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Large-scale production of gene therapeutics comprising equine infectious anaemia virus (EIAV) -based lentiviral vectors (LVs) would benefit from the development of producer cell lines enabling the generation of larger quantities of vector than achievable by transient systems. Such cell lines would contain three vector components (Gag/Pol, VSV-G envelope and genome expression constructs). As the vesicular stomatitis virus (VSV-G) envelope protein is cytotoxic, its expression must be regulated. It is also desirable to regulate Gag/Pol expression to minimise metabolic burden on the cell. The Tet repressor (TetR) system was selected to regulate expression of VSV-G and Gag/Pol, necessitating the introduction of a fourth construct, encoding TetR, into the cell line. We have generated an inducible packaging cell line that shows tight control of the packaging components, and high-titre vector production on transient transfection of the EIAV genome. The cell line is stable for at least 7 weeks in the absence of selective pressure. To verify that this packaging cell line can support the generation of producer cell lines it was transfected stably with an EIAV genome cassette encoding ProSavin; a gene therapeutic for Parkinson's disease. Producer cell lines were generated, which on induction, yielded ProSavin with titres comparable to the transient system.

Galectin-1 has been implicated in the development of skeletal muscle, being maximally expressed at the time of myofiber formation. Furthermore, in the presence of exogenous galectin-1, mononuclear myoblasts show increased fusion in vitro. In the current study, we have used the galectin-1 null mouse to elucidate the role of galectin-1 in skeletal muscle development and regeneration. Myoblasts derived from the galectin-1 mutant showed a reduced ability to fuse in vitro. In galectin-1 null mutants, there was evidence of a delay in muscle fiber development at the neonatal stage and muscle fiber diameter was reduced when compared with wild-type at the adult stage. Muscle regeneration was also compromised in the galectin-1 mutant with the process being delayed and a reduced fiber size being maintained. These results, therefore, show a definitive role for galectin-1 in fusion of myoblasts both in vitro, in vivo, and in regeneration after recovery from induced injury.

Ovine interferon tau (oIFN-tau) is an embryonic protein of critical importance in the establishment of pregnancy in the sheep. We have produced recombinant (r) oIFN-tau using a baculovirus expression system and demonstrated the biological activity of the protein produced. Bombyx mori larvae were infected with B. mori nuclear polyhedrosis virus (BmNPV), modified by inserting a cDNA coding for oIFN-tau downstream of the strong polyhedron promoter. Following infection, antiviral activity of the haemolymph rose to a maximum of 3.6 x 10(8) u/mL (equivalent to 3 mg roIFN-tau/mL) by day 5, when haemolymph was collected and stored frozen. Control haemolymph, collected from uninfected insects at an equivalent time, contained no antiviral activity. The roIFN-tau was partially purified by gel filtration column chromatography and the presence of roIFN-tau confirmed by western blotting. The biological activity of the partially purified roIFN-tau was tested in ewes. Treatment with roIFN-tau caused a significant delay in luteolysis confirming biological potency. The results demonstrate that this system can be successfully used to produce large quantities of roIFN-tau.

There is now evidence from in vivo and in vitro studies that the rate of Schwann cell generation is regulated by the balance of two opposing signals, beta neuregulins and endothelins. The beta neuregulins promote the development of precursors to Schwann cells whereas endothelins retard it through an action on endothelin-B receptors. The present work has shown additional controls of this transition, and implicates AP-2 transcription factors, in particular AP-2 alpha, as negative regulators of Schwann cell generation. We found that both AP-2 alpha and AP-2 gamma are present in early embryonic nerves, whereas AP-2 beta was not. Isoform-specific analysis of AP-2 alpha showed that isoform 3 was most abundant with isoforms 1 and 2 present in lesser amounts; isoform 4 was absent. Maximal AP-2 alpha and AP-2 gamma mRNA expression occurred at embryonic day (E) 12/13 in the mouse and at E14/15 in the rat, which correlates with the presence of Schwann cell precursors in the nerve. In both rats and in mice, in vivo and in vitro, downregulation of AP-2 alpha mRNA and protein coincided with one of the main steps in Schwann cell development, the precursor-Schwann cell transition. Moreover, Schwann cell generation was delayed if this downregulation was prevented by enforced expression of AP-2 alpha in precursors. These studies suggest that AP-2 is involved in the control of the timing of Schwann cell development.

