An Assessment of Surgical Experience among Obstetric and Gynaecology SpR Trainees.

Changes in gynaecological practice have resulted in a significant reduction in surgical exposure for trainees. We have attempted to assess surgical experience among obstetric and gynaecology SpR's in Ireland using an anonymous on-line questionnaire. Trainees were asked to assess their own ability to perform a variety of general gynaecological procedures. There was a 97% response rate (29/33 trainees). There were 11 trainees who were in the final or penultimate year of the scheme. This group were analysed separately to assess competency rates in those approaching the end of the scheme. They were subdivided in to those who have completed one year in a general hospital doing pure gynaecology and those who have not. Approximately half of this group (6/11) had completed a pure gynaecology year. All of these trainees deemed themselves competent to perform all general gynaecological procedures listed, with the exception of trans-urethral tape procedures, for which 3/6 reported the requirement of direct supervision. Only 2/6 deemed themselves competent to perform a total laparoscopic hysterectomy. Year 4/5 trainees who had not completed a pure gynaecology year displayed significantly lower competency rates for most of the procedures. With the current changes in gynaecological practice, these results highlight the importance of dedicated gynaecological surgical training.

Circulating pro- and anticoagulant levels in normal and complicated primigravid pregnancies and their relationship to placental pathology.

We aimed to compare the changes in factor VIII:C, antithrombin, protein C, protein S and fibrinogen in a cohort of low-risk primigravida who developed maternal or fetal complications to those who had uncomplicated pregnancies and to correlate these findings with placental pathology. This is a case-control study of 170 cases and 122 controls selected from a prospective cohort of 1,011 low-risk primigravida. Significantly elevated levels of factor VIII:C and significantly decreased levels of antithrombin were seen in women who developed pre-eclampsia (p <0.001), placental infarction (p < 0.001) or had infants with a birth weight < 3rd centile (p < 0.001). Placental villous dysmaturity was significantly associated with raised factor VIII:C (p < 0.001). Women who developed pre-eclampsia showed elevated fibrinogen at 14 weeks (p = 0.03). Significantly higher than normal pregnancy levels of factor VIII:C, in tandem with significantly lower antithrombin levels associated with certain adverse pregnancy outcomes, may be related to underlying placental insufficiency. This is supported by associated placental findings.

Vulval cancer, 1992-2002 audit--multidisciplinary care and prognostic factors.

Vulval carcinoma is becoming increasingly common. Thirty-four cases of vulval carcinoma were treated from 01/01/1992-31/12/2002. The mean age was 67, range (18-90). The presenting complaints were "a lump" (76%)(25/33), "itch" (49%)(16/33), "discomfort" (30%)(10/33) and postmenopausal bleeding (21%)(7/33). Most patients presented with stage 1 or 2 disease (73%) (n = 24/33). The majority (97%) (32/33) underwent surgical treatment. Five-year survival was 61% (17/28), (disease-free survival 76% (13/17)). There were 12 cases of local/regional recurrence. Survival rates deteriorated with stage of disease. Lymph-node results, lowered survival from 79% (11/14), if negative, to 17% (1/6) if positive. Age >70 reduced survival from 69% (11/16) to 50% (6/12). We conclude that age, the stage of disease, and lymph-node status were important prognostic factors. The favourable outcomes reflect muItidisciplinary care--combining clinical examinations with regular home contact with specialist nurses, by telephone.

Screening for Chlamydia trachomatis in asymptomatic women attending outpatient clinics in a large maternity hospital in Dublin, Ireland.

BACKGROUND: Chlamydia trachomatis can cause a sexually transmitted infection, which, untreated, may result in considerable morbidity. METHODS: A prevalence study was conducted for C trachomatis using nucleic acid amplification technology in asymptomatic women, and certain risk factors that may be used to direct future screening strategies were assessed. RESULTS: The study population comprised 945 asymptomatic women, of whom 783 were attending antenatal clinics, 91 were attending infertility clinics and 71 were attending family planning clinics. An overall C trachomatis prevalence of 3.7% (35/945) was found, with the highest prevalence of 11.2% (22/196) in Irish single women aged <25 years. Logistic regression analysis showed that single status and age <25 years were independent, statistically significant predictors of C trachomatis infection. CONCLUSION: These results support routine screening of asymptomatic women who are sexually active and aged <25 years. An opportunist active screening of all sexually active women independent of age should be additionally considered if resources permit.

