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Biomedical subjects

L Regan

Publications and source records attributed to L Regan.

At least 91 records · Page 5Linked to original sources
PubMed1999

Bone density changes in pregnant women treated with heparin: a prospective, longitudinal study.

Heparin plus aspirin significantly improves the live birth rate of women with primary antiphospholipid syndrome. Osteopenia is a major concern of long-term heparin therapy. We studied prospectively the bone mineral density (BMD) changes during pregnancy and the puerperium in 123 women with primary antiphospholipid syndrome treated with low-dose aspirin and subcutaneous low-dose heparin (46 women took unfractionated heparin and 77 took low-molecular-weight heparin). Lumbar spine, neck of femur and forearm BMD were measured, using dual energy X-ray absorptiometry, at 12 weeks gestation, immediately postpartum and 12 weeks postpartum. The mean heparin duration was 27 weeks (range 22-29). During pregnancy, BMD decreased by 3.7% (P < 0.001) at the lumbar spine and by 0.9% (P < 0.05) at the neck of femur with no significant change at the forearm. Lactation was associated with a significant decrease in the lumbar spine and neck of femur BMD. There was no significant difference in BMD changes between the two heparin preparations. No woman suffered a symptomatic fracture. Long-term heparin treatment during pregnancy is associated with a small but significant decrease in BMD at the lumbar spine and neck of femur. This decrease is similar to that previously reported to occur in untreated pregnancies.

Absorptiometry, Photon↗
PubMed1999

Pregnancy complications in women with recurrent miscarriage associated with antiphospholipid antibodies treated with low dose aspirin and heparin.

OBJECTIVE: To study the obstetric course of women with a history of recurrent miscarriage associated with antiphospholipid antibodies, lupus anticoagulant and anticardiolipin antibodies, treated with low dose aspirin and low dose heparin. DESIGN: Prospective observational study. SETTING: University based tertiary referral clinic. POPULATION: One hundred and fifty pregnant women with a history of recurrent miscarriage associated with persistently positive tests for antiphospholipid antibodies. METHODS: Lupus anticoagulant was detected using the dilute Russell's viper venom time together with a platelet neutralisation procedure. IgG and IgM anticardiolipin antibodies were detected using a standardised enzyme linked immunosorbent assay. An IgG anticardiolipin level > or = 5 per litre units and an IgM anticardiolipin level > or = 3 per litre units was considered positive. Aspirin (75 mg daily) was commenced at the time of a positive pregnancy test and heparin (5000 units subcutaneously 12 hourly, or enoxaparin 20 mg daily) was started when fetal heart activity was demonstrated on ultrasound. Treatment was stopped at the time of miscarriage or at 34 weeks of gestation. RESULTS: One hundred and seven pregnancies (71%) resulted in a live birth. Forty-one pregnancies (27%) miscarried, the majority in the first trimester. One woman had a stillbirth, and one a premature baby who died in the neonatal period. One pregnancy was terminated for a fetal anomaly. Gestational hypertension complicated 17% (18/108) of ongoing pregnancies and antepartum haemorrhage 7% (8/108). Twenty-six babies (24%) were delivered before 37 weeks of gestation. Fifty women (46%) were delivered by caesarean section. The median birthweight of all live born infants was 3069 g (range 531-4300); however 15% (16/108) of the infants were small for gestational age. CONCLUSION: Combination treatment with aspirin and heparin leads to a high live birth rate among women with recurrent miscarriage and antiphospholipid antibodies. However, successful pregnancies are prone to a high risk of complications during all trimesters. Close antenatal surveillance and planned delivery of these pregnancies in a unit with specialist obstetric and neonatal intensive care facilities are indicated.

Abortion, Habitual↗
PubMed1999

The C677T MTHFR gene mutation is not predictive of risk for recurrent fetal loss.

We have investigated the potency of the C677T mutation in the methylene tetrahydrofolate reductase (MTHFR) gene as a genetic risk factor in women with a history of early (</=12 weeks gestation) and/or late (>12 weeks gestation) recurrent miscarriage (three or more consecutive pregnancy losses). Fifty-seven of the total 173 (32. 9%) patients were heterozygous for the MTHFR mutation, 14/173 (8.1%) were homozygous (allele frequency 0.25). The prevalence of the MTHFR mutation in these women did not differ significantly from that in the control group of parous women with uneventful pregnancies, where 30/67 (44.8%) were heterozygous and 6/67 (9.0%) homozygous for the mutation (allele frequency 0.31; odds ratio for homozygous T/T 0.90, 95% CI 0.30-2.4). There was no association between the trimester of pregnancy loss and MTHFR genotype. We conclude that the C677T MTHFR mutation is not a risk predictor in women with a history of early or late recurrent miscarriage.

