Maternal and infant outcome after caesarean section without recorded medical indication: findings from a Swedish case-control study.

OBJECTIVE: To compare maternal complications and infant outcomes for women undergoing elective caesarean sections based on a maternal request and without recorded medical indication with those of women who underwent spontaneous onset of labour with the intention to have a vaginal birth. DESIGN: Retrospective register study. SETTING: Sweden; Medical Birth Register used for data collection. METHODS: A case-control study of 5877 birth records of women undergoing caesarean sections without medical indication and a control group of 13 774 women undergoing births through spontaneous onset of labour. The control group was further divided into women who actually had a vaginal birth and women who ended up with an emergency caesarean section. RESULTS: Maternal complications occurred more frequently among women undergoing caesarean section with odds ratios (OR) for bleeding complications of 2.5 (95% CI 2.1-3.0) in the elective caesarean group and 2.0 (95% CI 1.5-2.6) in the emergency caesarean group. The OR for infections was 2.6 in both groups. Breastfeeding complications were most common in women having an elective caesarean section: 6.8 (95% CI 3.2-14.5). Infant outcomes showed a higher incidence of respiratory distress with an OR of 2.7 (95% CI 1.8-3.9) in the elective caesarean section group compared with infants born by emergency caesarean section. The risk of hypoglycaemia was at least twice as high for infants in the caesarean group. CONCLUSIONS: Caesarean sections without medical indication as well as emergency caesarean sections were associated with higher risks for maternal and infant morbidity.

Few fathers-to-be prefer caesarean section for the birth of their baby.

The objective of this study was to investigate prospective fathers' preferences for caesarean section and associated factors. Data were collected by means of a questionnaire given in mid-pregnancy to 1105 fathers-to-be in northern Sweden. In total, 6.4% of fathers preferred a caesarean section. The factors associated with a preference for caesarean section were a wish to plan the date of the baby's birth [prevalence ratio (PR) 6.0], a previous negative birth experience (PR 8.6) and previous experience of a caesarean section (PR 5.7).

Characterization of stable complexes involving apolipoprotein E and the amyloid beta peptide in Alzheimer's disease brain.

Genetic evidence suggests a role for apolipoprotein E (apoE) in Alzheimer's disease (AD) amyloidogenesis. Here, amyloid-associated apoE from 32 AD patients was purified and characterized. We found that brain amyloid-associated apoE apparently exists not as free molecules but as complexes with polymers of the amyloid beta peptide (A beta). Brain A beta-apoE complexes were detected irrespective of the apoE genotype, and similar complexes could be mimicked in vitro. The fine structure of purified A beta-apoE complexes was fibrillar, and immunogold labeling revealed apoE immunoreactivity along the fibrils. Thus, we conclude that A beta-apoE complexes are principal components of AD-associated brain amyloid and that the data presented here support a role for apoE in the pathogenesis of AD.

Purification and characterization of the RNase H domain of HIV-1 reverse transcriptase expressed in recombinant Escherichia coli.

The ribonuclease H (RNase H) domain of human immuno-deficiency virus (HIV-1) reverse transcriptase has been produced with the aim of providing sufficient amounts of protein for biophysical studies. A plasmid vector is described which directs high level expression of the RNase H domain under the control of the lambda PL promoter. The domain corresponds to residues 427-560 of the 66 kDa reverse transcriptase. The protein was expressed in Escherichia coli and was purified using ion-exchange and size exclusion chromatography. The purified protein appears to be in a native-like homogeneous conformational state as determined by 1H-NMR spectroscopy and circular dichroism measurements. HIV-protease treatment of the RNase H domain resulted in cleavage between Phe-440 and Tyr-441.

A recombinant Escherichia coli heat-stable enterotoxin (STa) fusion protein eliciting anti-STa neutralizing antibodies.

