH4+ and 1.0 mol of CO2. The reactions proceeded under aerobic or anoxic conditions and were presumed to be hydrolytic. Data indicate that the same pathway occurred in another pseudomonad and a strain of Klebsiella pneumoniae.

Biological treatment specific for an industrial wastewater containing s-triazines.

Mixed cultures of bacteria grew in medium containing real s-triazine wastes as nitrogen source. About 80% of the s-triazine waste could be degraded as determined by HPLC and by measurements of dissolved nitrogen. The culture required an added carbon source in order to degrade s-triazines. A temperature optimum near 40 degrees C was observed and a salt concentration above about 4% markedly retarded growth and the degradation of s-triazines. This system was examined as a biological treatment for wastes from syntheses of s-triazines.

Bacterial degradation of N-cyclopropylmelamine. The steps to ring cleavage.

The s-triazine cyclopropylmelamine (N-cyclopropyl-1,3,5-triazine-2,4,6-triamine) was degraded to about 6 mol of NH4+/mol of substrate by a mixture of two bacteria (strains A and D, both Pseudomonas spp.) Only strain A grew with cyclopropylmelamine as sole and limiting source of nitrogen. The organism obtained 2 mol of nitrogen/mol of substrate and excreted a product that was identified as cyclopropylammelide [6-cyclopropylamino-1,3,5-triazine-2,4(1 H,3 H)-dione]. Proteins in extracts from strain A were separated on a Sephadex G-200 column. Cyclopropylmelamine was found to be deaminated in two separable steps to cyclopropylammelide via cyclopropylammeline [4-amino-6-cyclopropylamino-1,3,5-triazine-2(1 H)-one], which was identified. Strain D could not utilize cyclopropylmelamine or cyclopropylammeline, but could utilize cyclopropylammelide (or homologue) as sole and limiting source of nitrogen and obtain about 4 mol of nitrogen/mol of substrate. Proteins in cell extracts from strain D were separated on a DEAE-cellulose column. Alkylammelides were degraded quantitatively by one enzyme fraction to 1 mol of cyanuric acid plus 1 mol of alkylamine/mol of substrate. The specific activities of enzymes in extracts of the two strains were as high as the activities observed during growth. The three activities studied in the two strains were all active under aerobic and oxygen-free conditions. The reactions appear to be hydrolytic, yielding 2 mol of NH4+ plus 1 mol of cyclopropylamine and 1 mol of cyanuric acid/mol of substrate.

2-Chloro-4-amino-1,3,5-triazine-6(5H)-one: a new intermediate in the biodegradation of chlorinated s-triazines.

Pseudomonas sp. strain A grew with 2-chloro-1,3,5-triazine-4,6-diamine as the sole and growth-limiting source of nitrogen. The substrate was utilized quantitatively and concomitantly with growth and with excretion of a product which was identified as 2-chloro-4-amino-1,3,5-triazine-6(5H)-one. The reaction yielded 1 mol of organic product and 1 mol of NH4+ per mol of substrate.