Background and Methods: The Scottish Adjuvant Tamoxifen Trial (main trial) was initiated in April 1978 to assess the effect of tamoxifen given to patients with breast cancer immediately after mastectomy (or mastectomy plus radiation therapy) (adjuvant arm) or only after the patients had had a relapse (control arm); 1323 patients were randomly assigned (667 to the adjuvant arm and 656 to the control arm). Results have been reported for the follow-up period from 2.5 through 8 years. In this article, we report updated results after a median follow-up of 15 years. If agreeable and eligible, patients who were disease free at 5 years in the adjuvant arm of the main trial were entered into a duration trial and randomly assigned either to stop taking tamoxifen (169 patients) or to continue taking it indefinitely until relapse or death (173 patients). For this update, we analyzed information on death, recurrence, survival, and other malignancies for all but 21 of the 560 living patients from the original and duration trials to determine the probabilities of total survival, systemic relapse of disease, and death from breast cancer. All statistical tests are two-sided., Results: The beneficial effect of adjuvant tamoxifen given for 5 years on the probability of total survival (P =.006), systemic relapse (P =.007), and death from breast cancer (P =.002) has been maintained through 15 years. No additional benefit was observed in those randomly assigned to continue taking tamoxifen beyond 5 years., Conclusion: Information from this study suggests that, if adjuvant tamoxifen is given to women with operable breast cancer, it need not be for more than 5 years.

Inherited retinal degenerations such as retinitis pigmentosa (RP) are characterized by progressive loss of photoreceptors, apparently by apoptosis, and our recent report of increased secreted Frizzled-related protein-2 (SFRP2) in RP retinas suggests altered Wnt signalling may be a component of the degenerative process. The present study shows that levels of SFRPI, SFRP3 and SFRP5 mRNAs are also abnormal in RP, giving rise to idiosyncratic expression patterns. In highly degenerative retinas, the SFRP proteins localize mainly to the inner limiting membrane, but in a well-preserved retina SFRPI and SFRP5 are notably localized to the surviving photoreceptors. Together with increased c-jun mRNA expression in all cases examined, these results support the notion that disruptions of Wnt network signalling are involved retinal neurodegeneration.

In the rd/rd mouse model of inherited retinal degeneration, the majority of photoreceptors die apoptotically between postnatal age (P)10 and 20 days, during which period the inner retina appears morphologically unaffected. To examine mRNA changes associated with the degeneration, we performed differential screening of 588 arrayed murine cDNAs using probes reverse-transcribed from P8 predegenerative and control mouse retinal RNAs. We detected altered expression of the gene encoding nm23-M2, a member of the family of nucleoside diphosphate kinases implicated in diverse processes including metastasis suppression and transcriptional regulation. Retinal nm23 mRNA levels increased during degeneration while control levels decreased over age-matched time-points. In situ hybridization showed the high level of expression at P20 in rd/rd was concentrated in the retinal ganglion cells. Previous studies have indicated upregulation of the stress-response related gene alphaB-crystallin in the rd/rd inner retina, and increased nm23 levels may be a component of this response to photoreceptor loss and altered retinal architecture., (Copyright 2000 Academic Press.)

Purpose: Inherited retinal degenerations such as retinitis pigmentosa (RP) are characterized by progressive death of the photoreceptors due to apoptosis. To identify changes in gene expression associated with the degenerative state in RP retinas, expression profiling of apoptosis-related genes was performed using a gridded array technique., Methods: Total RNAs from RP and control retinas were used to generate radiolabeled cDNA probes to screen gridded membrane arrays of 205 apoptosis-related genes. Reverse transcription-polymerase chain reaction was used to generate probes corresponding to differentially expressed genes for Northern blot analysis and for mRNA in situ hybridization studies of retinal cryosections. Fluorescence immunocytochemistry was performed on retinal sections using available antibodies., Results: By expression profiling, we identified upregulated expression of the mRNA for secreted Frizzled-related protein-2 (SFRP2) in RP retina in comparison with control. By Northern blot analysis, SFRP2 mRNA levels were 2- to 20-fold higher in RP samples than in controls. The localization of SFRP2 mRNA by in situ hybridization varied according to the degree of degeneration, from stratified in relatively well-preserved retinas to diffuse in the highly degenerative state. By immunofluorescence, SFRP2 protein in RP retinas was found mainly to colocalize with the cell adhesion and signal transducing protein beta-catenin., Conclusions: SFRPs can regulate apoptosis in vitro and appear to interact with the Wnt/Frizzled signaling pathway, which includes routes to apoptotic activation. Increased SFRP2 expression in RP retinas suggests that an altered pattern of Wnt signal transduction may be a step in the degenerative process linking causal mutations with eventual photoreceptor demise.

