Labour outcome of women with successful external cephalic version: a prospective study.

Labour outcome for women following successful external cephalic version (ECV) was evaluated in a prospective case control study. ECV was successfully performed on 93 women in a dedicated ECV clinic at St Mary's Hospital, Portsmouth between 2004 and 2006. In the study group, 50% (n = 93) of women had a successful ECV. There were 103 women in the control group. There was no significant difference in the rate of instrumental deliveries between the study and the control groups. The rate for caesarean section was higher in the ECV group (18.2, n = 93) compared with the control group (7.7, n = 103; p < 0.028). When compared with the control group, the study group had a higher epidural rate (p < 0.016), oxytocin augmentation (p < 0.001), induction of labour rate (p < 0.009), electronic fetal monitoring (EFM) (p < 0.0001), abnormal fetal heart rate (p < 0.014) and birth in main unit (p < 0.001). Our data provide useful guidance to women undergoing ECV.

Real-time kinetics of ligand/cell surface receptor interactions in living cells: binding of epidermal growth factor to the epidermal growth factor receptor.

We describe a system for extending stopped-flow analysis to the kinetics of ligand capture and release by cell surface receptors in living cells. While most mammalian cell lines cannot survive the shear forces associated with turbulent stopped-flow mixing, we determined that a murine hematopoietic precursor cell line, 32D, is capable of surviving rapid mixing using flow rates as great as 4.0 mL/s, allowing rapid processes to be quantitated with dead times as short as 10 ms. 32D cells do not express any endogenous epidermal growth factor (EGF) receptor or other ErbB family members and were used to establish monoclonal cell lines stably expressing the EGF receptor. Association of fluorescein-labeled H22Y-murine EGF (F-EGF) to receptor-expressing 32D cells was observed by measuring time-dependent changes in fluorescence anisotropy following rapid mixing. Dissociation of F-EGF from EGF-receptor-expressing 32D cells was measured both by chase experiments using unlabeled mEGF and by experiments in which equilibrium was perturbed by dilution. Comparison of these dissociation experiments showed that little, if any, ligand-induced dissociation occurs in the chase dissociation experiments. Data from a series of association and dissociation experiments, performed at various concentrations of F-EGF in the nanomolar range and at multiple cell densities, were simultaneously analyzed using global analysis techniques and fit to a two independent receptor-class model. Our analysis is consistent with the presence of two distinct receptor populations having association rate constants of k(on1) = 8.6 x 10(6) M(-1) s(-1) and k(on2) = 2.4 x 10(6) M(-1) s(-1) and dissociation rate constants of k(off1) = 0.17 x 10(-2) s(-1) and k(off2) = 0.21 x 10(-2) s(-1). The magnitudes of these parameters suggest that under physiological conditions, in which cells are transiently exposed to nanomolar concentrations of ligand, ligand capture and release may function as the first line of regulation of the EGF receptor-induced signal transduction cascade.

Stimulation of mitogenic pathways through kinase-impaired mutants of the epidermal growth factor receptor.

Two residues have been shown to be critical for the kinase activity of the receptor for epidermal growth factor (EGF): lysine-721, which functions in the binding of ATP by correctly positioning the gamma-phosphate for phosphoryl transfer, and aspartate-813, which functions as the catalytic base of the kinase. Mutation of either of these two residues has been shown to disrupt kinase activity of the receptor. However, studies performed in different laboratories had suggested that while EGF receptors mutated at lysine-721 are unable to stimulate significant increases of [(3)H]thymidine incorporation into DNA in response to EGF treatment, cells expressing EGF receptors mutated at aspartate-813 do stimulate significant incorporation of [(3)H]thymidine into DNA in response to EGF. In the present study, EGF receptors mutated at lysine-721 or aspartate-813 (K721R and D813A, respectively), as well as wild-type EGF receptors, were expressed in the same cellular background, Chinese hamster ovary cells, and side-by-side experiments were performed to investigate possible signaling-related differences. Our results indicate that while there are measurable differences in the abilities of the two mutant receptors to stimulate [(3)H]thymidine incorporation between 20 and 24 h after addition of EGF, these differences cannot be correlated with significant differences in EGF-stimulated tyrosine phosphorylation of mutant EGF receptor and endogenous ErbB2, the extent of receptor internalization, EGF-stimulated ion uptake, stimulation of SHC activity, or receptor association with Grb2. Flow cytometric data suggest that populations of cells expressing either kinase-impaired mutant EGF receptor progress similarly into S phase in response to addition of EGF. These observations suggest that D813A and K721R retain similar ability to stimulate mitogenic signaling events through transactivation of ErbB2 with only subtle temporal differences, and they emphasize the importance of expressing mutant receptors in an identical cellular context to make valid comparisons of functions.

