Analysis of preterm deliveries below 35 weeks' gestation in a tertiary referral hospital in the UK. A case-control survey

Background

Preterm birth remains a major public health problem and its incidence worldwide is increasing. Epidemiological risk factors have been investigated in the past, but there is a need for a better understanding of the causes of preterm birth in well defined obstetric populations in tertiary referral centres; it is important to repeat surveillance and identify possible changes in clinical and socioeconomic factors associated with preterm delivery. The aim of this study was to identify current risk factors associated with preterm delivery and highlight areas for further research.

Findings

We studied women with singleton deliveries at St Michael's Hospital, Bristol during 2002 and 2003. 274 deliveries between 23-35 weeks' gestation (preterm group), were compared to 559 randomly selected control deliveries at term (37-42 weeks) using standard statistical procedures. Both groups were >80% Caucasian. Previous preterm deliveries, high maternal age (> 39 years), socioeconomic problems, smoking during pregnancy, hypertension, psychiatric disorders and uterine abnormalities were significantly associated with preterm deliveries. Both lean and obese mothers were more common in the preterm group. Women with depression/psychiatric disease were significantly more likely to have social problems, to have smoked during pregnancy and to have had previous preterm deliveries; when adjustments for these three factors were made the relationship between psychiatric disease and pregnancy outcome was no longer significant. 53% of preterm deliveries were spontaneous, and were strongly associated with episodes of threatened preterm labour. Medically indicated preterm deliveries were associated with hypertension and fetal growth restriction. Preterm premature rupture of the membranes, vaginal bleeding, anaemia and oligohydramnios were significantly increased in both spontaneous and indicated preterm deliveries compared to term controls.

Conclusions

More than 50% of preterm births are potentially preventable, but remain associated with risk factors such as increased uterine contractility, preterm premature rupture of the membranes and uterine bleeding whose aetiology is unknown. Despite remarkable advances in perinatal care, preterm birth continues to cause neonatal deaths and long-term morbidity. Significant breakthroughs in the management of preterm birth are likely to come from research into the mechanisms of human parturition and the pathophysiology of preterm labour using multidisciplinary clinical and laboratory approaches.

     

A new thermoactive pullulanase from Desulfurococcus mucosus: cloning, sequencing, purification, and characterization of the recombinant enzyme after expression in Bacillus subtilis

The gene encoding a thermoactive pullulanase from the hyperthermophilic anaerobic archaeon Desulfurococcus mucosus (apuA) was cloned in Escherichia coli and sequenced. apuA from D. mucosus showed 45.4% pairwise amino acid identity with the pullulanase from Thermococcus aggregans and contained the four regions conserved among all amylolytic enzymes. apuA encodes a protein of 686 amino acids with a 28-residue signal peptide and has a predicted mass of 74 kDa after signal cleavage. The apuA gene was then expressed in Bacillus subtilis and secreted into the culture fluid. This is one of the first reports on the successful expression and purification of an archaeal amylopullulanase in a Bacillus strain. The purified recombinant enzyme (rapuDm) is composed of two subunits, each having an estimated molecular mass of 66 kDa. Optimal activity was measured at 85 degrees C within a broad pH range from 3.5 to 8.5, with an optimum at pH 5.0. Divalent cations have no influence on the stability or activity of the enzyme. RapuDm was stable at 80 degrees C for 4 h and exhibited a half-life of 50 min at 85 degrees C. By high-pressure liquid chromatography analysis it was observed that rapuDm hydrolyzed alpha-1,6 glycosidic linkages of pullulan, producing maltotriose, and also alpha-1,4 glycosidic linkages in starch, amylose, amylopectin, and cyclodextrins, with maltotriose and maltose as the main products. Since the thermoactive pullulanases known so far from Archaea are not active on cyclodextrins and are in fact inhibited by these cyclic oligosaccharides, the enzyme from D. mucosus should be considered an archaeal pullulanase type II with a wider substrate specificity.     

Bioactivity screening of partially desulfated low-molecular-weight heparins: a structure/activity relationship study

A series of size-defined low-molecular-weight heparins were generated by regioselective chemical modifications and profiled for their in vitro and in vivo activities. The compounds displayed reduced anti-coagulant activity, demonstrated varying affinities toward angiogenic growth factors (fibroblast growth factor-2, vascular endothelial growth factor and stromal cell-derived factor-1α), inhibited the P-selectin/P-selectin glycoprotein ligand-1 interaction and, notably, exhibited anti-tumor efficacy in a murine melanoma experimental metastasis model. Our results demonstrate that modulating specific sequences, especially the N-domains (-NS or -NH(2) or -NHCOCH(3)) in these polysaccharide sequences, has a major impact on the participation in a diverse range of biological activities. These results also suggest that the 6-O-sulfates, but not the 2-O-sulfates, critically affect the binding of a desulfated derivative to certain angiogenic proteins as well as its ability to inhibit P-selectin-mediated B16F10 melanoma metastases. Furthermore, N-desulfation followed by N-acetylation regenerates the affinity/inhibition properties to different extents in all the compounds tested in the in vitro assays. This systematic study lays a conceptual foundation for detailed structure function elucidation and will facilitate the rational design of targeted heparan sulfate proteoglycan-based anti-metastatic therapeutic candidates.     