A recombinant fusion protein consisting of native Escherichia coli heat-stable enterotoxin (STa) and a dimer of a synthetic IgG-binding fragment (ZZ), derived from Staphylococcus aureus protein A was produced in E. coli. The fusion protein (ZZSTa) was secreted in large quantities into the growth medium and recovered by affinity chromatography on IgG-Sepharose. Rabbits immunized with the fusion protein responded by producing high serum levels of anti-STa antibodies that also effectively neutralized STa toxicity in infant mice. The fusion peptide ZZSTa had a substantially decreased toxicity as compared with native STa. A polymeric form of ZZSTa separated by size fractionation was about 100 times less toxic than the monomeric fusion protein, yet both forms had the same capacity to induce neutralizing antibodies. This suggests that modified non-toxic forms of ZZSTa with retained immunogenicity may be produced and tested for their usefulness as functional components in a vaccine against diarrhoea caused by enterotoxigenic E. coli.

A Versatile Route to 2-Substituted Cyclic 1,3-Dienes via a Copper(I)-Catalyzed Cross-Coupling Reaction of Dienyl Triflates with Grignard Reagents.

A general synthesis of 2-substituted cyclic 1,3-dienes in two steps from alpha,beta-unsaturated ketones has been developed. Formation of a dien-2-yl triflate followed by a copper(I)-catalyzed cross-coupling reaction with a Grignard reagent gives 2-substituted dienes in fair to excellent yields. Alkyl, aryl, and allyl Grignard reagents can be used.

Protective effect of vaccination with recombinant proteins from Streptococcus equi subspecies equi in a strangles model in the mouse.

A mouse model resembling Streptococcus equi subspecies equi infection in the horse, strangles, was used to assess the protective effect of vaccination with selected recombinant proteins from S. equi subsp. equi. After challenge the infection was monitored by weight loss and by nasal colonisation with S. equi subsp. equi. Vaccination with a collagen-binding protein (CNE) and a collagen-like protein (SclC) resulted in protective antibodies, whereas a novel fibronectin-binding protein (FNEB) did not. Co-administration of CNE with EAG, a poorly immunogenic alpha2-macroglobulin-, albumin- and immunoglobulin G-binding protein, resulted in a significant synergistic effect and enhanced the protective immune response against EAG.

Design of protecting groups for the beta-carboxylic group of aspartic acid that minimize base-catalyzed aspartimide formation.

With the objectives of developing new protecting groups for the beta-carboxyl group of aspartic acid that are resistant to base-catalyzed aspartimide formation and of evaluating the importance of sterical factors in the design of such protecting groups, four new alkyl ester derivatives of aspartic acid were synthesized. The beta-3-pentyl, beta-4-heptyl, beta-2,6-dimethyl-4-heptyl and the recently described beta-2,4-dimethyl-3-pentyl esters of Boc-aspartic acid were incorporated into model peptides, and the resin-bound protected peptides were treated with 20% piperidine for 10 h. The levels of aspartimide-related side products were compared with the previously reported beta-cyclohexyl, beta-menthyl and beta-2-adamantyl esters of aspartic acid. The results show that bulky, acyclic, aliphatic protecting groups (in particular the 2,4-dimethyl-3-pentyl ester) are significantly more resistant to base-catalyzed aspartimide formation than comparably rigid cyclic alkyl esters that under the same reaction conditions form several-fold more aspartimide-related side products. Using elevated temperatures to overcome difficult couplings leads to the formation of significant amounts of aspartimide when aspartic acid is protected with the cyclohexyl group, but the 2,4-dimethyl-3-pentyl protecting group offers excellent protection under these conditions. The use of the 2,4-dimethyl-3-pentyl protecting group will allow the use of orthogonally removable base-labile protecting groups in Boc chemistry and suggests a design of protecting groups for other nucleophile-sensitive trifunctional amino acids in both Boc and Fmoc chemistry.

Thrombin is inactivated by mast cell secretory granule chymase.

In a recent paper we demonstrated that cultures of murine adherent peritoneal cells expressed cell surface-associated serine protease activity that specifically inactivated thrombin by cleaving the enzyme into defined proteolytic fragments (Pejler, G., and Seljelid, R. (1992) J. Biol. Chem. 267, 3136-3142). In the present report, the purification and further characterization of the thrombin-inactivating serine protease is described. The serine protease is shown to be expressed by mast cells. Purification of the thrombin-inactivating serine protease by a combination of anion-exchange chromatography and Superdex 75 chromatography showed that the enzyme had an apparent molecular mass of 28 kDa. N-terminal sequence analysis of the purified protein demonstrated 100% identity of the thrombin-inactivating serine protease with the secretory granule chymases: murine mast cell protease 3 and murine mast cell protease 4. The serine protease showed chymotrypsin-like substrate specificity. The thrombin-inactivating activity was markedly enhanced by optimal concentrations of heparin.