beta neuregulins (also called NDF, GGF, ARIA, and heregulins) are neuron-derived molecules that are likely to be responsible for Schwann cell precursor survival, proliferation, and maturation in vivo and in vitro. Although the receptors to which beta neuregulins bind have been defined, little is known about the transcription factors these important ligands activate. Using antibodies, quantitative imaging methods and Western blotting, we show that beta neuregulin induces a high level of phosphorylation of the transcription factor cyclic AMP response element binding protein (CREB) on Ser-133 in cultured rat Schwann cells and that the phosphorylation is prolonged over several hours. In contrast, neurotrophins, CNTF, FGF-2, EGF, and TGF beta induce little or no phosphorylation of CREB despite the fact that receptors for these factors are present on Schwann cells. As expected CREB phosphorylation was detected following cAMP elevation, and it was also induced by elevation of cytoplasmic Ca2+, endothelin 1, and PDGF-BB. The signal was lower than that seen in response to beta neuregulin, and transient, unlike the sustained CREB activation induced by beta neuregulin. Our results suggest that the sustained phosphorylation of CREB on Ser-133 may contribute to the broad spectrum of effects that beta neuregulins have on cells of the Schwann cell lineage and that the CREB pathway may be important for transduction of neuregulin signals in Schwann cells.

Although basic helix-loop-helix (bHLH) proteins play an important role in transcriptional control in many cell types, the role of HLH proteins in Schwann cells has yet to be assessed. In this study, we have analyzed the expression of the dominant negative HLH genes, Id1 to Id4 and the class A gene REB, during Schwann cell development. We found that mRNA derived from these genes was present in the Schwann cell lineage throughout development including embryonic precursors and mature cells. The mRNA levels were not significantly regulated during development. Nevertheless, by using antibodies against the four different Id proteins, we found clear regulation of some of these genes at the protein level, in particular Id 2, 4, and REB, both in amount and nuclear/cytoplasmic localization. All these proteins are found in the nuclei of Schwann cell precursors but are not seen in nuclei of Schwann cells of newborn nerves. We observed extensive overlap in Id expression, especially in Schwann cell precursors that co-expressed all four Id proteins and REB. We also showed that Id 1 and 2 were up-regulated as Schwann cells progressed through the cell cycle. These data indicate that HLH transcription factors act as regulators of Schwann cell development and point to the existence of as yet unidentified cell type-specific bHLH proteins in these cells.

The interaction of cells with the extracellular matrix plays a critical role in morphogenesis and cell differentiation. To define how Schwann cells might interact with the extracellular matrix, we chose to study the expression of the laminin/collagen receptor alpha1beta1 integrin during nerve development in the rat from embryonic day 14 to maturity. We found that this integrin is expressed predominantly on mature non-myelin-forming cells and only at very low levels on myelin-forming cells. Significant levels of this integrin were not detected on Schwann cell precursors or embryonic Schwann cells in vivo. Experiments using transected and crushed sciatic nerve showed that alpha1beta1 integrin expression is regulated at least in part by axonal contact. Furthermore, Schwann cell culture experiments showed that alpha1beta1 integrin levels are strongly upregulated by transforming growth factor-beta(s) and phorbol esters.

The entire coding region of an ovine endometrial oxytocin receptor (OTR) cDNA was generated by PCR, subcloned into the SV40 major late promoter expression vector pSVLJ and transiently expressed in Cos-7 cells. A specific OTR antagonist, 125I-labelled d(CH2)5 [Tyr(Me)2,Thr4,Tyr-NH2(9)]-vasotocin (OTA), was used to describe the binding kinetics of the expressed receptor which had a Kd of 4.5 nM and Bmax of 2.4 nM/mg protein (6.8 x 10(5) receptor molecules/transfected cell). The functional properties of the expressed OTR were determined by measuring oxytocin-induced phosphoinositide (PI) hydrolysis. Oxytocin increased PI turnover in OTR transfected cells fourfold in excess of residual endogenous activity, and stimulated phospholipase C (PLC) activity in a dose- and time-dependent manner, confirming that the expressed OTR cDNA was functional. Arginine vasopressin also stimulated PI turnover in a dose-dependent manner; thresholds of responses to oxytocin and arginine vasopressin were 10(-9) M and 10(-7) M respectively. OTA did not increase PI turnover and competitively inhibited the oxytocin-induced response. Direct activation of the pathway by aluminium fluoride and guanosine (3'-O-thio)-triphosphate (GTP gamma S) confirmed that the OTR was G-protein linked. Co-incubation of GTP gamma S with oxytocin shifted the PI-response threshold from 10(-7) M to 10(-9) M and significantly increased the level of response, suggesting that maximum PI turnover was agonist-dependent. The G-protein involved in mediating the signal transduction pathway was pertussis toxin-insensitive and, therefore, probably a member of the Gq subfamily. The PLC inhibitor, U73122, had no effect on oxytocin-induced PI turnover, consistent with the response in endometrial tissue. These data suggest that the signalling pathway mediated by expressed OTR is similar to that attributed to OTR occupancy in ovine endometrium.

