Intended and unintended consequences of abortion law reform: perspectives of abortion experts in Victoria, Australia.

INTRODUCTION: In Victoria, Australia, abortion was decriminalised in October 2008, bringing the law in line with clinical practice and community attitudes. We describe how experts in abortion service provision perceived the intent and subsequent impact of the 2008 Victorian abortion law reform. METHODS: Experts in abortion provision in Victoria were recruited for a qualitative semi-structured interview about the 2008 law reform and its perceived impact, until saturation was reached. Nineteen experts from a range of health care settings and geographic locations were interviewed in 2014/2015. Thematic analysis was conducted to summarise participants' views. RESULTS: Abortion law reform, while a positive event, was perceived to have changed little about the provision of abortion. The views of participants can be categorised into: (1) goals that law reform was intended to address and that have been achieved; (2) intent or hopes of law reform that have not been achieved; (3) unintended consequences; (4) coincidences; and (5) unfinished business. All agreed that law reform had repositioned abortion as a health rather than legal issue, had shifted the power in decision making from doctors to women, and had increased clarity and safety for doctors. However, all described outstanding concerns; limited public provision of surgical abortion; reduced access to abortion after 20 weeks; ongoing stigma; lack of a state-wide strategy for equitable abortion provision; and an unsustainable workforce. CONCLUSION: Law reform, while positive, has failed to address a number of significant issues in abortion service provision, and may have even resulted in a 'lull' in action.

An analysis of the interactions between the Sem-5 SH3 domain and its ligands using molecular dynamics, free energy calculations, and sequence analysis.

The Src-homology-3 (SH3) domain of the Caenorhabditis elegans protein Sem-5 binds proline-rich sequences. It is reported that the SH3 domains broadly accept amide N-substituted residues instead of only recognizing prolines on the basis of side chain shape or rigidity. We have studied the interactions between Sem-5 and its ligands using molecular dynamics (MD), free energy calculations, and sequence analysis. Relative binding free energies, estimated by a method called MM/PBSA, between different substitutions at sites -1, 0, and +2 of the peptide are consistent with the experimental data. A new method to calculate atomic partial charges, AM1-BCC method, is also used in the binding free energy calculations for different N-substitutions at site -1. The results are very similar to those obtained from widely used RESP charges in the AMBER force field. AM1-BCC charges can be calculated more rapidly for any organic molecule than can the RESP charges. Therefore, their use can enable a broader and more efficient application of the MM/PBSA method in drug design. Examination of each component of the free energy leads to the construction of van der Waals interaction energy profiles for each ligand as well as for wild-type and mutant Sem-5 proteins. The profiles and free energy calculations indicate that the van der Waals interactions between the ligands and the receptor determine whether an N- or a Calpha-substituted residue is favored at each site. A VC value (defined as a product of the conservation percentage of each residue and its van der Waals interaction energy with the ligand) is used to identify several residues on the receptor that are critical for specificity and binding affinity. This VC value may have a potential use in identifying crucial residues for any ligand-protein or protein-protein system. Mutations at two of those crucial residues, N190 and N206, are examined. One mutation, N190I, is predicted to reduce the selectivity of the N-substituted residue at site -1 of the ligand and is shown to bind similarly with N- and Calpha-substituted residues at that site.

Maintenance of caspase-3 proenzyme dormancy by an intrinsic "safety catch" regulatory tripeptide.

Caspase-3 is synthesized as a dormant proenzyme and is maintained in an inactive conformation by an Asp-Asp-Asp "safety-catch" regulatory tripeptide contained within a flexible loop near the large-subunit/small-subunit junction. Removal of this "safety catch" results in substantially enhanced autocatalytic maturation as well as increased vulnerability to proteolytic activation by upstream proteases in the apoptotic pathway such as caspase-9 and granzyme B. The safety catch functions through multiple ionic interactions that are disrupted by acidification, which occurs in the cytosol of cells during the early stages of apoptosis. We propose that the caspase-3 safety catch is a key regulatory checkpoint in the apoptotic cascade that regulates terminal events in the caspase cascade by modulating the triggering of caspase-3 activation.