Inactivation of thrombin by a complex between rat mast-cell protease 1 and heparin proteoglycan.

Rat peritoneal mast cells were shown to inactivate thrombin rapidly. The thrombin-inactivating activity was purified to homogeneity by a combination of anion-exchange chromatography and h.p.l.c. on a Superdex 75 column. The purified thrombin inactivator had an apparent molecular mass of 29 kDa and an N-terminal amino acid sequence identical to rat mast-cell protease 1 (RMCP-1). After labelling of the mast cells in vivo with 35SO4(2-), RMCP-1 was recovered in a macromolecular complex with [35S]heparin proteoglycans. Dissociation of RMCP-1 from the heparin proteoglycans by Superdex 75 chromatography in the presence of 2 M NaCl resulted in a marked loss of the thrombin-inactivating activity displayed by the enzyme. When RMCP-1 was reconstituted with either endogenous [35S]heparin proteoglycans or standard pig mucosal heparin, the enzyme regained its thrombin-inactivating properties. Affinity chromatography of endogenous [35S]heparin on matrix-linked RMCP-1 demonstrated that all of the heparin molecules contained high-affinity binding sites for the mast-cell protease. In contrast, the endogenous mast-cell heparin showed low affinity for antithrombin, a protease inhibitor involved in the regulation of coagulation enzymes.

An optimized thymidylate kinase assay, based on enzymatically synthesized 5-[125I]iododeoxyuridine monophosphate and its application to an immunological study of herpes simplex virus thymidine-thymidylate kinases.

The biological synthesis and purification of 5-[125I]iododeoxyuridine monophosphate (IdUMP) are described. The specificity of IdUMP as substrate in the thymidylate monophosphate kinase (TMPK) assay is demonstrated, and a 100-fold gain in sensitivity as compared to the conventional TMPK assay is shown. TMPK measurements of isozymes derived from herpes simplex virus (HSV)-infected cells, uninfected cells, and tumor biopsies were performed. The results showed a significant difference in dependence of phosphate donor concentration present for TMPK activity from HSV-infected cells compared to the corresponding activity from uninfected cells, while only a minor difference in pH optima was observed for these enzyme activities. The increased sensitivity made it possible to detect and quantify HSV TMPK-blocking antibodies (ab) present in human sera. Sera from HSV ab-positive individuals were found to block the two HSV TMPKs to varying degrees and with different specificities. The immunological relationship between the TMPK and thymidine kinase (TK) induced by HSV-1 and HSV-2, respectively, was studied by comparing the capacities of different sera to block the two enzymatic activities. The results showed that the capacity to block HSV-1 TK and TMPK was proportional for all of the sera studied, while sera that preferentially blocked only the HSV-2 TMPK or HSV-2 TK were found. It was concluded that the HSV-2 TMPK and TK activities are less related than the corresponding activities for HSV-1 and that the HSV-2 enzyme activities are mediated by different catalytic sites.

Copper inhibits the protease from human immunodeficiency virus 1 by both cysteine-dependent and cysteine-independent mechanisms.

The protease of the human immunodeficiency virus is essential for replication of the virus, and the enzyme is therefore an attractive target for antiviral action. We have found that the viral protease is inhibited by approximately stoichiometric concentrations of copper or mercury ions. Inactivation by Cu2+ was rapid and not reversed by subsequent exposure to EDTA or dithiothreitol. Direct inhibition by Cu2+ required the presence of cysteine residue(s) in the protease. Thus, a synthetic protease lacking cysteine residues was not inhibited by exposure to copper. However, addition of dithiothreitol as an exogenous thiol rendered even the synthetic protease susceptible to inactivation by copper. Oxygen was not required for inactivation of either the wild-type or the synthetic protease. These results provide the basis for the design of novel types of protease inhibitors.

Experimental evidence supporting a CuIII intermediate in cross-coupling reactions of allylic esters with diallylcuprate species.

