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The effect of combinations of a mammalian target of rapamycin (mTOR) inhibitor, temsirolimus, and an estrogen receptor-α (ERα) antagonist, ERA-923, on breast carcinoma in culture and in a xenograft model has been studied. Phase III trials are underway using temsirolimus for several cancers. ERA-923 was studied in a phase I trial for tamoxifen refractory metastatic breast cancer and was shown to have good safety profiles. Combination of noninhibitory doses of temsirolimus with suboptimal doses of ERA-923 synergistically inhibited the growth of MCF-7 cells. Synergy was found across a wide range of doses and could also be achieved by combining temsirolimus with other antiestrogens such as raloxifene and 4-hydroxytamoxifen. In vivocombination of temsirolimus and ERA-923 at certain doses and schedules completely inhibited tumor growth, while individual agents were only partially effective. Although the mechanism underlying the synergism remains to be understood, the results were associated with the ability of temsirolimus to block the transcriptional activity mediated by ERα as well as an increase in G1 arrest when it was combined with ERA-923. Results demonstrated for the first time that the combination of temsirolimus and a pure antiestrogen has excellent anticancer activity in preclinical models and, therefore, may have clinical use in treating hormone-dependent tumors.

Choline is an essential nutrient in methylation, acetylcholine and phospholipid biosynthesis, and in cell signaling. The demand by an embryo or fetus for choline may place a pregnant woman and, subsequently, the developing conceptus at risk for choline deficiency. To determine whether a disruption in choline uptake and metabolism results in developmental abnormalities, early somite staged mouse embryos were exposed in vitro to either an inhibitor of choline uptake and metabolism, 2-dimethylaminoethanol (DMAE), or an inhibitor of phosphatidylcholine synthesis, 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3). Cell death following inhibitor exposure was investigated with LysoTracker Red and histology. Embryos exposed to 250–750 μM DMAE for 26 hr developed craniofacial hypoplasia and open neural tube defects in the forebrain, midbrain, and hindbrain regions. Embryos exposed to 125–275 μM ET-18-OCH3 exhibited similar defects or expansion of the brain vesicles. ET-18-OCH3-affected embryos also had a distended neural tube at the posterior neuropore. Embryonic growth was reduced in embryos treated with either DMAE (375, 500, and 750 μM) or ET-18-OCH3 (200 and 275 μM). Whole mount staining with LysoTracker Red and histological sections showed increased areas of cell death in embryos treated with 275 μM ET-18-OCH3 for 6 hr, but there was no evidence of cell death in DMAE-exposed embryos. Inhibition of choline uptake and metabolism during neurulation results in growth retardation and developmental defects that affect the neural tube and face. Teratology 64:114–122, 2001. © 2001 Wiley-Liss, Inc.

beta-catenin and plakoglobin are members of the armadillo family of proteins and were first identified as components of intercellular adhering junctions. In the adherens junction beta-catenin and plakoglobin serve to link classical cadherins to the actin-based cytoskeleton. In the desmosome plakoglobin links the desmosomal cadherins, the desmogleins and the desmocollins, to the intermediate filament cytoskeleton. beta-catenin is not a component of the desmosome. Previously we have shown that the central armadillo repeat region of plakoglobin is the site for desmosomal cadherin binding. We hypothesized that the unique amino- and/or carboxyl-terminal ends of beta-catenin may regulate its exclusion from the desmosomal plaque. To test this hypothesis we used chimeras between beta-catenin and plakoglobin to identify domain(s) that modulate association with desmoglein 2. Chimeric constructs, each capable of associating with classical cadherins, were assayed for association with the desmosomal cadherin desmoglein 2. Addition of either the N- or C-terminal tail of beta-catenin to the armadillo repeats of plakoglobin did not interfere with desmoglein 2 association. However, when both beta-catenin amino terminus and carboxyl terminus were added to the plakoglobin armadillo repeats, association with desmoglein 2 was diminished. Removal of the first 26 amino acids from this construct restored association. We show evidence for direct protein-protein interactions between the amino- and carboxyl-terminal tails of beta-catenin and propose that a sequence in the first 26 amino acids of beta-catenin along with its carboxyl-terminal tail decrease its affinity for desmoglein and prevent its inclusion in the desmosome.