Characterization of plant beta-ureidopropionase and functional overexpression in Escherichia coli.

Pyrimidine bases are rapidly catabolized in growing plant tissues. The final enzyme of the catabolic pathway, beta-ureidopropionase (beta-UP; EC 3.5.1.6), was partially purified from the shoots of etiolated maize (Zea mays) seedlings. The enzyme had a K(m) for beta-ureidopropionate (the substrate derived from uracil) of 11 microM. Only one enantiomer of racemic beta-ureidoisobutyrate (derived from thymine) was processed with a K(m) of 6 microM. The enzyme was inactivated by dialysis against 1,10-phenanthroline and activity could be partially restored by addition of Zn(2+). Maize beta-UP was very sensitive to inactivation by iodoacetamide. This could be prevented by addition of substrate, indicating the presence of an active site Cys. The enzyme was strongly inhibited by short chain aliphatic acids and aryl propionates, the most potent inhibitor of which was 2-(2, 6-dinitrophenoxy)-propionate (I(50) = 0.5 microM). A gene for Arabidopsis beta-UP encodes a polypeptide of 405 amino acids and has about 55% homology with the enzymes from other eukaryotic organisms. Several highly conserved residues link the plant beta-UP with a larger class of prokaryotic and eukaryotic amidohydrolases. An Arabidopsis cDNA truncated at the N terminus by 14 residues was cloned and overexpressed in Escherichia coli. The recombinant enzyme (43.7 kD) was soluble, functional, and purified to homogeneity with yields of 15 to 20 mg per 30 g fresh weight of E. coli cells. The recombinant enzyme from Arabidopsis and the native enzyme from maize had molecular masses of approximately 440 kD, indicating the enzyme is a decamer at pH 7.

Inhibition of Diabrotica Larval Growth by Patatin, the Lipid Acyl Hydrolase from Potato Tubers.

Patatin, the nonspecific lipid acyl hydrolase from potato (Solanum tuberosum L.) tubers, dose-dependently inhibits the growth of southern corn rootworm (SCR) and western corn rootworm when fed to them on artificial diet. The 50% growth reduction levels are somewhat cultivar dependent, ranging from 60 to 150 [mu]g/g diet for neonate SCR larvae. A single patatin isoform also inhibits larval growth. Neonate SCR continuously exposed to patatin are halted in larval development. Treatment with di-isopropylfluorophosphate essentially eliminates patatin's phospholipase, galactolipase, and acyl hydrolase activities. SCR growth inhibition is eliminated also, indicating that patatin's serine hydrolase activity is responsible for the observed activities. Patatin-mediated phospholipolysis is highly pH and cultivar dependent, with specific activities up to 300-fold less at pH 5.5 than at pH 8.5. Esterase or phospholipase activities do not correlate with insect growth inhibition. Galactolipase activity, being cultivar and pH independent, correlates significantly with SCR growth inhibition. Insect-growth inhibition of patatin is significantly reduced with increased dietary cholesterol levels. In conclusion, patatin represents a new class of insect-control proteins with a novel mode of action possibly involving lipid metabolism.

Maize ribosome-inactivating protein (b-32). Homologs in related species, effects on maize ribosomes, and modulation of activity by pro-peptide deletions.

The ribosome-inactivating protein (RIP) from maize (Zea mays L.) is unusual in that it is produced in the endosperm as an inactive pro-form, also known as b-32, which can be converted by limited proteolysis to a two-chain active form, alpha beta RIP. Immunological analysis of seed extracts from a variety of species related to maize showed that pro/alpha beta forms of RIP are not unique to maize but are also found in other members of the Panicoideae, including Tripsacum and sorghum. Ribosomes isolated from maize were quite resistant to both purified pro- and alpha beta maize RIPs, whereas they were highly susceptible to the RIP from pokeweed. This suggests that the production of an inactive pro-RIP is not a mechanism to protect the plant's own ribosomes from deleterious action of the alpha beta RIP. RIP derivatives with various pro-segments removed were expressed at high levels in Escherichia coli. Measurement of their activity before and after treatment with subtilisin Carlsberg clearly identified the 25-amino acid intradomain insertion, rather than the N- or C-terminal extensions, as the major element responsible for suppression of enzymatic activity. A RIP with all three processed regions deleted had activity close to that of the native alpha beta form.