The YRD motif is a major determinant of substrate and inhibitor specificity in T-cell protein-tyrosine phosphatase.

We have studied T-cell protein-tyrosine phosphatase (TCPTP) as a model phosphatase in an attempt to unravel amino acid residues that may influence the design of specific inhibitors. Residues 48--50, termed the YRD motif, a region that is found in protein-tyrosine phosphatases, but absent in dual-specificity phosphatases was targeted. YRD derivatives of TCPTP were characterized by steady-state kinetics and by inhibition studies with BzN-EJJ-amide, a potent inhibitor of TCPTP. Substitution of Asp(50) to alanine or Arg(49) to lysine, methionine, or alanine significantly affected substrate hydrolysis and led to a substantial decrease in affinity for BzN-EJJ-amide. The influence of residue 49 on substrate/inhibitor selectivity was further investigated by comparing subsite amino acid preferences of TCPTP and its R49K derivative by affinity selection coupled with mass spectrometry. The greatest effect on selectivity was observed on the residue that precedes the phosphorylated tyrosine. Unlike wild-type TCPTP, the R49K derivative preferred tyrosine to aspartic or glutamic acid. BzN-EJJ-amide which retains the preferred specificity requirements of TCPTP and PTP1B was equipotent on both enzymes but greater than 30-fold selective over other phosphatases. These results suggest that Arg(49) and Asp(50) may be targeted for the design of potent and selective inhibitors of TCPTP and PTP1B.

Female genital mutilation--experience of The Royal Women's Hospital, Melbourne.

This study was performed to improve our knowledge and understanding of the needs of women affected by female genital mutilation. We looked at the types of complications of these practices which present to a large metropolitan women's hospital in order to determine how we can appropriately treat and support affected women. This was an observational study of women from countries with a high prevalence of female genital mutilation who presented to the Royal Women's Hospital between October, 1995 and January, 1997. Fifty-one patients with a past history of female genital mutilation who were attending the hospital for antenatal or gynaecological care consented to participate in the study. We found that 77.6% of women identified as having had female genital mutilation had undergone infibulation. More than 85% of the women in our study reported a complication of the procedure. The major complications were dyspareunia, apareunia and urinary tract infections; 29.4% of these women required surgery to facilitate intercourse. In our study group there was no difference in Caesarean section rates between the women who had previously delivered in Australia compared with those who had delivered in Africa. Women who have had a female genital mutilation procedure have specific needs for their care which present challenges to both their general practitioners and obstetrician/gynaecologists. These women have significant complications related to their procedure including social and psychosexual problems which require sympathetic management.

Structure-based design of COX-2 selectivity into flurbiprofen.

Comparative computer modeling of the X-ray crystal structures of cyclooxygenase isoforms COX-1 and COX-2 has led to the design of COX-2 selectivity into the nonselective inhibitor flurbiprofen. The COX-2 modeling was based on a postulated binding mode for flurbiprofen and took advantage of a small alcove in the COX-2 active site created by different positions of the Leu384 sidechain between COX-1 and COX-2. The design hypothesis was tested by synthesis and biological assay of a series of flurbiprofen analogs, culminating in the discovery of several inhibitors having up to 78-fold selectivity for COX-2 over COX-1.

Substituted furans as inhibitors of the PDE4 enzyme.

The synthesis and in vitro activity of a series of substituted furans as a novel structural class of PDE4 inhibitors is described. Comparison of emetic threshold with known PDE4 inhibitors is presented.

The interaction of arginine 106 of human prostaglandin G/H synthase-2 with inhibitors is not a universal component of inhibition mediated by nonsteroidal anti-inflammatory drugs.

