During pregnancy in rats, a state of hypothalamic leptin resistance develops that facilitates the hyperphagia characteristic of pregnancy. Pseudopregnant rats are also hyperphagic but have a normal hypothalamic response to leptin. These animals have an identical hormone profile to the first half of pregnancy, but do not form a placenta, allowing the effects of hormones of maternal origin to be distinguished from placental hormones. The aim of this experiment was to examine whether extending pseudopregnancy (PSP), to provide a more prolonged exposure to pregnancy-like maternal hormones, is able to induce a loss of leptin responsiveness in the brain, as occurs during pregnancy.

Sprague-Dawley rats were mated with intact or vasectomised males. Intracerebroventricular (icv) cannulae were implanted 1-3 days later and on day 9 pseudopregnant rats received three blank silastic implants (40 mm long) or implants containing progesterone. Blank-implanted rats resumed estrous cyclicity by approximately day 12, whereas progesterone treatment extended PSP beyond 18 days. On day 13 of pregnancy and extended PSP, rats were fasted for 24 hours and then injected icv with leptin (4 μg in 2 μl) or vehicle. Food intake was measured 24 hours later. Leptin significantly suppressed post-fasting food intake in both sham and progesterone-treated PSP rats compared with vehicle-injected rats (17.0 ± 3.01 vs 24.9 ± 1.51 g (p < 0.001, n = 11), and 24.7 ± 1.19 vs 29.2 ± 1.17 g (p < 0.05, n = 16), respectively). There was no significant difference in food intake between pregnant rats given vehicle (27.11 ± 1.82 g, n = 8) or leptin (24.1 ± 1.34 g, n = 8), demonstrating central leptin resistance in pregnant rats.

During extended PSP there is an increase in food intake, as has previously been observed during pregnancy. These animals responded to central leptin, in contrast to pregnant rats, suggesting that placental-derived hormones may contribute to the changing hypothalamic response to leptin observed during pregnancy.

Routine adult-infant bedsharing remains controversial as some situations increase the risk of Sudden Infant Death Syndrome. Higher rectal temperatures in bedsharing infants may contribute to the risk. We investigated infants in the natural setting of their own home to compare thermal characteristics of bedsharing and cot-sleeping situations and the potential hazards.

Overnight video and physiological data was collected in family homes from 40 healthy, routine bedsharing infants (5-27 weeks) and 40 routine cot-sleeping infants matched for age and season of study. Physiological recordings included infant rectal, shin and room temperature. Overnight mean rectal, shin and room temperatures were calculated for each 30 minute epoch. Effective thermal insulation for the bedding and clothing of each infant was calculated by using the average measured thickness of typical New Zealand bedding and clothing of similar types. A log of infant sleep positions, the number of episodes of face covering and awakenings, total awake time, number of feeding sessions and infant movements were also recorded.

The mean rectal temperature 2 hours after sleep onset for bedshare infants was 36.79°C and for cot-sleeping infants, 36.75oC [difference 0.05°C (95% CI: -0.03 to 0.14)]. The rate of change thereafter was higher in the bedshare group than the cot group [0.04°C vs 0.03°C/h (difference 0.01, 0.00 to 0.02)]. Bedshare infants had a higher shin temperature at 2 hours [36.41 vs 35.59°C (difference 0.82, 0.15 to 1.48)] and a higher rate of change [0.04 vs -0.01°C/h (difference 0.13, 0.08 to 0.19)]. Bedsharing infants had more bedding. Face covering events were more common and bedshare infants woke and fed more frequently than cot infants (mean wake times/night: 4.6 vs 2.5).

Healthy bedshare infants at home experience warmer thermal conditions than those of cot sleeping infants but they thermoregulate adequately to maintain normal core temperature.

Attempts have been made to create conditionally replicating adenoviruses (Ads) that selectively replicate in tumour cells. A number of the strategies employed have focused on the finding that > 50% of human tumours lack the p53 protein and utilise p53 deficiency as criteria for selectivity, but this approach has had limited success.