Background:Reported sexual assault is increasing, and the diverse immediate and longer term needs of the victim are usually met by exposure to a number of healthcare professionals often in different locations, involving delays and travel, increasing the trauma for the victim. Objectives:To set up a centre to address the immediate and longer term needs of the sexual assault victim and review issues arising during the development of the service.Methods:Description of setting up the service in the genitourinary medicine department of Kings College Hospital, south London, and the aspects of care offered.Results:The number of victims referred by police increased from 15 in 1992 to 58 in 1996. In 1996, 55 female and three male victims were seen. 23 different police stations brought victims for examination; mean age of the victim was 27 years (range 14-60), median time between assault and examination was 22 hours (range 3 hours-3 months); 23% had genital injuries, 59% had other physical injury, and 11% needed further hospital care. 71% accepted screening for sexually transmitted infection (STI), 21% had an STI diagnosed, 16% of the women required emergency contraception, 26% received prophylactic antibiotics, and 58% saw a health adviser. 70% had a follow up appointment arranged of which 50% attended.Conclusion:The high uptake of STI screening, emergency contraception, health adviser consultation, and follow up supports the concept of a comprehensive integrated system to meet the disparate needs of the victim while still obtaining the necessary forensic evidence. The wide catchment area of service users indicates gaps in services available for the assault victim. Earlier genitourinary involvement after sexual assault is becoming increasingly pertinent in relation to HIV prophylaxis.

This study describes the timecourse of expression of low-affinity serotonin uptake sites in the developing craniofacial region of the mouse embryo. Whole mouse embryos were incubated in the presence of various serotonergic compounds followed by immunocyto-chemical localization of serotonin (5-HT) and its binding protein. In the gestational day 9 embryo (3–5 somites), 5-HT uptake was observed in the myocardium of the heart, the visceral yolk sac and foregut. A specific and transient pattern of 5-HT uptake was observed in the hindbrain neuroepithelium from day 9.5–11, where it was localized in rhombomeres 2–5 in the day 9.5 embryo. By day 10, when rhombomeres were no longer evident, uptake was present in the dorso-lateral neuroepithelium surrounding the fourth ventricle (rhombic lip; cerebellar anlage). Uptake of 5-HT was initially observed in the surface epithelium of the craniofacial region at day 10 (20–25 somites) and was greatly increased at day 11. The invaginating lens, nasal placode epithelium and otocyst also took up 5-HT at day 11. During these stages a 45 kD serotonin-binding protein (SBP) was expressed in craniofacial mesenchyme, and became progressively restricted to regions subjacent to epithelial uptake sites. These staining patterns were shown to be specific for 5-HT and SBP by their absence in embryos stained using preabsorbed antisera. The timecourse of these patterns are correlated with critical events in craniofacial morphogenesis including (1) onset of inductive epithelial-mesenchymal interactions, (2) invagination and fusion of placodal structures, (3) presence of rhombomeres, and (4) regions of low proliferative activity.

Two cell-cell junctions, the adherens junction and the desmosome, are prominent in epithelial cells. These junctions are composed of transmembrane cadherins which interact with cytoplasmic proteins that serve to link the cadherin to the cytoskeleton. One component of both adherens junctions and desmosomes is plakoglobin. In the adherens junction plakoglobin interacts with both the classical cadherin and with alpha-catenin. Alpha-catenin in turn interacts with microfilaments. The role plakoglobin plays in the desmosome is not well understood. Plakoglobin interacts with the desmosomal cadherins, but how and if this mediates interactions with the intermediate filament cytoskeleton is not known. Here we compare the domains of plakoglobin that allow it to associate with the desmosomal cadherins with those involved in interactions with the classical cadherins. We show that three sites on plakoglobin are involved in associations with the desmosomal cadherins. A domain near the N terminus is unique to the desmosomal cadherins and overlaps with the site that interacts with alpha-catenin, suggesting that there may be competition between alpha-catenin and the desmosomal cadherins for interactions with plakoglobin. In addition, a central domain is shared with regions used by plakoglobin to associate with the classical cadherins. Finally, a domain near the C terminus is shown to strongly modulate the interactions with the desmosomal cadherins. This latter domain also contributes to the association of plakoglobin with the classical cadherins.

