Maura Grealy

Cellular longevity: role of apoptosis and replicative senescence.

Cellular longevity refers to the lifespan of an individual cell. Normal cells have a finite lifespan and typically die by undergoing apoptosis, or enter into a state of irreversible growth arrest, termed replicative senescence, at the end of that lifespan. The lifespan of a cell is a balance between pro-survival/anti-apoptotic and pro-apoptotic death-promoting factors. The role of heat shock proteins, Bcl-2 family members, antioxidant molecules, and telomere length and telomerase activity in the regulation of apoptosis and replicative senescence, will be discussed.

Endocrine and ovarian responses and pregnancy rates in dairy cows following the administration of a gonadotrophin releasing hormone analog at the time of artificial insemination or at mid-cycle post insemination

Gonadotrophin releasing hormone analogue (GnRH-A) was administered to lactating dairy cows at the time of first artificial insemination (AI), or 12 days later and the effect on serum concentration of luteinizing hormone (LH) and progesterone, follicle populations, time of ovulation and pregnancy rate was measured. GnRH-A at AI induced a secondary LH surge, suppressed serum concentrations of progesterone (P<0.05) on Days 3–5 and did not affect the interval to ovulation. GnRH-A on Day 12 after AI induced a surge of LH, increased serum concentration of progesterone (P<0.05), decreased the number of large (≥ 10 mm diameter) follicles (P<0.05) and increased the inter-oestrus interval (P<0.06). Pregnancy rate in 1661 cows was not affected by administration of GnRH-A at AI, 12 days later or following AI at the first repeat. In conclusion, treatment of lactating dairy cows at pasture in a temperate climate with GnRH-A, either at AI or on Day 12 after AI, altered endocrine responses and ovarian follicle populations, but did not affect pregnancy rate.

Plakoglobin has both structural and signalling roles in zebrafish development.

Plakoglobin, or gamma-catenin, is found in both desmosomes and adherens junctions and participates in Wnt signalling. Mutations in the human gene are implicated in the congenital heart disorder, arrhythmogenic right ventricular cardiomyopathy (ARVC), but the signalling effects of plakoglobin loss in ARVC have not been established. Here we report that knockdown of plakoglobin in zebrafish results in decreased heart size, reduced heartbeat, cardiac oedema, reflux of blood between heart chambers and a twisted tail. Wholemount in situ hybridisation shows reduced expression of the heart markers nkx2.5 at 24 hours post fertilisation (hpf), and cmlc2 and vmhc at 48 hpf, while there is lack of restriction of the valve markers notch1b and bmp4 at 48 hpf. Wnt target gene expression was examined by semi-quantitative RT-PCR and found to be increased in morphant embryos indicating that plakoglobin is antagonistic to Wnt signalling. Co-expression of the Wnt inhibitor, Dkk1, rescues the cardiac phenotype of the plakoglobin morphant. beta-catenin protein expression is increased in morphant embryos as is its colocalisation with E-cadherin in adherens junctions. Endothelial cells at the atrioventricular boundary of morphant hearts have an aberrant morphology, indicating problems with valvulogenesis. Morphants also have decreased numbers of desmosomes and adherens junctions in the intercalated discs. These results establish the zebrafish as a model for ARVC caused by loss of plakoglobin function and indicate that there are signalling as well as structural consequences of this loss.

Epidermal growth factor receptor and ligands in elongating bovine blastocysts.

Preimplantation development depends on multiple interactions between mother and embryo. The Epidermal Growth Factor Receptor (EGF‐R) and its ligands are potential components of the embryo‐maternal cross‐talk. Employing RT‐PCR, in situ hybridization, and immunohistochemistry, we investigated on mRNA and protein level the expression of EGF‐R, Epidermal Growth Factor (EGF), Transforming Growth Factor alpha (TGF‐α), and Heparin‐binding EGF‐like Growth Factor (HB‐EGF) in spherical and elongating bovine blastocysts between day 13 and day 16 of gestation, and in endometrium at day 13 of gestation. EGF‐R mRNA and protein were detected in trophoblast and endoderm cells of all blastocyst stages that were studied, and in luminal and some glandular epithelial cells of the endometrium at day 13. EGF protein was detected in both blastocysts and endometrial epithelium. TGF‐α transcripts and protein were present in blastocysts prior to and after elongation and in uterine glandular and luminal epithelium at day 13 of gestation. HB‐EGF mRNA and protein was shown in the endoderm, and the protein also was detected immunohistochemically in about 45% of the blastocysts. This presence of the EGF receptor‐ligand system in the endometrium and the preimplantation embryo at the time of blastocyst elongation suggests an important role for these growth factors during bovine preimplantation development. Mol. Reprod. Dev. 51:402–412, 1998.