The reaction between an allylic ester and a magnesium diallylcuprate, or an allylic Grignard reagent in combination with a catalytic amount of a copper salt, has been studied. These reactions yield a mixture of homo- and cross-coupled 1,5-diene products. The product ratios obtained are close to those expected for a reaction proceeding via a triallylcopper(III) intermediate consisting of three equivalent allyl groups bound to copper. When the reaction is performed with a stoichiometric amount of a preformed diallylcuprate, a homo-coupling/cross-coupling ratio larger than that predicted for a CuIII intermediate is observed. However, on dilution this ratio decreases and becomes close to the predicted ratio. The deviation from the predicted homo-coupling/cross-coupling ratio was accounted for by an olefin-induced homo-coupling, as demonstrated in control experiments. The possibility of the allylic ligands to coordinate to the metal center in a eta3 or eta1 fashion provides an opportunity for the stabilization of the intermediate CuIII species.

Study of the alkylation propensity of cations generated by acidolytic cleavage of protecting groups in Boc chemistry.

The alkylation of cysteine residue by different classes of carbonium ions, derived from the cleavage of side chain protective groups in anhydrous HF, was investigated. It was found that side chain protection as beta-2,4-dimethylpent-3-yl ester (Dmp) or 2,4-dimethylpent-3-yloxycarbonyl (Doc) groups resulted in more than seven-fold lower level of alkylated byproducts. This makes Dmp and Doc protection of amino acid side chain during solid phase synthesis particularly valuable in the synthesis of peptides containing cysteine residues or other functional groups prone to alkylation by carbonium ions.

Dual labeling of a binding protein allows for specific fluorescence detection of native protein.

Fluorescence resonance energy transfer has been investigated in the context of specific detection of unlabeled proteins. A model system based on the staphylococcal protein A (SPA)-IgG interaction was designed, in which a single domain was engineered to facilitate site-specific incorporation of fluorophores. An Asn23Cys mutant of the B domain from SPA was expressed in Escherichia coli and subsequently labeled at the introduced unique thiol and at an amino group, using N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (1,5-IAEDANS) and succinimidyl 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoate (NBD-X, SE), respectively. Biosensor analysis of purified doubly labeled protein showed that high-affinity binding to the Fc region of IgG was retained. The fluorescence emission spectrum of the doubly labeled protein showed a shift in the relative emission of the two fluorophores in the presence of Fc3(1) fragments, which bind specifically to the B domain. In addition, the fluorescence emission ratio 480/525 nm was shown to increase with increasing concentration of Fc3(1), whereas the presence of a control protein did not affect the emission ratio over the same concentration range.

Allylic phosphates and allylic phosphinates as electrophiles in efficient silylcupration reactions of acetylenes.

Atom-efficient stoichiometric silylcupration reactions of acetylenes followed by electrophilic trapping of the intermediate vinylcopper species with allylic phosphates have been developed. The reaction sequence was also carried out with the use of a catalytic amount of CuCN employing of both allylic phosphates and allylic phosphinates as electrophiles. The methods developed provide an easy access to silylated 1,4-diene systems.

Identification of the proteolytic thrombin fragments formed after cleavage with rat mast cell protease 1.

We have previously identified rat mast cell protease 1 (RMCP-1), a chymotrypsin-like secretory granule serine protease, as a potent inactivator of thrombin. The present study outlines the cleavage pattern obtained after degradation of thrombin by RMCP-1. The cleavage sites in thrombin were identified by N-terminal amino acid sequence analysis of recovered thrombin fragments. Incubation of thrombin with RMCP-1 resulted in the rapid formation of a 37-kDa fragment, due to cleavage of the Phe1G-Gly1F bond in the thrombin A chain (numbering of amino acid residues according to topological equivalencies with chymotrypsinogen). Further incubation resulted in cleavage of the Trp148-Thr149 bond in the B chain, along with the formation of fragments of 27 kDa and 15 kDa. When the RMCP-1/thrombin mixtures were incubated further, successive degradation of the 37-kDa, 27-kDa and 15-kDa fragments was observed, along with cleavage of the Tyr117-Ile118 bond in the B chain and the formation of fragments of 12, 9 and 6 kDa. No residual thrombin activity was detected after the degradation process had proceeded to this stage. Heparin was shown to markedly enhance the rate of thrombin degradation by RMCP-1.