Using the rat sciatic nerve as a model for the study of Schwann cell differentiation we have identified a Schwann cell precursor, a distinct cell type present in developing nerves at a time when they are projecting to their target tissues. These cells develop into Schwann cells over a relatively short time in vivo. In vitro, they can generate Schwann cells if they are cultured in neuron-conditioned medium or in the presence of neu-differentiation factors (NDF) (neuregulins, heregulins, glial growth factor), a recently discovered family of growth factors expressed at high levels in neurons. Thus neu-differentiation factors may be important neuro-glia signalling molecules in the Schwann cell lineage. Later stages in the development of Schann cells, such as differentiation towards a myelin phenotype, can be studied using cultured Schwann cells. These cells dedifferentiate both in vivo and in vitro when they are deprived of axonal contact. Elevation of intracellular cyclic AMP levels in the absence of cell division causes high levels of expression of Po, the major myelin glycoprotein. TGF beta s and FGFs suppress this induction, while IGFs promote it.

A bovine trophoblast interferon (IFN-tau) gene promoter sequence (-450 to +26 bp relative to the transcription start site) led to expression of reporter gene (CAT) constructs transfected into L929 (murine fibroblast) or JAR (human choriocarcinoma) cells. Expression depended on the presence of an exogenous (SV40) enhancer. Poly(I)(C) activated endogenous IFN production in L929 and JAR cells but had no consistent effect on CAT expression. Similar results were obtained in L929 cells with inactivated Newcastle disease virus. There was no 'priming' effect of exogenous Type I IFN. Deletion mutants revealed sites exerting negative control on expression between - 338 and - 247 bp, and between - 150 and - 71 bp; these regions contained sequences resembling previously identified negative regulatory domains. In the absence of viral inducibility it is proposed that negative regulation contributes towards the stringent control of expression characteristic of IFN-tau genes.

Myelination by Schwann cells is likely to be regulated in vivo by positive and negative epigenetic factors. In vitro, the positive regulation of myelin differentiation, in particular expression of the major myelin protein Po, can be mimicked by cAMP elevating agents, while serum, transforming growth factor (TGF) beta s, and fibroblast growth factor (FGF)2 have been shown to exert a negative effect on this differentiation. Growth factors which promote Po induction have not, however, been identified previously. Using a forskolin concentration (0.4 microM) which alone produces little Po mRNA or protein induction, we show that insulin-like growth factor (IGF)-I, IGF-II and high concentrations of insulin promote high levels of Po induction, although in the absence of forskolin they have no effect. Another event related to Schwann cell differentiation, induction of galactocerebroside expression in response to cAMP analogues, is also potentiated by IGFs. In a different context, IGFs regulate Schwann cell DNA synthesis. We find that in defined medium forskolin plus FGF2, TGF beta or platelet-derived growth factor (PDGF) BB causes minimal DNA synthesis in the absence of IGFs and that IGFs act as potent mitogens under these conditions. IGFs also potentiate DNA synthesis induced by beta isoforms of neu-differentiation factors (NDFs), although in this case considerable DNA synthesis occurs even in the absence of IGF. These results show that IGFs can act as powerful stimulators of both proliferation and differentiation in Schwann cells, and that the total growth factor input determines which of these pathways IGFs will promote.

We have examined both how the molecular phenotype of Schwann cells in vitro is regulated by transforming growth factor beta (TGF-beta), using immunohistochemistry and immunoblotting, and the distribution of TGF-beta 2 and 3 in embryonic and mature nerves and ganglia, using immunohistochemistry and in situ hybridisation. We find that TGF-beta 2 and -3 upregulate expression of the neural cell adhesion molecules NCAM and L1. In TGF-beta-treated cultures, in addition to the 140 and 120 kD isoforms known to be present in Schwann cells, small amounts of the 180 kD isoform can be detected. TGF-beta s also block cAMP-induced expression of the lipid antigens galactocerebroside (GalC) and O4, in addition to blocking expression of protein zero (P0), the major peripheral myelin glycoprotein, as previously shown. Using antibodies specific to TGF-beta 2 and -3, respectively, we confirm the presence of these proteins in myelin-forming Schwann cells and show also that TGF-beta 2 and -3 are clearly expressed by peripheral glia that are not involved in myelination. This includes Schwann cell precursors, embryonic Schwann cells, non-myelin-forming Schwann cells and satellite cells from adult nerves and ganglia, and neonatal Schwann cells in purified cultures without neurones. In situ hybridisation with a digoxygenin-labelled riboprobe reveals a strong TGF-beta 3 mRNA signal in Schwann cells, satellite cells, and some neurones. Schwann cells in culture also secrete TGF-beta in a latent form, whereas purified cultures of dorsal root ganglion neurones from 1-day-old rats secrete active TGF beta during the first 48 h in culture.