An analytical approach to the measurement of equilibrium binding constants: application to EGF binding to EGF receptors in intact cells measured by flow cytometry.

In ligand binding studies, ligand depletion often limits the accuracy of the results obtained. This problem is approached by employing the simple observation that as the concentration of receptor in the assay is reduced, ligand depletion is also reduced. Measuring apparent K(D)'s of a ligand at multiple concentrations of receptor with extrapolation to infinitely low receptor concentration takes ligand depletion into account and, depending on the binding model employed, yields a K(D) within the defined limits of accuracy. We apply this analysis to the binding of epidermal growth factor (EGF) to the EGF receptor expressed in intact 32D cells, using a homogeneous fluorescein-labeled preparation of EGF and measuring binding by flow cytometry. Binding isotherms were carried out at varying cell densities with each isotherm fit to the generally applied model with two independent binding sites. Examination of the variation in the K(D)'s versus cell density yields a high-affinity site that accounts for 18% of the sites and a lower affinity site that accounts for the remainder. However, further examination of these data suggests that while consistent with each individual isotherm, the simple model of two independent binding sites that is generally applied to EGF binding to the EGF receptor is inconsistent with the changes in the apparent K(D)'s seen across varying cell densities.

A prospective audit of external cephalic version at term: are ultrasound parameters predictive of outcome?

This prospective study was designed to audit our provision of external cephalic version (ECV) but also to assess the relative importance of various ultrasound markers as indicators of a successful outcome. Data were collected between November 1998 and November 1999. There were 102 breech presentations at term and 47 women had an external cephalic version. We had an overall success rate of 55% with no adverse outcomes. Parity, a non-extended breech presentation and posterior placenta were all independently associated with a higher success rate. As only 49% of women with an uncomplicated breech presentation at term agreed to have an ECV the audit has prompted us to assess how we might improve the uptake of our service. Given that fetal position and placental site seem to influence outcome, we would suggest that a basic ultrasound scan should form part of the procedure. The audit has confirmed the value of ECV in reducing the incidence of breech presentation at term. With the increasing caesarean section rate for this obstetric problem it is important that all units in the United Kingdom are able to provide this service.

Seasonal changes in sex and adrenal steroid hormones of gopher tortoises (Gopherus polyphemus).

We sampled a population of gopher tortoises (Gopherus polyphemus) from May to October 1997 to determine seasonal cycles of steroid hormones (testosterone, T; 17beta-estradiol, E; and progesterone, P) and related them to observations of mating behavior. In males, plasma T levels peaked in July and August and remained elevated through October. This coincides with the reported time of peak mating and spermatogenesis, indicating that males display an associated pattern of reproduction. In females, E levels were high in September and October. Plasma T levels in females were elevated in May, decreased to basal levels in June and July, and rose again in August and September. Elevated E and T levels correspond to the reported time of peak vitellogenic activity, indicating that females also display an associated cycle. Plasma P in females remained basal throughout the active season, suggesting that ovulation occurs in late winter. We also determined levels of corticosterone (B) to assess the influence of capture stress on tortoises and correlated B levels with tortoise activity patterns and sex steroid levels. We found no seasonal variation in levels of B in males or females. Plasma B levels were not correlated with levels of T or E, but were positively correlated with female P levels. Further, we found no relationship between plasma B levels in males and mean distance moved, mean number of burrows used, or mean home range size. However, there was a significant negative correlation between plasma B levels and male body size. In females, there was no relationship between B levels and mean distance moved, but B levels were significantly negatively correlated with the number of burrows females occupied. Lastly, there was no relationship between levels of B and the number of minutes required to obtain blood from an animal. However, B levels increased with the length of time that a tortoise spent in a trap, suggesting that trapped tortoises do exhibit capture stress.

An audit of conservative surgery for endometriosis in a district general hospital 1995-1998.