Identification of the molecular target of small molecule inhibitors of HDL receptor SR-BI activity

Scavenger receptor, class B, type I (SR-BI), controls high-density lipoprotein (HDL) metabolism by mediating cellular selective uptake of lipids from HDL without the concomitant degradation of the lipoprotein particle. We previously identified in a high-throughput chemical screen of intact cells five compounds (BLT-1-5) that inhibit SR-BI-dependent lipid transport from HDL, but do not block HDL binding to SR-BI on the cell surface. Although these BLTs are widely used to examine the diverse functions of SR-BI, their direct target(s), SR-BI itself or some other component of the SR-BI pathway, has not been identified. Here we show that SR-BI in the context of a membrane lipid environment is the target of BLT-1, -3, -4, and -5. The analysis using intact cells and an in vitro system of purified SR-BI reconstituted into liposomes was aided by information derived from structure-activity relationship (SAR) analysis of the most potent of these BLTs, the thiosemicarbazone BLT-1. We found that the sulfur atom of BLT-1 was crucially important for its inhibitory activity, because changing it to an oxygen atom resulted in the isostructural, but essentially inactive, semicarbazone derivative BLT-1sc. SAR analysis also established the importance of BLT-1's hydrophobic tail. BLTs and their corresponding inactive compounds can be used to explore the mechanism and function of SR-BI-mediated selective lipid uptake in diverse mammalian experimental models. Consequently, BLTs may help determine the therapeutic potential of SR-BI-targeted pharmaceutical drugs.     

Differential production of prostaglandin E(2) in male and female mice subjected to thermal injury contributes to the gender difference in immune function: possible role for 15-hydroxyprostaglandin dehydrogenase

We have previously reported a macrophage-mediated gender difference in postburn immunosuppression, which was dependent upon elevated levels of circulating 17beta-estradiol (E(2)) and, in part, interleukin-6. Herein we examined the role of prostaglandin E(2) (PGE(2)), a potent suppressor of cell-mediated immunity. Circulating levels of PGE(2) were significantly elevated in females but not males at 10 days postburn (P < 0.01), and indomethacin treatment fully restored the delayed-type hypersensitivity and splenocyte proliferative responses of thermally injured females. While there was no difference in cyclooxygenase-2 protein expression in the lungs and liver of thermally injured male and female mice, there was a marked decrease in the protein expression of 15-hydroxyprostaglandin dehydrogenase in females. These data demonstrate that PGE(2) is a critical mediator of immunosuppression in thermally injured female mice and that the increase in circulating PGE(2) is derived, in part, from decreased degradation and clearance of PGE(2).     

Intact-protein-based high-resolution three-dimensional quantitative analysis system for proteome profiling of biological fluids

The substantial complexity and vast dynamic range of protein abundance in biological fluids, notably serum and plasma, present a formidable challenge for comprehensive protein analysis. Integration of multiple technologies is required to achieve high-resolution and high-sensitivity proteomics analysis of biological fluids. We have implemented an orthogonal three-dimensional intact-protein analysis system (IPAS), coupled with protein tagging and immunodepletion of abundant proteins, to quantitatively profile the human plasma proteome. Following immunodepletion, plasma proteins in each of paired samples are concentrated and labeled with a different Cy dye, before mixing. Proteins are subsequently separated in three dimensions according to their charge, hydrophobicity, and molecular mass. Differences in the abundance of resolved proteins are determined based on Cy dye ratios. We have applied this strategy to profile the plasma proteome for changes that occur with acute graft-versus-host disease (GVHD), following allogeneic bone marrow transplantation (BMT). Using capillary HPLC ESI Q-TOF MS, we identified 75 proteins in the micromolar to femtomolar range that exhibited quantitative differences between the pre- and post-GVHD samples. These proteins included serum amyloid A, apolipoproteins A-I/A-IV, and complement C3 that are well-known acute-phase reactants likely reflecting the post-BMT inflammatory state. In addition, we identified some potentially interesting immunologically relevant molecules including vitamin D-binding protein, fetuin, vitronectin, proline-rich protein 3 and 4, integrin-alpha, and leukocyte antigen CD97. IPAS provides a combination of comprehensive profiling and quantitative analysis, with a substantial dynamic range, for disease-related applications.     