Between January 1980 and October 1983, 417 patients were randomized for mastectomy followed by axillary node sampling or full axillary clearance. The aim of the study was to determine whether a standard 'four-node' axillary sample, followed by careful dissection of removed tissue, could accurately indicate the extent of local treatment required. Axillary radiotherapy was given only to patients with histological involvement of sampled nodes and not to any having axillary clearance. The incidence of involved nodes was similar for both groups, as were distant relapse and survival rates. Currently 62.6 percent are alive after clearance and 65.0 percent after sampling. A non-significant increase in the rate of locoregional relapse was observed for those treated by axillary node clearance, this being due mainly to increased relapse on the unirradiated chest wall (clearance 21 percent versus sampling 12 percent in patients with node-positive disease). There was only a minor difference in axillary relapse, favouring axillary clearance (3.0 versus 5.4 percent). In patients with operable breast cancer, mastectomy with axillary node sampling gives equal control to mastectomy with axillary node clearance but, as morbidity is greater, surgical clearance of the axilla is the preferred option.

Objective: To determine any cardiac or vascular morbidity associated with long term treatment with tamoxifen given after mastectomy for primary breast cancer., Design: Cohort study using linkage between database of a randomised trial and statistics of Scottish hospital inpatients to identify episodes of cardiac and vascular morbidity., Setting: NHS hospitals in Scotland., Subjects: 1312 women who had undergone mastectomy for breast cancer and who were randomised either to a treatment group to receive adjuvant tamoxifen or to a control group to be given tamoxifen only on first relapse of disease. Maximum duration of tamoxifen treatment was 14 years. Total woman years of follow up were 9943., Main Outcome Measures: Randomised and observational comparisons of risk (expressed as hazard ratios) of myocardial infarction, other cardiac event, cerebrovascular disease, or thromboembolic event according to treatment allocated and between nonusers, former users, and current users of tamoxifen., Results: Use of tamoxifen was associated with lower rates of myocardial infarction. Hazard ratio for women in control group was 1.92 (95% confidence interval 0.99 to 3.73) compared with women allocated to adjuvant treatment. The association was stronger for current use: hazard ratio for non-users was 3.49 (1.52 to 8.03) compared with current users. Current users of tamoxifen, however, had higher rates of thromboembolic events:hazard ratio for non-users was 0.40 (0.18 to 0.90) compared with current users., Conclusions: Our results provide further evidence that tamoxifen reduces the risk of myocardial infarction. Thromboembolic events should be carefully monitored in trials of tamoxifen, particularly those of prophylactic treatment, in which tamoxifen is given to healthy women.

A sheep endometrial oxytocin receptor (OTR) cDNA (1.5 kb) was isolated from a lambda-ZAP library using a reverse transcription-PCR product probe generated from oestrous endometrial mRNA. The sheep OTR cDNA shared an overall similarity of 82% with human OTR cDNA, 85% with pig OTR cDNA and 76% with rat OTR cDNA. The encoded receptor was a 391 amino acid polypeptide 94% similar to human OTR, 94% similar to pig OTR and 93% similar to rat OTR. The sheep OTR contained two additional amino acids compared with human OTR which were located in the highly GC-rich third intracytoplasmic loop. This region is thought to be associated with G protein coupling and signal transduction. Expression of the cDNA in Cos-7 cells and measurement of oxytocin-induced phosphoinositide turnover confirmed that it coded for a functional product. The affinity of the expressed receptor was comparable with that observed for the in vivo receptor.