The three-dimensional cocrystal structures of ovine prostaglandin G/H synthase-1 (PGHS-1) with S-flurbiprofen and murine PGHS-2 with S-flurbiprofen and indomethacin reveal that the carboxylate acid groups of these nonsteroidal anti-inflammatory drugs (NSAIDs) form a salt bridge with the guanidinium group of Arg120 in PGHS-1 and Arg106 in PGHS-2. Mutagenesis studies confirmed that the Arg120 residue of PGHS-1 is critical for binding of substrate and inhibitors through ionic interactions of its guanidinium group with the carboxylate moieties of arachidonic acid and certain NSAIDs. We report here that the analogous R106E substitution in human PGHS-2 results in a catalytically active enzyme with a 30-fold higher Km value for arachidonic acid. Comparison of the inhibition of hPGHS-2(R106E) with wild-type hPGHS-2 by 11 structurally diverse selective and nonselective PGHS inhibitors revealed a 0-1000-fold decrease in inhibitory potency on the mutant enzyme. The loss of inhibitory potency of NSAIDs on hPGHS-2(R106E) could not be correlated with the presence or absence of a carboxylate functional group in the inhibitor, as was demonstrated previously for the PGHS-1(R120E) mutant, or with the selective or nonselective nature of the PGHS inhibitor. The decreases in the inhibitory potencies on hPGHS-2(R106E) by the carboxylate-containing NSAIDs flurbiprofen, indomethacin, meclofenamic acid, and diclofenac on hPGHS-2(R106E) were 965-, 48-, 5.5-, and 4.5-fold, respectively. The nonuniversal requirement for interaction of the carboxylate group of certain NSAIDs with the Arg106 residue in hPGHS-2 is supported by the observation that the methyl ester derivative of indomethacin was a more potent inhibitor than indomethacin on both hPGHS-2 and hPGHS-2(R106E). The greatest loss of potency for inhibition of hPGHS-2(R106E) was observed with the hPGHS-2-selective sulfonamide-containing inhibitors NS-398 and flosulide. The PGHS-2-selective inhibitor DuP697 and a desbromo-sulfonamide analogue of DuP697 displayed equivalent potency on hPGHS-2(R106E) and hPGHS-2. The change in inhibitory potency of NS-398 on hPGHS-2(R106E) was due to a difference in the kinetics of inhibition, with NS-398 displaying time-dependent inhibition of hPGHS-2 but time-independent inhibition of PGHS-2(R106E). The time-dependent inhibition of hPGHS-2 by DuP697 was not affected by the presence of the R106E mutation. We conclude that the Arg106 residue of hPGHS-2 is involved in binding arachidonic acid and certain NSAIDs, but interactions with Arg106 are not a universal requirement for inhibition by either carboxylate-containing NSAIDs or PGHS-2-selective inhibitors.

Conversion of prostaglandin G/H synthase-1 into an enzyme sensitive to PGHS-2-selective inhibitors by a double His513 --> Arg and Ile523 --> val mutation.

Modeling of the active site of prostaglandin G/H synthase-2 (PGHS-2) onto PGHS-1 utilizing the known crystal structure of PGHS-1 shows that the only residues impinging directly on the active site that were not conserved in the two enzymes are His513 and Ile523 of PGHS-1 (Arg499 and Val509 of PGHS-2). These residues of human PGHS-1 were each mutated to the corresponding PGHS-2 residues (His513 --> Arg and Ile523 --> Val) and a double mutant (His513 --> Arg,Ile523 --> Val) containing both residues was also constructed. The mutant enzyme forms were expressed in COS-7 cells, and their properties were compared with those of the normal isoforms using microsomal membranes. The mutated enzyme forms all had apparent Km values within 1.4-fold that of the wild type enzyme, and the specific activity of the mutants were within 2-fold of that of PGHS-1. DuP697, NS-398, DFU, and SC-58125 are selective PGHS-2 inhibitors that act as time-dependent inhibitors of PGHS-2 and rapidly reversible competitive inhibitors of PGHS-1. The single Ile523 --> Val mutation increased the sensitivity to each of these selective inhibitors with most of the effect detected using instantaneous inhibition assays, except for DuP697, whose potency was further increased by preincubation with the enzyme. The double PGHS-1 His513 --> Arg, Ile523 --> Val mutant became more sensitive to inhibition by NS-398 and DFU than the single IV mutant, and time-dependent inhibition was observed. In contrast, the single HR mutation did not increase the sensitivity to inhibition by the selective PGHS-2 inhibitors. The potency of a selective PGHS-1 inhibitor, L-745,296, was decreased 5- and 13-fold in the HR and HR-IV mutants, respectively. All the results indicate that mutations of His513 and Ile523 residues of PGHS-1 can strongly increase sensitivity to selective PGHS-2 inhibition and restore time-dependent inhibition. They also suggest that the corresponding Arg499 and Val509 residues of PGHS-2 are essential determinants in differentiating between the interaction of nonselective NSAIDs and selective PGHS-2 inhibitors and their mechanism of action.