Proteolysis of the 85-kilodalton crystalline cysteine proteinase inhibitor from potato releases functional cystatin domains.

The protein crystals found in potato (Solanum tuberosum L.) tuber cells consist of a single 85-kD polypeptide. This polypeptide is an inhibitor of papain and other cysteine proteinases and is capable of binding several proteinase molecules simultaneously (P. Rodis, J.E. Hoff [1984] Plant Physiol 74: 907-911). We have characterized this unusual inhibitor in more detail. Titrations of papain activity with the potato papain inhibitor showed that there are eight papain binding sites per inhibitor molecule. The inhibition constant (Ki) value for papain inhibition was 0.1 nM. Treatment of the inhibitor with trypsin resulted in fragmentation of the 85-kD polypeptide into a 32-kD polypeptide and five 10-kD polypeptides. The 32-kD and 10-kD fragments all retained the ability to potently inhibit papain (Ki values against papain were 0.5 and 0.7 nM, respectively) and the molar stoichiometries of papain binding were 2 to 3:1 and 1:1, respectively. Other nonspecific proteinases such as chymotrypsin, subtilisin Carlsberg, thermolysin, and proteinase K also cleaved the 85-kD inhibitor polypeptide into functional 22-kD and several 10-kD fragments. The fragments obtained by digestion of the potato papain inhibitor with trypsin were purified by reverse-phase high-performance liquid chromatography, and the N-terminal amino acid sequence was obtained for each fragment. Comparison of these sequences showed that the fragments shared a high degree of homology but were not identical. The sequences were homologous to the N termini of members of the cystatin superfamily of cysteine proteinase inhibitors. Therefore, the inhibitor appears to comprise eight tandem cystatin domains linked by preteolytically sensitive junctions. We have called the inhibitor potato multicystatin (PMC). By immunoblot analysis and measurement of papain inhibitory activity, PMC was found at high levels in potato leaves (up to 0.6 microgram/g fresh weight tissue), where it accumulated under conditions that induce the accumulation of other proteinase inhibitors linked to plant defense. PMC may have a similar defensive role, for example in protecting the plant from phytophagous insects that utilize cysteine proteinases for dietary protein digestion.

Characterization of a genomic sequence coding for potato multicystatin, an eight-domain cysteine proteinase inhibitor.

A gene coding for potato multicystatin (PMC), the crystalline inhibitor of cysteine proteases which is found in tubers, was isolated and characterized. The deduced polypeptide product of this genomic sequence is 757 amino acids long and has a molecular mass of 86,778 Da. It consists exclusively of eight closely related domains, with 53-89% identity of residues. Each repeated unit is homologous to the cystatin superfamily of cysteine protease inhibitors. To date, no other member of this family has been found to contain so many inhibitor domains in one polypeptide. Eight introns are proposed in the 3.5 kb of genomic DNA coding for PMC, one in each cystatin unit. There is a family of 4 to 6 such large genes in potato, while in pea and maize the homologues are much smaller, and probably code for single-domain cystatins. PMC transcripts are abundant in tubers, but scarce in undamaged leaves or stems of field-grown potatoes. The tuber messages are derived from at least four genes (including the cloned example). The pattern of gene expression, as well as the properties of the protein, suggest that PMC has a role in the plant's defense system.

Characterization and molecular cloning of a proenzyme form of a ribosome-inactivating protein from maize. Novel mechanism of proenzyme activation by proteolytic removal of a 2.8-kilodalton internal peptide segment.