The effects of operation (lower-limb amputation) on the growth of the Lewis lung tumour and its metastases were studied. The role of C. parvum in counteracting these effects was investigated. Anaesthesia alone or with amputation did not affect primary tumour growth. C. parvum depressed this growth. Anaesthesia did not affect the number of pulmonary metastases, but amputation caused a significant increase. C. parvum inhibited metastases and completely counteracted the effects of operation on them. Large doses of cortisone acetate significantly increased metastases but small doses had no effect. Experiments with adrenalectomized mice suggested the effects of operation were due to non-specific stress.

The level of peripheral blood platelets was determined after i.v. injection of Corynebacterium parvum in normal C57BL mice and in those bearing the Lewis lung carcinoma. Twenty minutes after injection of a formalin-killed active strain (CN6134, (CN6134, which inhibited tumour metastases) or a killed inactive strain (CN 5888, which did not inhibit metastases) the number of circulating blood platelets was reduced by 50%. The level of platelets returned to control values by 8 h after the active, and by approximately 3 days after the inactive strain. The active strain alone caused a second and prolonged fall in platelet numbers, from approximately 16 h to 21 days after injection. Heparin given 3 X weekly to these mice restored the platelet count to normal values by 10 days after injection of active-strain C. parvum. The level of platelets in tumour-bearing mice was essentially similar to that in normal mice. Possible causes of the thrombocytopenia and the significance of platelets in metastasis are discussed.

An investigation was made into the effect of Corynebacterium parvum therapy on cerebral tumours in mice. I.v. C. parvum caused a slight but significant increase in the survival of BALB/c mice injected intracerebrally (i.c.) with not more than 50 Meth A cells. C. parvum was most effective if given on the same day or 5 days after tumour. If this interval was increased there was no effect. Multiple i.v. injections were no more effective than a single dose. I.v. C. parvum had no influence on the survival of C57BL mice injected i.c. with Lewis tumour cells, and had little effect on the induction of i.c. or s.c. tumours by methylcholanthrene. It was concluded that C. parvum therapy was of little use in the treatment of cerebral tumour in mice. The clinical implications of these findings are discussed.

The murine dose of i.v. C. parvum (466 microgram) was compared with a single, low, human-equivalent dose of 70 microgram and with repeated weekly low doses. All treatments increased the antibody titre against C. parvum (CP). However, repeated doses stimulated a much higher titre than single doses. In all treated animals spleen weight peaked at 2 weeks and then fell. A single low dose caused a 3-fold increase, a single high dose or multiple low doses a 6-fold increase. Liver weight changes followed a similar pattern. Hepatosplenomegaly was prolonged by multiple doses. The effects of these treatments on Lewis tumour metastases were studied. A single high dose and a single low dose on the day of tumour implantation (Day 0) were equally effective at inhibiting pulmonary metastases. Repeated low doses starting on Day 0 were no more effective than a single dose. The effect of CP on survival after primary-tumour excision on Day 10 was observed. Low dose CP on Day 7 doubled the harmonic mean of survival time. Repeated doses were no more effective than a single dose. Low-dose prophylaxis up to 2 weeks before tumour significantly inhibited metastases. However, when repeated low-dose prophylaxis was combined with a single low dose on Day 0, the anti-metastatic effect was abrogated. This neutralization of the anti-metastatic effect of CP given on Day 0 was found to persist after a 13-week treatment-free interval. Possible mechanisms for this phenomenon are discussed.

An actin-myosin complex located in apical regions of the neurectoderm has been postulated to play a role in neurulation. Numerous studies have documented the presence of microfilaments in this area and confirmed their composition as actin. By necessity, if such a contractile system is to exert a force, these filaments must be anchored in some way to the cell membrane. In this study, the presence of the actin-binding protein, spectrin (fodrin), is demonstrated in the neurectoderm of neurulating mouse embryos using antispectrin antibodies and indirect immunofluorescent techniques. The patterns of spectrin localization correlate with the previously reported regions of increased numbers of microfilaments and also with the morphology of the neural folds. Thus, during the initial stages of cranial fold elevation, a process reportedly dependent on increased glycosaminoglycan synthesis, little spectrin is present in the neuroepithelial cells. Later as the folds begin to converge toward the midline, deposition of the protein, as demonstrated by the intensity of fluorescence, is increased in the apices of these cells, and is most prominent in regions of greatest bending in the neural folds. Caudal neural fold regions show a similar pattern of staining. Thus, the hypothesis that a cytoskeletal system assists in neurulation is supported by these results, which for the first time demonstrate the presence of a putative actin-membrane attachment protein in a morphogenetically active system.