We have demonstrated that in the absence of p53, Ad infection leads to an increase in expression of p53-responsive genes. This indicates an Ad protein can mimic some of p53’s roles. We have explored the effects of Ad E1a on p53-target genes in the presence and absence of the Retinoblastoma protein (Rb). E1a expression plasmids were introduced into p53 null cells along with promoter reporter constructs of various p53-target genes, e.g. BAX, and target gene activation was measured.

We found that E1a can induce p53-target genes in the presence of Rb, suggesting that E1a can mimic some of p53’s functions. However no induction of these genes was observed in cells that have a mutant Rb. Furthermore, transfecting Rb back into these cells restored activation. This shows that Rb is required for E1a activation of p53-regulated genes. Subsequent bioinformatic analysis revealed that the transcription factor, Sp1, is common to all p53-responsive promoters tested. Additionally Sp1 binds to E1a, implicating its involvement in transcriptional activation of these promoters. Furthermore, mobility shift assays showed an increase in Sp1 bound to the BAX promoter in the presence of E1a.

Thus, E1a and Rb can co-operate with Sp1 to activate p53-target genes and may fulfill some of p53’s roles in p53 mutant or null cancer cells. This evidence may go some of the way to explain the limited success of attempts to target p53 deficiency as criteria for selectivity of conditionally replicating Ads.

Neonatal hypoxia-ischemia (HI) is a major cause of cerebral palsy. Neuronal cell death in the striatum of the brain contributes to the motor deficits of cerebral palsy. In our earlier study, combined antioxidant-hypothermia treatment significantly protected striatal neurons one week after neonatal HI in the rat. This study investigated the long-term efficacy of this combination.

To investigate long-term neuroprotection, male Sprague-Dawley rats received six, 12-hourly subcutaneous injections of the antioxidant S-PBN (N-tert-butyl-(2-sulfophenyl)-nitrone, 100 mg/kg, n = 12), or its diluent (n = 12), from postnatal (PN) day 7. HI was induced on PN 8 by right common carotid artery ligation under anaesthesia, followed 2.5 h later by exposure to 8% oxygen for 1.5 h. Diluent-treated pups were then exposed to normothermia (37˚C), and S-PBN-treated pups to hypothermia (26˚C), for 6 h. Serial 40 µm sections were cut through the right striatum of 12 week old rats and coded. The total number of striatal medium-spiny neurons was stereologically determined using the optical disector/Cavalieri method. S-PBN/hypothermia treatment significantly preserved medium-spiny neurons compared with diluent/normothermia (2,578,000 ± 155,000 versus 1,893,000 ± 192,000, mean ± SEM, p = 0.0083, 2-tailed Mann-Whitney U test).

A separate experiment investigated fine motor skills using the staircase test. Eight animals from each HI-exposed treatment group, and 7 normal control animals, were tested daily by a blinded observer, from 9-11 weeks-of-age. S-PBN/hypothermia prevented HI-induced, long-term motor deficits in the forelimb contralateral to the lesion. Specifically, S-PBN/hypothermia improved grasping (p = 0.031; repeated-measures ANOVA) and depth of reach (p = 0.015) compared to diluent/normothermia. HI-exposed, S-PBN/hypothermia-treated animals and normal animals did not differ in either measure (p = 0.503 and p = 0.547, respectively).

This is the first study to identify a treatment that offers persistent striatal neuroprotection and preservation of fine motor skills following neonatal HI in the rat.

Poly (alkylcyanoacrylate) (PACA) nanoparticles containing protein may be a useful system for the delivery of proteins, especially vaccine delivery. The aim was to investigate entrapment of a model protein, fluorescein isothiocyanate conjugated ovalbumin, (FITC-Ova) in nanoparticles prepared by interfacial polymerisation of microemulsions (water in oil (w/o) droplet and bicontinuous) and to study the effect that the type of microemulsion template and concentration of monomer and protein have on entrapment.