Inhibitor-induced changes in the intrinsic fluorescence of human cyclooxygenase-2.

The steady state tryptophan fluorescence of apo-human cyclooxygenase-2 (hCox-2) is quenched approximately 40%-50% by the slow binding inhibitors diclofenac, indomethacin, ketoprofen, NS-398, and DuP-697. The effects of these inhibitors on tryptophan fluorescence are both time and concentration dependent. Addition of each inhibitor results in a rapid fluorescence decrease, followed by a slower time dependent quenching. The slow, time dependent loss of fluorescence follows first-order kinetics, the rate constants for the process increasing with inhibitor concentration in a saturation-type manner. The rapid fluorescence loss also increases with increasing inhibitor concentration in the same manner. These results are consistent with the initial formation of a rapid equilibrium complex of enzyme and inhibitor (EI), followed by the slower formation of a tightly bound enzyme-inhibitor complex (EI*). The fluorescence of the EI complex is not significantly different from that of the EI* complex. The kinetic parameters of each inhibitor derived for this process (Ki and kon) are close to those obtained by determination of the rate constants for the onset of enzyme inhibition, thereby linking the fluorescence changes with inhibitor binding. The reversible inhibitors ibuprofen and docosahexaenoic acid do not quench the protein fluorescence but do decrease both the rate of the slow fluorescence loss and the magnitude of the initial rapid fluorescence decrease caused by the slow binding inhibitors, consistent with their competitive behavior. ASA-acetylated apo-hCox-2 shows the same fluorescence-quenching behavior in the presence of most of the above inhibitors. However, acetylation apparently blocks the binding of diclofenac, whereas the affinity of ibuprofen is increased. The effects of the collisional quenching agents iodide and acrylamide on both the native and inhibited enzyme are small (< 20% quenching at 0.3 M), showing that inhibitor binding does not result in an increased solvent accessibility of protein tryptophans. The cause of the inhibitor-induced quenching of the intrinsic apo-hCox-2 fluorescence is likely energy transfer to the bound inhibitor. Calculations based on the inhibitor-tryptophan distances in ovine Cox-1 indicate that the distances are within the required range for significant quenching to occur.

Mutation of serine-516 in human prostaglandin G/H synthase-2 to methionine or aspirin acetylation of this residue stimulates 15-R-HETE synthesis.

Prostaglandin G/H synthase (PGHS) is a key enzyme in cellular prostaglandin (PG) synthesis and is the target of non-steroidal anti-inflammatory agents. PGHS occurs in two isoforms, termed PGHS-1 and PGHS-2. These isoforms differ in several respects, including their enzymatic activity following acetylation by aspirin. While PG synthesis by both isoforms is inhibited by aspirin, 15-R-hydroxyeicosatetraenoic acid (15-R-HETE) synthesis by PGHS-2, but not PGHS-1, is stimulated by preincubation with aspirin. We have mutated the putative aspirin acetylation site of hPGHS-2, and expressed the mutants in COS-7 cells using recombinant vaccinia virus. Enzyme activity and inhibitor sensitivity studies provide evidence that Ser516 is the aspirin acetylation site of human PGHS-2 and that substitution of a methionine residue at this position can mimic the effects of aspirin acetylation on enzyme activity.

Topological similarities between a cyclic enkephalin analogue and a potent opiate alkaloid: a computer-modeling approach.