A comparison between static versus rotator culture systems and a variety of media (rat serum, new born calf serum, DMEM and Waymouth’s) was made in an attempt to promote in vitro growth of mouse embryos from the beginning of neurulation (headfold stage) to the closure of the neural tube and formation of the limb buds (48 h). The results demonstrate that good development can be achieved for 48 h using a rotator system and that 80 % of embryos cultured on rotators show growth and differentiation similar to that obtained for the same time period in vivo. Static cultures are less successful and embryos grown in this system show lower protein content and somite numbers than those maintained on rotators. Undiluted rat serum is superior to all other media tested and supports better growth and development as monitored by total protein and developmental abnormalities.

During the last twenty years, the UK has made steady progress in the understanding of the ‘lead in drinking water’issue and towards compliance with a lead standard which is more stringent than elsewhere in Europe.

Insects of the genus Wiseana(Lepidoptera: Hepialidae) are major agricultural pests in New Zealand. Singly enveloped nucleopolyhedroviruses (SNPVs) isolated from three of the seven described Wiseanaspecies have potential as biological control agents. As part of an effort to characterise the WiseanaSNPV genome the polyhedrin gene was cloned and the nucleotide sequence determined. The gene sequence was used, in conjunction with morphological and restriction endonuclease analysis, to compare isolates from different sites and species of Wiseana. Heterogeneity was detected within a single site, as well as between SNPV from separate Wiseanaspecies. The extent of divergence between the nucleotide sequences was small enough, however, to consider three SNPVs from W. signata, W. cervinataand W. umbraculataas different strains of a single SNPV species. This improves the likely practicability of developing a single viral agent to control this pest complex. In addition, the virus polyhedrin gene sequence was used to estimate the phylogenetic relatedness of a W. signataSNPV to 16 other NPV from diverse insect genera. These comparisons suggest the WiseanaSNPV was unique within the Baculoviridae, but was more closely related to the group II NPVs.

Corynebacterium parvum was labelled by growing live bacteria in the presence of [3H]thymidine. The bacteria were killed by formalin, washed thoroughly and resuspended at a concentration of 7 mg dry weight/ml. An activity of 1-6 X 10(5) ct/min/0-1 ml was obtained. The biological properties (inhibition of tumour growth and hepatosplenomegaly) of the labelled C. parvum were compared with those of commercially available vaccine, and were found to be similar. Labelled C. parvum was injected i.v., i.p., or s.c. into normal C57BL mice and the localization of activity determined at 4 h and 1,3,7 and 14 days after injection. After i.v. or i.p. injection, highest counts were recorded in the liver. Moderate activity was found in the spleen, lungs and small gut. After s.c. injection, the majority of radioactive label was detected at the site of injection and little found in other tissues. The distribution of injected C. parvum was also studied in mice bearing Lewis tumour, and was found to be similar to that in normal mice. Moderate amounts of labelled C. parvum were recovered from tumour. There appeared to be no relationship between the antitumour effect of C. parvum given by a particular route of injection and the concentration of C. parvum recovered from the tumour.

In mice, i.v. C. parvum induces intravascular coagulation. This is a prolonged reaction lasting up to 7 days. It results in thrombosis in hepatic vessels with consequent hepatic necrosis, and thrombosis in pulmonary and splenic vessels. This may be important in the assessment of the tumour-inhibitory activity of C. parvum.