Abortion, Habitual↗
PubMed1998

A chimeric protein C containing the prothrombin Gla domain exhibits increased anticoagulant activity and altered phospholipid specificity.

To determine the structural basis of phosphatidylethanolamine (PE)-dependent activated protein C (APC) activity, we prepared a chimeric molecule in which the Gla domain and hydrophobic stack of protein C were replaced with the corresponding region of prothrombin. APC inactivation of factor Va was enhanced 10-20-fold by PE. Protein S enhanced inactivation 2-fold and independently of PE. PE and protein S had little effect on the activity of the chimera. Factor Va inactivation by APC was approximately 5-fold less efficient than with the chimera on vesicles lacking PE and slightly more efficient on vesicles containing PE. The cleavage patterns of factor Va by APC and the chimera were similar, and PE enhanced the rate of Arg506 and Arg306 cleavage by APC but not the chimera. APC and the chimera bound to phosphatidylserine:phosphatidylcholine vesicles with similar affinity (Kd approximately 500 nM), and PE increased affinity 2-3-fold. Factor Va and protein S synergistically increased the affinity of APC on vesicles without PE to 140 nM and with PE to 14 nM, but they were less effective in enhancing chimera binding to either vesicle. In a factor Xa one-stage plasma clotting assay, the chimera had approximately 5 times more anticoagulant activity than APC on PE-containing vesicles. Unlike APC, which showed a 10 fold dependence on protein S, the chimera was insensitive to protein S. To map the site of the PE and protein S dependence further, we prepared a chimera in which residues 1-22 were derived from prothrombin and the remainder were derived from protein C. This protein exhibited PE and protein S dependence. Thus, these special properties of the protein C Gla domain are resident outside of the region normally hypothesized to be critical for membrane interaction. We conclude that the protein C Gla domain possesses unique properties allowing synergistic interaction with factor Va and protein S on PE-containing membranes.

Amino Acid Sequence↗
PubMed1998

The de novo design of a rubredoxin-like Fe site.

A redox center similar to that of rubredoxin was designed into the 56 amino acid immunoglobulin binding B1 domain of Streptococcals protein G. The redox center in rubredoxin contains an iron ion tetrahedrally coordinated by four cysteine residues, [Fe(S-Cys)4](-1),(-2). The design criteria for the target site included taking backbone movements into account, tetrahedral metal-binding, and maintaining the structure and stability of the wild-type protein. The optical absorption spectrum of the Co(II) complex of the metal-binding variant is characteristic of tetrahedral chelation by four cysteine residues. Circular dichroism and nuclear magnetic resonance measurements reveal that the metal-free and Cd(II)-bound forms of the variant are folded correctly and are stable. The Fe(III) complex of the metal-binding mutant reproduces the optical and the electron paramagnetic resonance spectra of oxidized rubredoxin. This demonstrates that the engineered protein chelates Fe(III) in a tetrahedral array, and the resulting center is similar to that of oxidized rubredoxin.

Amino Acid Sequence↗
PubMed1998

Aromatic rescue of glycine in beta sheets.

BACKGROUND: Glycine is an intrinsically destabilizing residue in beta sheets. In natural proteins, however, this destabilization can be 'rescued' by specific cross-strand pairing with aromatic residues. Here, we present an experimental study of this effect. RESULTS: Protein variants containing glycine and aromatic residues positioned across beta strands in both antiparallel and parallel orientations were studied. The pairing of glycine and phenylalanine across antiparallel strands resulted in a synergistic increase in protein stability. Dramatic differences in stability were observed for the parallel beta-sheet mutants, which were dependent upon the type of site occupied by glycine as well as the type of aromatic residue with which it was cross-strand paired. CONCLUSIONS: Experimental results from a series of mutants suggest a thermodynamic benefit for glycine-aromatic pairing across antiparallel beta strands, consistent with the prevalence of such pairs in natural proteins. We also demonstrate the specificity of glycine-aromatic interactions across parallel beta strands, which defines strand register.

Animals↗
PubMed1998

Antiphospholipid syndrome and pregnancy loss.

Antiphospholipid antibodies (aPL) are associated with recurrent miscarriage. Pregnancies that survive the first trimester risk developing pre-eclampsia, intrauterine growth retardation and fetal distress during labour. Pregnancy loss is initially caused by defective embryonic implantation and later by thrombosis of the placental vasculature. In women with aPL, thromboprophylaxis during pregnancy improves the live birth rate.