Ribosome-inactivating proteins (RIPs) are a widely distributed family of plant enzymes that are remarkably potent catalytic inactivators of eukaryotic protein synthesis. All RIPs described to date, including the A-chain of the plant cytotoxin ricin, are polypeptides of 25-32 kDa and share significant amino acid sequence homologies. We have characterized and cloned an RIP from maize (Zea mays). In contrast to previously described RIPs, we have found that maize RIP is synthesized and stored in the kernel as a 34-kDa inactive precursor (isoelectric point = 6.5). During germination, this neutral precursor is converted into a basic, active form (isoelectric point greater than 9) by limited proteolysis, which removes 25 amino acids (2.8 kDa) of net charge -6 from the center of the polypeptide chain. Additional processing also occurs at the amino and carboxyl termini of the polypeptide. The sequence of the internal processed region is unique and it is equivalent to an insertion centered around Thr-156 in the amino acid sequence of ricin toxin A-chain, i.e. in the center of the enzymatically active domain. The generation of an active enzyme by removal of a large amino acid segment from the middle of a precursor polypeptide chain represents a novel mechanism of proenzyme activation that is distinct from more conventional activation mechanisms involving NH2-terminal proteolytic processing. A two-chain active RIP (comprised of 16.5- and 8.5-kDa fragments that remain tightly associated) is produced from this processing event.

Two kunitz-type proteinase inhibitors from potato tubers.

Two proteinase inhibitors have been isolated from tubers of potato (Solanum tuberosum). Based on N-terminal amino acid sequence homologies, they are members of the Kunitz family of proteinase inhibitors. Potato Kunitz inhibitor-1 (molecular weight 19,500, isoelectric point 6.9) is a potent inhibitor of the animal pancreatic proteinase trypsin, and its amino terminus has significant homology to a recently characterized cathepsin D Kunitz inhibitor from potato tubers (Mares et al. [1989] FEBS Lett 251:94-98). Potato Kunitz inhibitor-2 (molecular weight 20,500, isoelectric point 8.6) is an inhibitor of the microbial proteinase subtilisin Carlsberg; its amino terminus is almost identical to an abundant 22 kilodalton protein from potato tubers (Suh et al. [1990] Plant Physiol 94:40-45) and has significant homology to other Kunitz-type subtilisin inhibitors from small grains. Both Kunitz inhibitors are abundant proteins of the cortex of potato tubers.

Halogenated protocatechuates as substrates for protocatechuate dioxygenase from Pseudomonas cepacia.

Substrates containing electron-withdrawing groups were reacted with protocatechuate 3,4-dioxygenase and oxygen. Haloprotocatechuates (5-fluoro-, 5-chloro-, 5-bromo-, 2-chloro-, and 6-chloroprotocatechuates) are oxygenated by the enzyme at rates 28- to 3000-fold lower than that with the native substrate. These lower rates are due to both deactivation of substrate to O2 attack, and to the formation of abortive enzyme-substrate (ES) complexes. Such ES complexes with haloprotocatechuates are spectrally distinct from the normal ES complex. 6-Chloroprotocatechuate produces changes more like those due to protocatechuate. The abortive ES complexes, when rapidly mixed with oxygen, decay to free enzyme and product monophasically, and the dependence of the rates on O2 concentration shows that a rate-limiting step precedes reaction with O2. Thus these complexes are rather unreactive toward O2, and the rate-limiting step in oxygenation is their conversion to active complexes. In contrast, the reaction of O2 with the enzyme and 6-chloroprotocatechuate is biphasic, the first phase being dependent on O2 concentration (2 X 10(4) M-1 S-1) and the second not (7 S-1). The intermediate formed after the first phase strongly resembles the second intermediate seen in the reaction of enzyme with protocatechuate and O2 (Bull, C., Ballou, D. P., and Otsuka, S., (1981) J. Biol. Chem. 256, 12681-12686), implying that the electron-withdrawing effect of the chlorine slows the O2 addition step considerably while the conversion to the second intermediate is hardly affected. When the enzyme cycles through several turnovers with 6-chloroprotocatechuate, an enzyme species is formed that resembles the unreactive ES complexes seen with the other haloprotocatechuates, indicating that a small amount of the unreactive complex is in equilibrium with the reactive complex and that during successive turnovers the enzyme is slowly converted into the unreactive form. The formation of this form correlates with the observation that in assays the rate of product formation gradually decreases with time.

Rapid reaction studies on the oxygenation reactions of catechol dioxygenase.