Previous studies have shown that the ketone body D,L,-beta-hydroxybutyrate was teratogenic to mouse embryos exposed in culture during the period of neurulation. Inhibition of closure of the cranial and caudal neuropores was the most frequently occurring defect and these abnormalities were thought to be the forerunner of anencephaly and spina bifida, respectively. However, additional studies demonstrated that embryos could recover morphologically from these effects if the ketone body was removed from the culture medium and if the recovery period was of sufficient duration. In an attempt to define further the phenomenon responsible for this recovery and to determine the extent of the recovery process, the present study examining the cross-sectional area, cell number, and mitotic index of cranial neuroepithelial cells was conducted in mouse embryos cultured from the early somite stage under one of the following conditions: 1) control medium for 60 h; 2) medium containing 32 mmol/1 D,L,-beta-hydroxybutyrate for 24 h followed by culture in control medium for an additional 36 h (recovery group); 3) medium containing 32 mmol/1 D,L,-beta-hydroxybutyrate for 60 h (continuously exposed group). The results indicate that although neural tube closure occurred in the recovery group, complete recovery was limited to the ventral regions of the forebrain and that the remainder of the prosencephalon as well as the rhombencephalon failed to undergo complete catch-up growth. Thus, cell numbers in these areas were approximately 70% of control values. Therefore, while the gross anatomical disturbances produced by the ketone body may be compensated for, histological alterations in the affected tissues remain. Ultimately, these data suggest that neurological deficits may be an outcome of ketone body exposure during the early stages of embryogenesis.

The possible involvement of the neurotransmitter serotonin (5-HT) in morphogenesis of the craniofacial region in the mouse embryo has been investigated using the method of whole-embryo culture. Day-12 embryos were incubated for 3–4 h in the presence of 5-HT or its precursors L-tryptophan (L-TRP) or 5-hydroxytryptophan (5-HTP), followed by fixation, sectioning and staining with a specific antiserum to 5-HT. Sites of 5-HT immunoreactivity were found in a variety of locations in tissues of the head and neck, which are either epithelia derived from the non-neural ectoderm or are non-neuronal midline brain structures. These sites include the surface epithelia of the head, face, nasal prominences, branchial arches, oral cavity and associated parts of the nasal epithelium, the epithelium covering the eye, parts of the otic vesicle, the epiphysis and roof of the diencephalon. With the exception of the oral cavity, sites of immunoreactivity for serotonin-binding protein were identified in the mesenchyme adjacent to these sites. This mesenchyme consists of ectodermally derived neural crest cells, which are known to receive inductive influences from the epithelia with which they interact during their migration through the craniofacial region. The presence of 5-HT uptake sites in epithelia and adjacent sites of SBP in the underlying mesenchyme raises the possibility that 5-HT might be involved in those epithelial-mesenchymal interactions known to be important for the development of structures in the craniofacial region.

Early somite (2—4) mouse embryos were explanted and then maintained in culture for 24 or 48 h intervals. Various types of media were tested and it was determined that rat serum supported normal growth over a period of 48 h, based on total protein analysis and histological comparisons with in vivo specimens. Other media including fetal calf serum and fetal calf serum + Waymouth's (1:1) supported some growth, but did not equal the success of using rat serum alone. During the 48 h culture period in rat serum, embryos developed to stages indistinguishable from embryos maintained for a similar time in vivo.

The effect of chlorambucil on the rates of DNA, RNA, and protein synthesis in mouse embryos was investigated using a system of whole embryo culture. Embryos were isolated on the 11th day of gestation (33 ± 3 somites) and grown in culture media for periods of 4–8 h. Reichert's membrane and most of the placental tissue was removed leaving only the amnion and visceral yolk-sac surrounding the embryo. In the presence of teratogenic doses of chlorabucil (15 μg/ml) the rate of DNA synthesis was significantly decreased at 4 and 8 h. RNA and protein synthesis were not inhibited at either of these times. A trend toward decreasing rates of protein synthesis at some time beyond 8 h was noted, but not tested.