Abortion, Habitual↗
PubMed1997

Randomised controlled trial of aspirin and aspirin plus heparin in pregnant women with recurrent miscarriage associated with phospholipid antibodies (or antiphospholipid antibodies)

OBJECTIVE: To determine whether treatment with low dose aspirin and heparin leads to a higher rate of live births than that achieved with low dose aspirin alone in women with a history of recurrent miscarriage associated with phospholipid antibodies (or antiphospholipid antibodies), lupus anticoagulant, and cardiolipin antibodies (or anticardiolipin antibodies). DESIGN: Randomised controlled trial. SETTING: Specialist clinic for recurrent miscarriages. SUBJECTS: 90 women (median age 33 (range 22-43)) with a history of recurrent miscarriage (median number 4 (range 3-15)) and persistently positive results for phospholipid antibodies. INTERVENTION: Either low dose aspirin (75 mg daily) or low dose aspirin and 5000 U of unfractionated heparin subcutaneously 12 hourly. All women started treatment with low dose aspirin when they had a positive urine pregnancy test. Women were randomly allocated an intervention when fetal heart activity was seen on ultrasonography. Treatment was stopped at the time of miscarriage or at 34 weeks' gestation. MAIN OUTCOME MEASURES: Rate of live births with the two treatments. RESULTS: There was no significant difference in the two groups in age or the number and gestation of previous miscarriages. The rate of live births with low dose aspirin and heparin was 71% (32/45 pregnancies) and 42% (19/45 pregnancies) with low dose aspirin alone (odds ratio 3.37 (95% confidence interval 1.40 to 8.10)). More than 90% of miscarriages occurred in the first trimester. There was no difference in outcome between the two treatments in pregnancies that advanced beyond 13 weeks' gestation. Twelve of the 51 successful pregnancies (24%) were delivered before 37 weeks' gestation. Women randomly allocated aspirin and heparin had a median decrease in lumbar spine bone density of 5.4% (range -8.6% to 1.7%). CONCLUSION: Treatment with aspirin and heparin leads to a significantly higher rate of live births in women with a history of recurrent miscarriage associated with phospholipid antibodies than that achieved with aspirin alone.

Abortion, Habitual↗
PubMed1997

An inverse correlation between loop length and stability in a four-helix-bundle protein.

BACKGROUND: The loops in proteins are less well characterized than the secondary structural elements that they connect. We have used the four-helix-bundle protein Rop as a model system in which to explore the role of loop length in protein folding and stability. RESULTS: A natural two-residue loop was replaced with a series of glycine linkers up to 10 residues in length. All 10 mutants are highly helical dimers that retain wild-type RNA-binding activity. As loop length is increased, the stability of Rop toward thermal and chemical denaturation is progressively decreased. CONCLUSIONS: All the mutants assume a wild-type-like structure, which suggests that the natural loop does not actively dictate the final protein fold. The strong inverse correlation observed between loop length and stability is well described by a simple polymer model in which the entropy of loop closure is the dominant energetic term. Our results emphasize the importance of optimization of loop length to successful protein design.

Cloning, Molecular↗
PubMed1997

Speeding up protein folding: mutations that increase the rate at which Rop folds and unfolds by over four orders of magnitude.

BACKGROUND: The dimeric four-helix-bundle protein Rop folds and unfolds extremely slowly. To understand the molecular basis for the slow kinetics, we have studied the folding and unfolding of wild-type Rop and a series of hydrophobic core mutants. RESULTS: Mutation of the hydrophobic core creates stable, dimeric, and wild-type-like proteins with dramatically increased rates of both folding and unfolding. The increases in rates are dependent upon the number and position of repacked residues within the hydrophobic core. CONCLUSIONS: Rop folds by a rapid collision of monomers to form a dimeric intermediate with substantial helical content, followed by a slow rearrangement to the final native structure. Rop unfolding is a single extremely slow kinetic phase. The slow steps of both folding and unfolding are dramatically increased by hydrophobic core replacements, suggesting that their main effect is to substantially decrease the energy of the transition state.

Amino Acid Sequence↗
PubMed1997

Transmuting alpha helices and beta sheets.

Protein architecture involves two main secondary structural classes: alpha helices and beta sheets. Some natural proteins alter their fold in response to changes in solution conditions or as a consequence of mutation. Here, we discuss recent attempts to induce such conformational changes by design: specifically, the motivation and success of efforts to change one protein fold into a different one in response to the 'Paracelsus Challenge'. The results of such efforts may provide a better understanding of the processes that underlie conformational plasticity in proteins.

Amino Acid Sequence↗
PubMed1997

Protein alchemy: changing beta-sheet into alpha-helix.

For most proteins the amino acid sequence determines the tertiary structure. The relative importance of the individual amino acids in specifying the fold, however, remains unclear. To highlight this, Creamer and Rose put forth the 'Paracelsus challenge': Design a protein with 50% sequence identity to a protein with a different fold. We have met this challenge by designing a sequence which retains 50% identity to a predominantly beta-sheet protein, but which now adopts a four helix bundle conformation and possesses the attributes of a native protein. Our results emphasize that a subset of the amino acid sequence is sufficient to specify a fold, and have implications both for structure prediction and design.

Amino Acid Sequence↗