We have shown previously that growth-associated protein 43 (GAP-43) is expressed by rat Schwann cells and is restricted to non-myelin-forming Schwann cells in vivo. Here we examined the regulation of GAP-43 using agents that are known to control Schwann cell differentiation in vitro. GAP-43 protein and mRNA levels are decreased by forskolin and other agents that elevate intracellular cAMP (and promote expression of the myelinating Schwann cell phenotype). We also found that expression of GAP-43 protein but not mRNA is down-regulated by transforming growth factor betas (TGF-beta s). Moreover, TGF-beta treatment of Schwann cells results in cell clumping, process retraction and disappearance of GAP-43 from the plasma membrane, revealing that GAP-43 is associated with the Golgi apparatus. This association was confirmed by partial overlap of GAP-43 with the trans-Golgi network marker (23c) and the disruption of the Golgi with brefeldin A or monensin leading to altered GAP-43 distribution. Golgi-associated GAP-43 appeared to have the same molecular weight as the plasma membrane-associated GAP-43. Thus these results show that GAP-43 expression in Schwann cells is subject to regulation by both extracellular and intracellular signalling molecules and that Schwann cell GAP-43 is often associated with the Golgi apparatus.

In order to identify the transcription factors that may be involved in the development and differentiation of rat Schwann cells we examined the expression of c-Jun, Jun B, Jun D and the cAMP response element binding protein (CREB) in vivo and in vitro. We found that CREB was expressed at high levels throughout nerve development by both Schwann cells and their precursors. Jun family members, on the other hand, were expressed only at low levels in a few nuclei of the developing nerve. After sciatic nerve transection, however, c-Jun levels were rapidly up-regulated in many Schwann cells of the distal stump but CREB, Jun B and Jun D levels were not affected. When nerve contact was resumed after crush injury c-Jun levels returned to control values. Interestingly, unlike the situation in vivo, when Schwann cells were removed from the nerve and cultured, levels of all three Jun family members were rapidly up-regulated. This also occurred in Schwann cell precursors. In other experiments we found that Schwann cell c-Jun, but not Jun B or Jun D, expression was down-regulated by the adenylate cyclase activator, forskolin. In addition, we show that the forskolin induced down-regulation of c-Jun is not necessary for Schwann cell proliferation or myelination to occur.

The sheep endometrial oxytocin receptor plays a central role in determining the time at which luteolysis occurs during the oestrous cycle, and in the events leading to the establishment of pregnancy (the maternal recognition of pregnancy). Expression of the receptor in the uterus is controlled by ovarian steroid hormones, and by trophoblast interferon (IFN-tau). We report here studies on the second messengers involved in the effect of IFN-tau, and on the structure and expression of the oxytocin receptor. The receptor is expressed in ovine endometrial explants during culture, when the explants are taken during the luteal phase of the cycle; this process is partially blocked by inhibitors of protein kinase C, or by down-regulation of protein kinase C. Therefore it is suggested that protein kinase C, rather than tyrosine kinases, is involved in the effect of IFN-tau on oxytocin receptor expression. Northern blotting shows that in common with uterine oxytocin receptor mRNA in other species, the message is heterogeneous. cDNA sequencing indicates the sheep uterine oxytocin receptor is at least 2 amino acids longer than those of other species, and expression of the receptor in Cos-7 cells induces oxytocin responsiveness in terms of phosphoinositide turnover.

In a trial conducted in southeast Scotland between April 1974 and December 1979, 332 women with invasive breast cancer of Stage I and II with histological evidence of node involvement, or who had operable or inoperable Stage III disease, were randomized, after primary local therapy (mastectomy, node biopsy and radiotherapy for all except the inoperable disease patients who underwent radiotherapy alone) to receive 12 4-weekly intravenous injections of 5-fluorouracil (5-FU), 700 mg/m2 or no systemic therapy. After a median follow-up of 15 years from randomization, no difference is shown between the two groups in terms of distant relapse (hazard ratio (HR) = 1.02; 95% CI 0.78-1.32), event free survival (HR = 1.23; 95% CI 0.97-1.56), or total survival (HR = 1.19; 95% CI 0.93-1.52). Locoregional relapse is significantly reduced by 5-FU administration (HR = 1.88; 95% CI 1.20-2.96). The results are similar for the trial as a whole or when mastectomy patients are considered alone. Toxicity was minimal with marrow suppression in only 19 of 147 patients receiving more than one injection; only five patients discontinued therapy due to nausea and vomiting. However, retrosternal pain occurred in 16 patients, nine of whom had their treatment curtailed as a result. Seventy-seven per cent of patients have died, the majority from breast cancer. Only 1.2% of deaths are considered attributable to cardiac causes of 5-FU is not associated with excess cardiac deaths in these patients.(ABSTRACT TRUNCATED AT 250 WORDS)

This paper describes the results of a national survey on the non-surgical treatment of early breast cancer in the UK relating to the management of ductal carcinoma in-situ (DCIS), and the rationale for the current status of the UK DCIS trial. The survey shows that there is considerable clinical uncertainty, which can only be resolved by the trial.