The pancreatic regenerating (reg) gene is proposed to be involved in pancreatic β-cell growth. Up- or down-regulation of reg gene expression has been shown to parallel variations in β-cell mass and function in the adult pancreas. In several species at least two nonallelic reg genes have been identified. In this study we investigated the expression of each individual reg gene (reg-I and reg-II) during embryogenesis in the mouse. Single mouse embryos were harvested at 8·5, 9, 10, and 12 days of development, homogenized and subjected individually to reverse transcription (RT)-PCR, with a single primer pair to amplify both reg-I and -II mRNAs. Southern blot analysis of the RT-PCR products revealed the presence of reg mRNA at day 9 of embryogenesis, just before the beginning of pancreatic organogenesis. Slot-blot analysis with internal oligonucleotide probes that specifically recognize reg-I or -II sequences demonstrated that only reg-I mRNA was present in day 9 and day 10 prepancreatic embryos. Reg-II mRNA was not detected until day 12, a stage corresponding to late organogenesis. RT-PCR for insulin mRNA from the same samples used for the amplification of reg mRNA showed that the earliest insulin expression occurred at day 8·5, and coincided with the onset of reg-I expression. Hybridization with gene-specific oligonucleotide probes revealed that only insulin-II mRNA was detectable at this time. Insulin-I mRNA was not detectable until day 12 and coincided with early reg-II expression. These results suggest that the two nonallelic reg genes and the two insulin genes are expressed differentially during early embryogenesis. Differential expression of reg-I and -II suggests that they may be induced by different and independent stimuli and have distinct functions.

Leukaemic cells from rats with a lymphoid (HRL) or myeloid (SAL) leukaemia were labelled with 125IUDR and injected i.v. into either normal or leukaemic syngeneic recipients. The fate of the injected cells was studied in terms of the radioactivity in various tissues at various times up to 24 h later. In normal animals the leukaemia cells were destroyed rapidly in the reticulo-endothelial (RE) system; immediately after injection most recoverable activity was in the lung, with smaller amounts in the blood, spleen and liver but by 24 h only 20-30% of the injected activity could be recovered. In leukaemic recipients with high numbers of blasts in the blood the amount of activity recoverable from the lungs and bone-marrow was markedly reduced, while that in the blood was doubled. Nonetheless, the overall rate at which radioactivity was eliminated was not significantly different from that found in normal rats, in spite of the fact that the RE system was extensively infiltrated by leukaemia cells.

Cocaine use has been associated with adverse developmental effects in humans. However, clinical reports both confirm and deny an association between cocaine use and malformations. Similarly, differences in species and strain, as well as route and timing of cocaine administration, have added to the difficulties in determining the teratogenicity of cocaine in animal models. This study was undertaken to compare the effects of dose, route, and timing of cocaine administration in ICR mice during early organogenesis. A single intraperitoneal (ip) administration of cocaine (≥60 mg/kg) on Day 9 of gestation (plug day = 1) produced maternal lethality. The predominant developmental effect of cocaine administration was an increase in the percentage of litters exhibiting an enlarged renal pelvis. Despite a high incidence of affected pups at these doses, the enlargement was not severe. These results, in agreement with previous reports, provide further evidence that the developing urogenital system is sensitive to cocaine administration. When cocaine was administered using a subcutaneous route, pup weights were greater and the incidence of enlarged renal pelvis was lower than when an ip route was used. To better mimic human binge cocaine abuse, the toxicity of a “split dose” was determined. A 60 mg/kg dose was administered using one administration of 60 mg/kg, two treatments of 30 mg/kg, or three administrations of 20 mg/kg with 1 hr separating the treatments. The incidence of enlarged renal pelvis was similar when cocaine was administered as one or two but was decreased when cocaine was administered as three treatments. Both the route and split-dose studies suggest that high-peak serum concentrations are required to perturb development. There were no differences in the incidence or severity of enlarged renal pelvis when cocaine was administered on Day 8, 9, or 10 or on all 3 days of gestation. This suggested that the increase in enlarged renal pelvis may not be a specific teratogenic effect of cocaine administration but may be a delay of normal development induced by cocaine exposure during this early period of organogenesis. To address this hypothesis, cocaine was administered on Day 9 using an ip route and the pups were allowed to be naturally born. In pups whose mothers received cocaine there was an increase in postnatal deaths and a trend toward a reduction in pup body weight/litter at Postnatal Day 21. However, when renal morphology was assessed on Postnatal Day 21 no abnormal kidneys were seen. This supports the hypothesis that enlarged renal pelvis produced by cocaine administration during early organogenesis represents a developmental delay and not a persistent teratogenic defect. These studies suggest that high peak cocaine concentrations are required to delay normal kidney morphogenesis in mice.