As conservative surgery for endometriosis is a relatively new introduction to our hospital we felt it would be of value to audit our results and compare these with results from published series. We sent postal questionnaires to 104 patients who had undergone surgery over the past 3 years to assess their response to treatment. We combined this with an additional questionnaire to patients who had a Laparoscopic uterine nerve ablation (LUNA) procedure. We received replies from 81% of the patients with 81% having symptom improvement following their operation. Eighty-seven per cent of patients who had LUNA returned the questionnaire with 64% having some symptom improvement following surgery. On the basis of our results we will continue to offer conservative surgery for endometriosis as the best primary treatment but have some reservations about the addition of LUNA in patients with endometriosis.

Presumptive Sry-negative XX sex reversal in a llama with multiple congenital anomalies.

Multiple congenital abnormalities of the external genitalia consistent with XX sex reversal were detected in a juvenile llama. The llama had a typical female karyotype (74, XX) and did not have a Y chromosome, but a minute chromosome was detected. To determine whether a piece of Y chromosome containing the Sry gene might be located in a small translocation, DNA analysis by polymerase chain reaction was performed; the Sry gene was not detected. Histologic examination revealed ovarian tissue, whereas testicular tissue was not found. External genitalia were partially masculinized, indicating that the urogenital sinus, genital tubercle, and genital swellings had been exposed to androgens during development, although the dam had not received exogenous androgens. Testicular tissue in the ovaries may have been undetected or had regressed prior to birth, as has been reported in sex reversal in mice.

Endogenous lipid (cholesterol) pneumonia associated with bronchogenic carcinoma in a cat.

An 11-year-old, female domestic longhair was presented for dyspnea, vomiting, and left forelimb lameness. A mass in the left caudal lung lobe was seen on thoracic radiographs. The mass was resected during thoracotomy, and histopathology confirmed a diagnosis of endogenous lipid pneumonia. The cat recovered slowly from surgery and was euthanized 11 days following discharge because of persistent respiratory difficulties. Necropsy findings included lipid pneumonia and bronchogenic carcinoma, with probable tumor metastasis to the small intestine, spleen, kidney, and left triceps muscle.

Unliganded epidermal growth factor receptor dimerization induced by direct interaction of quinazolines with the ATP binding site.

Receptor dimerization is critical for signaling by the epidermal growth factor receptor (EGFR) tyrosine kinase. This occurs after binding of the receptor's extracellular domain by ligand or bivalent antibodies. The role of other receptor domains in dimerization is less clear, and there are no examples of dimers induced by direct perturbation of the EGFR kinase domain. Submicromolar concentrations of AG-1478 and AG-1517, quinazolines specific for inhibition of the EGFR kinase, induced reversible receptor dimerization in vitro and in intact A431 cells. Consistent with the inhibitory effect of quinazolines on receptor kinase activity, the dimers formed lacked a detectable Tyr(P) signal. Quinazoline-induced EGFR dimerization was abrogated in vitro by ATP and the ATP analog adenyl-5'-yl imidodiphosphate. Receptors with a single-point mutation in the ATP binding site as well as wild-type EGFR with a covalent modification of the ATP site failed to dimerize in response to AG-1478 and AG-1517. These data suggest that EGFR dimerization can be induced by the interaction of quinazolines at the ATP site in the absence of receptor ligand binding. In SKBR-3 cells, the quinazolines induced the formation of inactive EGFR/ErbB-2 heterodimers, potentially sequestering ErbB-2 from interacting with other coreceptors of the ErbB family. Structural studies of the quinazoline interaction with the EGFR tyrosine kinase domain should allow for an analysis of receptor-specific chemical features required for binding to the ATP site and disruption of signaling, a strategy that can be perhaps applied to other tumor cell receptor systems.

Brain cytochrome oxidase subunit complementary DNAs: isolation, subcloning, sequencing, light and electron microscopic in situ hybridization of transcripts, and regulation by neuronal activity.