The oxytocin receptor, a seven transmembrane domain, G protein-linked receptor molecule, plays a central role in determining the endocrine function of the ruminant uterine endometrium. During nonpregnant cycles the control of this molecule by circulating steroid hormones leads to regression of the corpora lutea. The kinetics of the mechanisms involved determine the time at which luteolysis occurs, and therefore the length of the oestrous cycle. In pregnancy, secretions of the trophoblast block endometrial oxytocin receptor gene expression and lead to luteal maintenance. An understanding of the molecular mechanisms involved in the steroidal control of oxytocin receptor gene expression will provide an explanation for the relative constancy of oestrous cycle lengths in non-pregnant animals. Unravelling the way in which trophoblast products block expression of the oxytocin receptor gene will lead to a better understanding of the reasons for the high rate of embryonic loss in domestic ruminants.

This report gives the responses of general surgeons in Scotland to two questionnaires. Satisfactory rates were obtained: 82% for the more detailed survey in 1988 and 62% for the second survey (1991), where nonrespondents were not followed up. In 1988 the rationale was the poor participation in the Scottish breast conservation trial while the 1991 survey further investigated the diversity of surgical opinion identified in the first. The limited trial support in 1988 was mainly due to reluctance to accept all treatment options. The majority were prepared to consider trial participation although 47% believed this compromised doctor-patient relationships. Although breast-conserving therapy was widely supported, many different views on management were encountered, both in the degree of influence of specific factors and in the decisions taken in relation to them. We believe these surveys have re-inforced a need for management guidelines, particularly as around half the patients with symptomatic breast cancer were being treated in non-specialist units.

The trophoblast of the developing ruminant conceptus secretes large quantities (up to 100 micrograms, or 10(7) IU, per 24 h) of a number of characteristic Type I interferons. Secretion (gene expression) commences at Day 8 or 9 (in the sheep; Day 10 in cattle) and ends at about Day 22 (Day 25). The function of this material is in the inhibition of uterine prostaglandin secretion, and hence in the maintenance of the corpus luteum (the 'maternal recognition of pregnancy'). Such rapid onset and cessation of gene transcription begs questions about the function of trophoblast interferon gene promoters, and a limited number of studies have now been carried out with reporter gene constructs, with inconclusive results. It seems unlikely, however, that viral responsiveness accounts for the phenomenon, as in adult interferon genes, although the trophoblast interferons are highly active antiviral agents.

A synthetic 45-mer oligonucleotide corresponding to part of the ovine endometrial oxytocin receptor cDNA was hybridized to sections of ovine uterus collected from 40 ewes at different stages during the oestrous cycle, the first 3 weeks of pregnancy and seasonal anoestrus. The quantity of oxytocin receptor mRNA was measured as the optical density (OD) value on autoradiographs using image analysis. Message first appeared in the luminal epithelium on days 14-15 of the cycle, increasing to a peak OD of 0.48 at oestrus and decreasing again between days 2 and 5. Oxytocin receptor mRNA in the superficial glands, deep glands and caruncular stroma increased between day 15 and oestrous to peak OD values of 0.17, 0.11 and 0.11 respectively, declining again by day 2 and reaching basal values (OD < 0.015) by day 5. Hybridization to the myometrium tended to rise from a mean OD value of 0.01 on days 2-15 to a peak of 0.03 +/- 0.01 (mean +/- S.E.M.) on days 0-1, but the change was not significant. In pregnant ewes there was no detectable oxytocin receptor mRNA on days 14-15 in any region, but hybridization to the luminal epithelium was present in two of three ewes on day 21. In anoestrous ewes oxytocin receptor mRNA concentrations in all areas of the endometrium were approximately half those measured at oestrus. Optical density readings for oxytocin receptor mRNA in the various uterine compartments were compared with measurements of oxytocin receptors in the same regions as assessed by binding studies using the 125I-labelled oxytocin antagonist d(CH2)5[Tyr(Me)2,Thr4,Tyr-NH2(9)]-vasotocin (125I-labelled OTA). In the endometrium, receptor mRNA and 125I-labelled OTA binding patterns changed in parallel, and both sets of measurements were significantly correlated (P < 0.01). In the myometrium, a significant increase in 125I-labelled OTA binding occurred at oestrus; this was not accompanied by a similar increase in oxytocin receptor mRNA hybridization. This study helps to confirm that the previously identified cDNA clone is derived from the ovine oxytocin receptor, as patterns of oxytocin receptor mRNA expression in the endometrium closely resembled those of oxytocin binding. Maximum expression and binding both occurred at oestrus, suggesting that regulation of the oxytocin receptor gene in the uterus occurs principally at the transcriptional, rather than at the translational, level. Failure to detect a significant increase in myometrial mRNA expression at oestrus may indicate that the endometrial and myometrial oxytocin receptors are of different isoforms.