Nanoparticles were prepared by dissolving 100-600 mg ethyl 2-cyanoacrylate monomer in 300-1800 mg chloroform and slowly adding this mixture to a microemulsion. Polymerisation was performed at 4oC and stirring overnight. FITC-Ova (0.1-5 mg) was added to the water component. Particle size and polydispersity index of the resulting nanoparticles were measured by photon correlation spectroscopy and morphology was observed by field-emission cryo-scanning electron microscopy. Determination of the entrapment efficiency was carried out by measuring the fluorescence intensity of the supernatant after spinning down the nanoparticles.

Entrapment of protein was up to 95% and was influenced by the amount of monomer and protein used, whereas entrapment of FITC-Ova increased with increasing monomer concentration from 42% (100 mg monomer) up to 95% (600 mg monomer). Entrapment of FITC-Ova dropped upon increasing the protein and keeping the monomer constant and might be explained by an exceedance of the loading capacity of the nanoparticles between 0.1-0.5 mg FITC-Ova. The type of microemulsion also influenced the entrapment and using equal quantities of protein and monomer led to entrapment of 30% (300 mg monomer) when using a bicontinuous microemulsion compared to 75% with a w/o droplet microemulsion, suggesting that the larger water domains in the bicontinuous microemulsion offered less possibility for interaction of protein with the monomer during polymerisation.

Due to their particulate character and high entrapment efficiency for proteins, PACA nanoparticles render an interesting system for the delivery of vaccines.

Atherosclerosis, the pathological entity that lies behind significant morbidity and mortality in the developed world, is often thought of as disease of old age. In fact pathological evidence has determined that the atherosclerotic process begins at a very early age, if not in utero. More recently, epidemiological evidence has linked the in utero condition with cardiovascular events later in life. The effect of in utero growth restriction in the brown norway (BN) rat was examined. The BN rat spontaneously develops elastic tissue defects in the abdominal aorta, which are known to play an integral role in atherosclerotic plaque initiation and progression.

Sixty-three dams were used in the study. The uterine arteries in the ligation group (21 dams) were ligated on day 18 of pregnancy, which decreased pup weight at 72 hours in males and females by 14% when compared to no surgery pups (P < 0.05). Dissected aortas were opened longitudinally, pinned flat and stained with Haematoxylin, permitting the visualisation and counting of elastic tissue defects under a light microscope. In both sexes, the abdominal aortas from the ligation group had double the number of elastic tissue defects (p < 0.05) compared to the no surgery group at eight weeks of age. At sixteen weeks of age male ligation animals still have 60% (p = 0.0006) more defects than the no surgery group, however this difference is no longer evident in females. This increase in defect numbers is not accompanied by any significant change in blood pressure (as assessed by conscious tail-cuff or unconscious intra-arterial methods).

This study supports the hypothesis that links the in utero environment to cardiovascular disease later in life. It appears that a even a moderate insult, such as this, can permanently alter blood vessel structure, leading to the formation of increased numbers of elastic tissue defects.

Ovarian follicles are nests of cells where female gametes (oocytes) are surrounded by their support cells (the granulosa cells). The molecular mechanisms that underlie the initiation of follicle growth and differentiation are poorly understood. Basic fibroblast growth factor (bFGF) is a molecule that has been implicated in the growth and differentiation of granulosa cells in culture since the 1970s. In this study human ovarian biopsies were used to investigate bFGF gene expression during small follicle development.

Ovarian biopsies were obtained from healthy fertile women, ranging in age between 25 - 34 years, undergoing tubal ligation for fertility control. Small follicles of different developmental sizes were isolated from 1 mm3 biopsies by laser capture microdissection (LCM). Follicles from 12 µm sections were classified into three different developmental categories (small non-growing follicles, primary follicles, or small secondary follicles) by microscopic criteria. The oocytes and granulosa cells of each follicle category were independently isolated for each patient via LCM. RNA was isolated from each sample and reverse transcribed. Analysis of bFGF gene expression was carried out using real-time PCR. After normalising bFGF gene expression to the housekeeping gene 18S, expression levels for different follicle populations were compared within each patient. All patients showed decreasing expression of bFGF mRNA as the follicles matured (n = 7; p < 0.05).