The cyclic enkephalin analogue H-Tyr-cyclo[-D-N delta-Orn-Gly-Phe-Leu-] (1-c) and the rigid narcotic alkaloid 7 alpha-[(1R)-1-hydroxy-1-methyl-3-phenylpropyl]-6,14- endo-ethenotetrahydrooripavine (PEO) were studied by using computer graphics methods to investigate potential geometrical congruencies of their respective pharmacophoric elements. Particular emphasis was placed on the relative spatial disposition of the tyramine moiety and the additional aromatic ring that occurs in both molecules. A three-dimensional vector map was generated defining the locus of the C21 aromatic ring for all those conformers of PEO having up to 10 kcal above the minimum energy conformer. A systematic conformational search on the cyclic peptide afforded four allowable sets of conformers whose side chain of the phenylalanine residue coincided with the vector map of PEO. Local energy minima for the peptide within the revised mutual vector space were found and subjected to bimolecular energy refinement with correspondingly local energy minima for the opiate alkaloid. Several low-energy conformers of the cyclic peptide were identified that permitted a good fit with the alkaloid provided that the tyramine moiety of the respective molecules does not coincide. In the designated conformations the basic nitrogen of the former occupies a distinct geometrical locus, and the side chain of the leucine residue has no structural correlate in the alkaloid.

Analysis of the outcome of in vitro fertilization in relation to the timing of human chorionic gonadotropin administration by the duration of estradiol rise in stimulated cycles.

A retrospective analysis was carried out to assess the outcome of ovarian stimulation on in vitro fertilization when human chorionic gonadotropin (hCG) was administered after 5, 6, or 7 days of continuously rising plasma estradiol (E2). There was no significant difference in the number and size of large follicles in each group although the number of small follicles (less than 15 mm in diameter) decreased significantly after 7 days of E2 rise. After hCG injection in the 7-day group, the E2 level fell below the previous day's value in 40% of patients, whereas a similar fall was observed in only 16% of patients in the 5- and 6-day groups. In those cycles where a luteinizing hormone surge occurred, most surges were detected during the seventh day of E2 rise. The pregnancy rate was 31% when hCG was given after 6 days of rising E2, 21% after 5 days, and 14% after 7 days. In patients achieving pregnancy in the 6-day group, 53% of embryos were derived from leading follicles. In the 7-day group, only 15% of embryos associated with pregnancies were derived from leading follicles. These results strongly suggest that in stimulated cycles, hCG should be administered after 6 days of continuously rising E2. It is therefore postulated that 6 days of rising E2 represents a mean optimal period for follicular growth and oocyte maturation in stimulated cycles.

Gestation sac size in in-vitro fertilization pregnancies.

The gestation sac size in pregnancies resulting from in-vitro fertilization (IVF) and embryo transfer have been compared with those in spontaneous pregnancies. Small-for-dates gestational sac sizes were found in 36% of the IVF pregnancies. This proportion held for both singleton and multiple pregnancies. With increasing gestation beyond 8 weeks the gestation sac volume increasingly approached normal. In contrast to spontaneous conceptions, IVF pregnancies had a low rate of pregnancy loss once fetal heart movements were demonstrated, when the gestation sac size was small-for-dates. Small sac size in an IVF pregnancy may lead to the misdiagnosis of a failed pregnancy.

Patient selection for in vitro fertilization: physical and psychological aspects.

The impact of maternal age, previous childbearing, and the type of underlying pathology on the success rate of an IVF program is explored. There is no difference in the pregnancy rate or in the abortion rate with increasing maternal age, and a similar result is seen in relation to previous childbearing. A reduced fertilization rate is seen in couples classified as having unexplained infertility, dyspermia, and male immobilizing sperm antibodies. The pregnancy rates in the first two groups are satisfactory once fertilization has occurred, but there have been no ongoing pregnancies if there are circulating sperm immobilizing antibodies present in either the male or the female. The psychological problems facing the infertile patient are discussed with special reference to in vitro fertilization.