We have cloned a 2.4-kb cDNA containing the complete coding sequence of ovine Mx from a lambda ZAP library constructed using RNA from the endometrium of a normal sheep on day 16 of pregnancy. Ovine Mx shows 80% similarity to human MxA. Human and mouse Mx are type-1 interferon (IFN)-induced genes that have previously been shown to confer resistance to influenza virus. The ovine Mx cDNA contains an open reading frame of 1962 nucleotides (nt) coding for a 653-amino-acid (aa) protein. The deduced translated sequence has consensus GTP-binding sites and similarity to the human MxA sequence (RKFLKERLARL) that has been shown to be essential for resistance against vesicular stomatitis virus (VSV). The presence of Mx mRNA was investigated by Northern blot analysis in the endometrium of non-pregnant sheep and between days 8 and 127 of pregnancy. Mx expression was detected at high levels between day 13 and day 20 of pregnancy. Furthermore, eightfold higher levels of Mx mRNA were detected in the pregnant versus the non-pregnant uterine horn in unilaterally pregnant sheep. Mx mRNA can be induced in sheep endometrium by ovine trophoblast interferon (IFN-tau).

During the development of the rat sciatic nerve extensive proliferation of glial cells occurs, and there is a very substantial rearrangement of the cytoarchitecture as axons and Schwann cells assume relationships which lead to the formation of the myelinated and unmyelinated axons characteristic of adult nerve. The maturation of Schwann cells from Schwann cell precursors and the matching of Schwann cell numbers to axons is an important part of this process. We have therefore studied the proliferation of Schwann cell precursors and Schwann cells during the development of the rat sciatic nerve from embryonic day 14 to postnatal day 28 by combining bromodeoxyuridine injections of rats with double-label immunohistochemical techniques. The results reveal that DNA synthesis occurs in both Schwann cell precursors and Schwann cells throughout early nerve development. The labelling index is already substantial at embryonic day 14, but from embryonic day 17, when essentially all the glial cells have converted from precursor to Schwann cell phenotype, it rises sharply, peaking between embryonic day 19 and 20 before declining precipitously in the early postnatal period. This rapid decline in DNA synthesis coincides with the appearance of the myelin protein P0, and in individual cells DNA synthesis is incompatible with the expression of P0 protein. Nonmyelin-forming Schwann cells, which mature later in development, continue to synthesize DNA until at least postnatal day 15, but by day 28 essentially all Schwann cells in the nerve are quiescent.

The polymerase chain reaction (PCR) was used to generate a 131 bp cDNA encoding part of the sheep endometrial oxytocin receptor. The nucleotide sequence of this cDNA was 93.8% identical to the human oxytocin receptor sequence in this region. When used to probe Northern blots of sheep endometrial RNA the PCR product identified a 6.7 kb mRNA which appeared and disappeared during the oestrous cycle in parallel with the oxytocin receptor molecule as measured by ligand binding. The sheep endometrial receptor mRNA was significantly larger than the human myometrial mRNA (4.7 kb). It is suggested that the cloned cDNA described here is an appropriate probe for use where it is required to measure sheep oxytocin receptor mRNA.

Recently it has been demonstrated that the growth-associated protein GAP-43 is not confined to neurons but is also expressed by certain central nervous system glial cells in tissue culture and in vivo. This study has extended these observations to the major class of glial cells in the peripheral nervous system, Schwann cells. Using immunohistochemical techniques, we show that GAP-43 immunoreactivity is present in Schwann cell precursors and in mature non-myelin-forming Schwann cells both in vitro and in vivo. This immunoreactivity is shown by Western blotting to be a membrane-associated protein that comigrates with purified central nervous system GAP-43. Furthermore, metabolic labeling experiments demonstrate definitively that Schwann cells in culture can synthesize GAP-43. Mature myelin-forming Schwann cells do not express GAP-43 but when Schwann cells are removed from axonal contact in vivo by nerve transection GAP-43 expression is upregulated in nearly all Schwann cells of the distal stump by 4 wk after denervation. In contrast, in cultured Schwann cells GAP-43 is not rapidly upregulated in cells that have been making myelin in vivo. Thus the regulation of GAP-43 appears to be complex and different from that of other proteins associated with nonmyelin-forming Schwann cells such as N-CAM, glial fibrillary acidic protein, A5E3, and nerve growth factor receptor, which are rapidly upregulated in myelin-forming cells after loss of axonal contact. These observations suggest that GAP-43 may play a more general role in the nervous system than previously supposed.