This finding suggests that bFGF gene expression is not positively correlated with follicle growth. Instead bFGF could have an inhibitory affect on follicle development. Alternatively the results presented here augment emerging evidence from granulosa cell cultures that bFGF may act as an anti-apoptotic survival factor.

Aberrant DNA repair and cancer aetiology are intimately linked. Loss of the ability to repair DNA often results in genomic instability and subsequent tumourigenesis. Likewise, a proportion of high-grade tumours typically over-express DNA repair proteins, such as Rad51. Rad51 is a central player in the repair of double-strand DNA breaks by homologous recombination (HR). Rad51 acts by catalysing the invasion of ssDNA from the break site into a homologous template. Recent evidence indicates that Adenoviral (Ad) proteins can interact with, and manipulate, components of the cell’s DNA repair machinery. With this in mind, we have investigated further the interplay between Ads and the DNA repair machinery.

By infecting human fibroblasts with wild-type Ad5, and using protein expression as a marker, we showed that Rad51 levels were markedly up-regulated upon infection. This effect was generalisable to a number of diverse cell types. Rad51 up-regulation has been demonstrated using an array of Ads housing mutations spanning the majority of their early regions. All but one mutant tested led to up-regulation of Rad51. The exceptional mutant, ts125, contains a single amino acid substitution in the DNA binding protein. Flow cytometry data derived from this mutant, coupled with southern and western analyses, directly tied Rad51 up-regulation to Ad DNA replication. Moreover, transient over-expression of Rad51 led to a 45-fold increase in Ad progeny.

We have shown for the first time that Ad specifically up-regulates Rad51 protein expression. Ts125 mutant analysis has coupled this up-regulation directly to Ad DNA replication. Furthermore, over-expression of Rad51 during infection yielded significant increases in the production of daughter virions. Collectively, these data suggest that Rad51 plays a direct role in the Ad replication strategy, increasing the efficiency of the replication process. Importantly, these novel findings may be exploited in the development of therapeutic Ads to target those tumours that over-express Rad51.

Motoneurons are irreplaceable cells, whose survival is controlled by multiple factors, at least some of which are unknown. Our group has previously used microarray analysis of motoneurons to identify novel regulators. A highly expressed growth factor receptor in the analysis was the type II anti-Müllerian hormone receptor (AMHRII). This receptor mediates the actions of AMH in vivo, in concert with the type Ia bone morphogenetic protein receptor (BMPRIa). AMH contributes to sex differentiation and certain functions of adult gonads. The presence of AMHRII in non-sexually-related neurons is without precedence. In this study, we have examined the existence of AMH-related molecules in motoneurons.

Adult spinal motoneurons were isolated from both male (n = 6) and female (n = 5) mice using laser capture microdissection. The copy numbers of AMHRII mRNA and other transforming growth factor (TGF)-beta superfamily mRNAs were quantified in non-amplified total RNA, using real-time PCR. The abundance of AMHRII transcripts (0.41 ± 0.04 % relative to glyceraldehydes-3-phosphate-dehydrogenase, mean ± SEM, n = 11, p < 0.0001; t-test) was significantly higher than other receptors, including RET (0.09 ± 0.014 %) and TGF-betaRII (0.01 ± 0.005%), which mediate the classical motoneuron survival factors. The ligand for AMHRII (AMH) was also present in the motoneurons (0.003 ± 0.0014%), with an abundance similar to that seen in the testes (0.001 ± 0.0003%). In contrast, other members of the TGF-beta superfamily, such as BMP2, BMP4 and BMP6 were undetectable. The levels of AMH and AMHRII in females were 78 ± 26.3% and 90 ± 4.2% of those in males respectively. AMH and AMHRII proteins were also present: a 63 kDa protein was detected in spinal cord extracts after immunoprecipitation with an antibody specific to AMHRII; AMH and AMHRII were also detected in motoneurons, using immunohistochemistry.

The colocalisation of AMH and AMHRII in motoneurons is consistent with AMH being an autocrine regulator of their functions. This hypothesis is currently being tested.