The reaction of oxygen with catechol 1,2-dioxygenase from Pseudomonas arvilla ATCC 23974 in complex with catechol, 4-methylcatechol, and 4-fluorocatechol has been studied using single turnover stopped flow spectrophotometry. Two sequential enzyme intermediates have been resolved and their visible spectra characterized by computer-assisted methods. These intermediates are spectrally similar to those observed in a similar study with protocatechuate dioxygenase (Bull, C., Ballou, D. P., and Otsuka, S. J. Biol. Chem. 256, 12681-12686 (1981), although the first intermediate seen with the latter enzyme was not observed in this study. The rate of formation of intermediate I is oxygen-dependent and also accelerated by electron-donating substituents on the C-4 of the substrate. This is consistent with the proposed substrate reduction of dioxygen to form a hydroperoxide. Intermediate I is thus suggested to be a 6-hydroperoxycyclohexa-3,5-diene-1-one. The decay of intermediate I is also accelerated by electron donors and is consistent with the rearrangement of intermediate hydroperoxide via an acyl migration mechanism. It is inconsistent with mechanisms involving nucleophilic attack at the carbonyl carbon. Intermediate II is proposed to be an enzyme-product complex based on the resemblance of its visible spectra to those of the benzoate complex of catechol 1,2-dioxygenase and enzyme-product complexes of protocatechuate dioxygenase. Careful 18O2-labeling experiments have shown that no label is lost to the solvent, implying that no free hydroxide forms during catalysis.

The characterization and magnetic properties of the azide and imidazole derivatives of Pseudomonas nitrite reductase.

Optical absorption, mcd, and epr spectroscopy have been used to characterize the azide and imidazole derivatives of oxidized Pseudomonas nitrite reductase. At pH 7.0 azide binds solely to heme d1 with an affinity constant, Kaff = 360 M-1, whereas imidazole binds to both hemes c and d1 with kaff = 35 and 55 M-1, respectively. Low-temperature mcd and epr spectroscopy indicate that c and d1 are low-spin ferrihemes in both derivatives, although the epr of the heme d1-azide component is very weak and requires explanation. Attempts to obtain a high-spin heme d1 in the intact enzyme using the weak field ligands fluoride and thiocyanate have proved unsuccessful. Electron paramagnetic resonance experiments involving an oxidized enzyme derivatives in which heme d1 is complexed by NO, and hence epr silent, have enabled unambiguous assignment of the epr spectrum of Pseudomonas nitrite reductase.

Studies on heme d1 extracted from Pseudomonas aeruginosa nitrite reductase.

Heme d1 has been extracted from Pseudomonas nitrite reductase. Imidazole, cyanide, and chloride-ferroheme, and CO, NO, cyanide, imidazole, and pyridine-ferroheme complexes have been prepared for study by UV/vis spectroscopy, and in some cass by epr and low-temperature mcd as well. Iron determinations have been carried out to assess extinction coefficients. Absorption spectra were used to monitor the transition of chloride-ferriheme d1 to an alkaline form of ferriheme d1 and a pka of 6.5 was determined for the process. The epr spectrum of chloride-ferriheme possessed the characteristic g = 6 signal of high spin (S = 5/2) iron, but the alkaline-ferriheme form gave no detectable epr signals. Electron paramagnetic resonance spectra were also obtained for cyanide and imidazole-ferriheme d1 and for NO-ferroheme d1. The imidazole complex gave signals that were very weak in comparison with the cyanide complex, but mcd measurements of imidazole-ferriheme d1 were consistent with it being a low-spin (S = 1/2) system. The epr signals of NO-ferroheme d1 were similar to those of the corresponding holo-enzyme complex. Reduction of alkaline-ferriheme d1 was found to be affected by the presence of oxygen, but under N2 give the same result with ascorbate and dithionite. Autoreduction of alkaline-ferriheme d1 was observed when placed under CO, and NO, atmospheres, or when treated with pyridine.

Electron paramagnetic resonance studies on Pseudomonas nitrosyl nitrite reductase. Evidence for multiple species in the electron paramagnetic resonance spectra of nitrosyl haemoproteins.