The role of interleukin 6 in interferon-gamma production in thermally injured mice

Following traumatic injury, patients suffer from compromised immunity increasing their susceptibility to infection. Previous studies from this laboratory demonstrated that female BALB/c mice subjected to a 15% total body surface area (TBSA) scald injury exhibit a decrease in cell-mediated immunity 10 days post-burn. Studies described herein revealed that concanavalin A (Con A; 2 microg/ml)-stimulated splenocytes from sham treated animals produced 3557+/-853 pg/ml of IFN-gamma while splenocytes from burn injured animals released two-fold more cytokine (P<0.05). To determine whether leukocyte production of IFN-gamma was under the influence of macrophages, splenic macrophage supernatants generated from burned animals were incubated with splenic lymphocytes from sham and burn animals. The amount of IFN-gamma released by lymphocytes from sham animals increased when cultured with macrophages from burned mice (P<0.05). This suggests that the increase in IFN-gamma production by unfractionated splenocytes in burned mice relative to sham treated animals is macrophage-dependent. Macrophage supernatants from burned mice released twice as much IL-6 as supernatants from sham animals (P<0.05), and when IL-6 was blocked in vivo, the amount of IFN-gamma production in burned mice decreased to sham levels (P<0.05). Thus, IL-6 mediates IFN-gamma production following burn.     

Pullulanase type I from Fervidobacterium pennavorans Ven5: cloning, sequencing, and expression of the gene and biochemical characterization of the recombinant enzyme

The gene encoding the type I pullulanase from the extremely thermophilic anaerobic bacterium Fervidobacterium pennavorans Ven5 was cloned and sequenced in Escherichia coli. The pulA gene from F. pennavorans Ven5 had 50.1% pairwise amino acid identity with pulA from the anaerobic hyperthermophile Thermotoga maritima and contained the four regions conserved among all amylolytic enzymes. The pullulanase gene (pulA) encodes a protein of 849 amino acids with a 28-residue signal peptide. The pulA gene was subcloned without its signal sequence and overexpressed in E. coli under the control of the trc promoter. This clone, E. coli FD748, produced two proteins (93 and 83 kDa) with pullulanase activity. A second start site, identified 118 amino acids downstream from the ATG start site, with a Shine-Dalgarno-like sequence (GGAGG) and TTG translation initiation codon was mutated to produce only the 93-kDa protein. The recombinant purified pullulanases (rPulAs) were optimally active at pH 6 and 80 degrees C and had a half-life of 2 h at 80 degrees C. The rPulAs hydrolyzed alpha-1,6 glycosidic linkages of pullulan, starch, amylopectin, glycogen, alpha-beta-limited dextrin. Interestingly, amylose, which contains only alpha-1,4 glycosidic linkages, was not hydrolyzed by rPulAs. According to these results, the enzyme is classified as a debranching enzyme, pullulanase type I. The extraordinary high substrate specificity of rPulA together with its thermal stability makes this enzyme a good candidate for biotechnological applications in the starch-processing industry.     

Phenol/cresol degradation by the thermophilic Bacillus thermoglucosidasius A7: cloning and sequence analysis of five genes involved in the pathway

Bacillus thermoglucosidasius A7 degraded phenol at 65 degrees C via the meta cleavage pathway. Five enzymes used in the metabolism of phenol were cloned from B. thermoglucosidasius A7 into pUC18. Nine open reading frames were present on the 8.1kb insert, six of which could be assigned a function in phenol degradation using database homologies and enzyme activities. The phenol hydroxylase is a two-component enzyme encoded by pheA1 and pheA2. The larger component (50kDa) has 49% amino acid identity with the 4-hydroxyphenylacetate hydroxylase of Escherichia coli, while the smaller component (19kDa) is most related (30% amino acid identity) to the styrene monoxygenase component B from Pseudomonas fluorescens. Both components were neccessary for activity. The catechol 2, 3-dioxygenase encoded by pheB has 45% amino acid identity with dmpB of Pseudomonas sp. CF600 and could be assigned to superfamily I, family 2 and a new subfamily of the Eltis and Bolin grouping. The 2-hydroxymuconic acid semialdehyde hydrolase (2HMSH), encoded by pheC, revealed the highest amino acid identity (36%) to the equivalent enzyme from Pseudomonas sp. strain CF600, encoded by dmpD. Based on sequence identity, pheD and pheE were deduced to encode the 2-hydroxypenta-2,4-dienoate hydratase (2HDH), demonstrating 45% amino acid identity to the gene product of cumE from Pseudomonas fluorescens and the acetaldehyde dehydrogenase (acylating) demonstrating 57% amino acid identity to the gene product of bphJ from Pseudomonas LB400.