The goal of the present study was to isolate, for the first time, cytochrome oxidase subunit genes from murine brain complementary DNA library and to characterize the expression of these genes from mitochondrial and nuclear sources at both light and electron microscopic levels. Brain subunit III (mitochondrial) shared 100% identity with that of murine L cells. Subunit VIa (nuclear) was known to have tissue-specific isoforms in other species: the ubiquitous liver isoform and the heart/muscle isoform. Our brain subunit VIa shared 93% homology with that of the rat liver and 100% identity with the recently reported murine liver isoform, which is only 62% identical to that of the rat heart isoform. In situ hybridization with riboprobes revealed messenger RNA labelling that was similar, though not identical, to that of cytochrome oxidase histochemistry. Monocular enucleation in adult mice induced a significant down-regulation of both subunit messages in the contralateral lateral geniculate nucleus. However, the decrease in subunit III messenger RNAs surpassed that of subunit VIa at all time periods examined, suggesting that mitochondrial gene expression is more tightly regulated by neuronal activity than that of nuclear ones. At the electron microscopic level, subunit III messenger RNA was localized to the mitochondrial compartment in both cell bodies and processes, while that of nuclear-encoded subunit VIa was present exclusively in the extramitochondrial compartment of somata and not of dendrites or axons. Surprisingly, the message was primarily associated with the rough endoplasmic reticulum, suggesting a novel pathway for its synthesis and trafficking. Our results indicate that the unique properties of neurons impose special requirements for subunits of a single mitochondrial enzyme with dual genomic origins. At sites of high energy demands (such as postsynaptic dendrites and some axon terminals), mitochondrial-encoded cytochrome oxidase subunits can be locally transcribed and translated, and they provide the framework for the subsequent importation and incorporation of nuclear-encoded subunits, which are strictly synthesized in the cell bodies. Dynamic local energy needs are met when subunits from the two genomic sources are assembled to form functional holoenzymes.

Identification of residues of the epidermal growth factor receptor proximal to residue 45 of bound epidermal growth factor.

A triple mutant of murine epidermal growth factor (mEGF), N1Q/H22Y/R45K-mEGF, was constructed by site-directed mutagenesis, expressed, purified, and characterized for use in an affinity cross-linking study to identify aminoacyl residues of the EGF receptor adjacent to a residue in the carboxyl-terminal domain of bound EGF thought to be important in distinguishing between EGF and transforming growth factor-alpha in their recognition by the receptor. Cyclization of Gln1 to form pyroglutamate (pE) limited the site of cross-linking in the mutant to Lys45, permitting identification of receptor residues that are proximal to this residue of bound EGF. The resulting N1pE/H22Y/R45K-mEGF was shown to be comparable to wild-type mEGF in receptor binding and stimulation of receptor autophosphorylation. 125I-Labeled N1pE/H22Y/R45K-mEGF was reacted with the heterobifunctional cross-linking reagent sulfo-N-succinimidyl-4-(fluorosulfonyl)benzoate, and the resulting modified EGF was incubated with A431 membrane vesicles bearing EGF receptors. Incubation resulted in specific cross-linking of the labeled N1pE/H22Y/R45K-mEGF to EGF receptors. The resulting cross-linked complex was then partially purified, denatured, reduced, and carboxyamidomethylated. Digestion with endoprotease LysC resulted in a unique radiolabeled peptide that could be immunoprecipitated using antibodies to mEGF. This immunoprecipitated fragment was purified by gel electrophoresis and subjected to microsequencing. The resulting sequence was matched to that of a LysC fragment of the receptor, which begins with Thr464 and is near the interface of receptor subdomains III and IV. Loss of signal at cycle 2 suggests that the point of attachment of cross-linked N1pE/H22Y/R45K is Lys465 of the receptor.

5'-(p-fluorosulfonylbenzoyl)-2'(or 3')-(methylanthraniloyl)adenosine, fluorescent affinity labels for adenine nucleotide binding sites: interaction with the kinase active site of the receptor for epidermal growth factor.