The e.p.r. spectra of reduced 14NO- and 15NO-bound Pseudomonas nitrite reductase have been investigated at pH 5.8 and 8.0 in four buffer systems. At pH 8.0, absorption spectra indicated that only the haem d1 was NO-bound, but, although quantification of the e.p.r. signals in all cases accounted for NO bound the the haem d1 in both subunits of the enzyme, the precise form of the signals varied with buffer and temperature. A rhombic species, with gx = 2.07, gz = 2.01 and gy = 1.96, represented in the low-temperature spectra seen in all the buffers was converted at high temperatures (approx. 200K) into a form showing a reduced anisotropy. Hyperfine splitting on the gz component of this rhombic signal indicated a nitrogen atom trans to NO and it is proposed that histidine provides the endogenous axial ligand for haem d1. At pH 5.8, absorption spectra indicated NO binding to both haems c and d1 and e.p.r. quantifications accounted for NO-bound haems c and d1 in both enzyme subunits. The e.p.r. spectra at pH 5.8 were generally similar to those at pH 8.0 with respect to g-values and hyperfine coupling constants, but were broader with less well defined hyperfine splittings. As at pH 8, rhombic signals present in spectra at low temperatures were converted to less anisotropic forms at high temperatures. The results are discussed in relation to work on model nitrosyl-protohaem complexes [Yoshimura, Ozaki, Shintani & Watanabe (1979) Arch. Biochem, Biophys. 193, 301-313]. No. e.p.r. signal was observed from oxidized NO-bound Pseudomonas nitrite reductase at pH 6.0, over the temperature range 6-100K.

A re-evaluation of some basic structural and functional properties of Pseudomonas cytochrome oxidase.

Determinations of iron content and dry-weight measurements on samples of Pseudomonas cytochrome oxidase were coupled with sodium dodecyl sulphate/polyacrylamide-gel-electrophoresis studies of both the native protein and covalently cross-linked oligomers in order to estimate the enzyme's molecular weight and spectral absorption coefficients. A value of epsilon(ox.) (410)=282x10(3) litre.mol(-1).cm(-1) was calculated for a dimeric protein molecule having a total molecular weight of 122000 (based on iron analysis). Steady-state kinetic observations of the enzyme-catalysed oxidation of reduced azurin by nitrite indicated a marked increase in enzyme inactivation as the pH was raised from 5.7 to 7.2. Since NO, a product of the nitrite reductase activity of Pseudomonas cytochrome oxidase, is known to bind to the enzyme, a study was undertaken to try to assess the potential of NO as a product inhibitor. Investigations showed that samples of the oxidized protein at pH values 4, 5 and 6 bound NO to both haem c and d(1) components, but oxidized enzyme samples at pH7 and above formed their reduced ligand-bound forms when placed under an atmosphere of the gas. Ascorbate-reduced enzyme samples at pH4, 5, 6 and 7 were also found to bind NO at both haem components, although at pH7 the rate of haem c binding was very slow. At pH8 and 9 only the ferrohaem d(1) bound NO. Titration experiments on the reduced protein over the pH range 5-7, with nitrite as a precursor of NO, showed that the haem d(1) had a much higher affinity than the haem c: experiments at pH5.2 and 5.9 with NO-equilibrated solutions revealed the same pattern of behaviour with the oxidized enzyme.

Some magnetic properties of Pseudomonas cytochrome oxidase.

The magnetic properties of the haem groups of Pseudomonas cytochrome oxidase and its cyanide-bound derivatives were studied in both the oxidized and reduced states by means of m.c.d. (magnetic circular dichroism) at low temperatures. In addition, the oxidized forms of the enzyme were also investigated by e.p.r. (electron-paramagnetic-resonance) spectroscopy, and a parallel study, using both e.p.r. and m.c.d., was made on Pseudomonas cytochrome c-551 to aid spectral assignments. For ascorbate-reduced Pseudomonas cytochrome oxidase, the temperature-independence of those features in the m.c.d. spectrum corresponding to the haem c, and the temperature-dependence of those signals corresponding to the haem d1, showed the former to be low-spin and the latter to be high-spin (s = 2). However, addition of cyanide to the reduced enzyme gave a form of the protein that was completely low-spin. The e.p.r. and m.c.d. sectra of oxidized Pseudomonas cytochrome oxidase and its cyanide derivative were consistent with the haem c and d1 components being low-spin in both cases. Pseudomonas cytochrome c-551 was found to be low-spin in both its oxidized and reduced redox states.