We have found that the epidermal growth factor (EGF) receptor kinase can utilize the fluorescent ATP derivative, methylanthraniloyl ATP, as a substrate. On the basis of this observation, together with our previous studies that showed that 5'-(p-fluorosulfonylbenzoyl)adenosine (5'-FSBAdo) is a highly specific affinity label for the ATP site of the kinase domain of the EGF receptor, we prepared new derivatives of 5'-FSBAdo, 5'-(p-fluorosulfonyl)-2'(or 3')-(methylanthraniloyl)adenosine (FSBMantAdo), as fluorescent affinity labels for adenine nucleotide binding sites, and in particular for the ATP site of the EGF receptor. The two products were purified by HPLC and were characterized by UV-Vis absorbance spectroscopy, mass spectrometry, nuclear magnetic resonance spectroscopy, and fluorescence spectroscopy. Incubation of membrane vesicles containing the EGF receptor with either the 2' or 3' derivative resulted in irreversible inhibition of the receptor kinase activity, as assessed by autophosphorylation assays. Preincubation of vesicles with AMP imidodiphosphate (AMPPNP), a hydrolysis-resistant ATP analog, prior to treatment with FSBMantAdo resulted in the protection of the receptor kinase activity' from FSBMantAdo inactivation. Steady state fluorescence spectra (with excitation at 360 nm) revealed a blue shift in the emission maximum of partially purified FSBMantAdo-labeled receptor (426 nm), as compared with the emission maximum of free FSBMantAdo (441 nm) in aqueous solution, suggesting that the receptor-bound label is in a relatively low polarity environment. These studies show that FSBMantAdo is a specific affinity label for the ATP site of the EGF receptor. FSBMantAdo may also prove useful as a fluorescent affinity label for other ATP binding sites.

Steric constraints in the recognition of peptide substrates for the epidermal growth factor receptor kinase.

Epidermal growth factor (EGF) stimulates cellular mitogenesis by binding to and activating its membrane-associated receptor. An important component of signal transduction by the activated receptor is the stimulation of an intrinsic tyrosyl residue-specific protein kinase, which selectively phosphorylates tyrosyl residues in the cytoplasmic tail of the receptor and in other cytoplasmic substrates. A recent study utilizing tyrsub, a new high affinity synthetic peptide substrate for the EGF receptor kinase, provided evidence that in peptide substrate binding, the tyrosyl residue plays the central role in recognition, with residues surrounding the tyrosyl residue contributing to stabilization of docking [Guyer et al. (1994) Arch. Biochem. Biophys. 312, 573-578]. A large body of previous work had identified acidic residues near the site of phosphorylation as most important for binding; therefore, other residues in tyrsub appeared to be promising sites for locating spectroscopic reporter groups. Since tyrsub has neutral residues -4 and +4 residues from the site of phosphorylation, we prepared two analogs of tyrsub, in each of which one of those residues was substituted with Cys. These cystyrsubs were found to be effectively phosphorylated by EGF receptor prepared from A431 cells, on stimulation with EGF, with high affinities [Km(app) = 40-50 microM.] Modification of the cystyrsubs with iodoacetamide had no deleterious effect on the ability of the peptide to be phosphorylated by the EGF receptor kinase, while the labeling by 5-iodoacetimidofluorescein completely abolished the productive interaction between the peptide and the EGF receptor. This unexpected failure of the fluorescently labeled peptides to be phosphorylated does, however, provide information on steric limitations to recognition of substrates by the EGF receptor kinase.

The treatment of incest offenders--a hypnotic approach: a brief communication.

Incest has become more prominent in public awareness over the past 15 years. The major focus of this interest has been on the incest survivor. The incest offender has received less attention. A hypnotic approach to treating incest offenders is outlined that involves a seven-stage approach. A case example is presented and future research directions suggested.

Peptide substrate recognition by the epidermal growth factor receptor.

The epidermal growth factor (EGF) receptor, like other protein tyrosine kinases, shows a preference for substrates having acidic residues in the vicinity of the tyrosyl residue that undergoes phosphorylation. We have developed a peptide substrate for the EGF receptor, termed tyrsub, which is based upon the highly acidic amino terminal sequence of human erythrocyte Band 3. Tyrsub possesses the lowest apparent Km(Km(app) = 32 microM) for phosphorylation by the EGF receptor of any peptide substrate reported to date. Using tyrsub, as well as analogs containing either Ser (sersub) or Phe (phesub) in place of Tyr, we investigated the relative importance of characteristics of the hydroxyaminoacyl residue in substrate recognition. Sersub was unable either to act as a substrate or serve as an effective inhibitor of tyrsub phosphorylation by the EGF receptor. Phesub was also unable to inhibit EGF-stimulable tyrsub phosphorylation, suggesting that the phenolic hydroxyl of the tyrosyl residue, rather than the aromatic ring, predominates in substrate recognition. These results indicate that for peptide substrates, at least, binding consists of two steps, recognition, in which the tyrosyl side chain plays the central role, and docking, in which residues surrounding the tyrosyl residue contribute to stabilizing binding interactions.