Insulin-like growth factor-2 regulates early neural and cardiovascular system development in zebrafish embryos

The insulin-like growth factor (IGF) family is essential for normal embryonic growth and development and it is highly conserved through vertebrate evolution. However, the roles that the individual members of the IGF family play in embryonic development have not been fully elucidated. This study focuses on the role of IGF-2 in zebrafish embryonic development. Two igf-2 genes, igf-2a and igf-2b, are present in the zebrafish genome. Antisense morpholinos were designed to knock down both igf-2 genes. The neural and cardiovascular defects in IGF-2 morphant embryos were then examined further using wholemount in situ hybridisation, TUNEL analysis and O-dianisidine staining. Knockdown of igf-2a or igf-2b resulted in ventralised embryos with reduced growth, reduced eyes, disrupted brain structures and a disrupted cardiovascular system, with igf-2b playing a more significant role in development. During gastrulation, igf-2a and igf-2b are required for development of anterior neural structures and for regulation of genes critical to dorsal-ventral patterning. As development proceeds, igf-2a and igf-2b play anti-apoptotic roles. Gene expression analysis demonstrates that igf-2a and igf-2b play overlapping roles in angiogenesis and cardiac outflow tract development. Igf-2b is specifically required for cardiac valve development and cardiac looping. Injection of a dominant negative IGF-1 receptor led to similar defects in angiogenesis and cardiac valve development, indicating IGF-2 signals through this receptor to regulate cardiovascular development. This is the first study describing two functional igf-2 genes in zebrafish. This work demonstrates that igf-2a and igf-2b are critical to neural and cardiovascular development in zebrafish embryos. The finding that igf-2a and igf-2b do not act exclusively in a redundant manner may explain why both genes have been stably maintained in the genome.

Secretion of growth hormone elicited by intravenous desipramine in the conscious, unrestrained rat.

1 Acute intravenous administration of either clonidine (Clon) (50 μg kg−1) or desipramine (DMI) (5 mg kg−1) elicited a pulse of growth hormone (GH) and corticosterone secretion in conscious, unrestrained rats. 2 The responses to DMI were similar to those with Clon, except that the GH pulse following DMI was delayed and was not dose‐dependent. 3 The GH response to DMI was inhibited by prior administration of idazoxan (1 mg kg−1) or yohimbine (0.5 mg kg−1), but not by atropine (10 μg kg−1), sulpiride (5 mg kg−1) or prazosin (1 mg kg−1). 4 The corticosterone secretion following DMI was not altered by prior atropine, sulpiride or prazosin, but was augmented by idazoxan (1 mg kg−1). 5 GH secretion was not influenced by atropine, sulpiride, prazosin or idazoxan given alone. Idazoxan or yohimbine given alone elicited significant secretion of corticosterone. 6 It is concluded that i.v. DMI caused an activation through indirect mechanisms of α2‐adrenoceptors specifically involved in hypothalamic‐pituitary regulation of GH release and also a distinct, independent and transient generalized activation of the pituitary‐adrenal axis.

Development of a seaweed derived platelet activating factor acetylhydrolase (PAF-AH) inhibitory hydrolysate, synthesis of inhibitory peptides and assessment of their toxicity using the Zebrafish larvae assay

The vascular inflammatory role of platelet activating factor acetylhydrolase (PAF-AH) is thought to be due to the formation of lysophosphatidyl choline and oxidized non-esterified fatty acids. This enzyme is considered a promising therapeutic target for the prevention of atherosclerosis and there is a need to expand the available chemical templates of PAF-AH inhibitors. This study demonstrated how natural PAF-AH inhibitory peptides were isolated and characterized from the red macroalga Palmaria palmata. The dried powdered alga was hydrolyzed using the food grade enzyme papain, and the resultant peptide containing fraction generated using RP-HPLC. Several oligopeptides were identified as potential PAF-AH inhibitors following bio-guided fractionation, and the amino acid sequences of these oligopeptides were confirmed by Q-TOF-MS and microwave-assisted solid phase de novo synthesis. The most promising PAF-AH inhibitory peptide had the amino acid sequence NIGK and a PAF-AH IC50 value of 2.32 mM. This peptide may constitute a valid drug template for PAF-AH inhibitors. Furthermore the P. palmata hydrolysate was nontoxic when assayed using the Zebrafish toxicity model at a concentration of 1mg/ml.

Neuroendocrine response to clonidine and 8-OH-DPAT in rats following chronic administration of desipramine or sertraline

1 Rats were administered either desipramine (DMI) or sertraline daily at doses 7.5 mg kg−1 or 10 mg kg−1, i.p., respectively and the effects on the functional state of hypothalamic neuroendocrine control mechanisms assessed by measurements of plasma hormones following acute drug challenge. The effects of treatment on gross behaviour and brain adrenoceptor density were also determined. 2 Both DMI and sertraline caused significant reduction in activity measured as ambulation and rearing at 14 days of treatment. 3 All animals were chronically cannulated after 14 days of treatment and tested for neuroendocrine response to acute i.v. clonidine (50 μg kg−1) or 8‐hydroxy‐2‐(di‐n‐propylamino)tetralin (8‐OH‐DPAT, 250 μg kg−1) after 21 or more days of treatment. 4 Rats treated with DMI but not sertraline showed a virtually complete suppression of the growth hormone (GH) secretion elicited by clonidine in controls, while the secretion of corticosterone was augmented. 5 Treatment with DMI but not sertraline led to a significantly greater 8‐OH‐DPAT‐induced secretion of prolactin than in the control rats, while the plasma concentrations of corticosterone following 8‐OH‐DPAT were not influenced by either DMI or sertraline treatment. 6 The density (but not the affinity) of cerebral cortical binding of [3H]‐dihydroalprenolol was significantly reduced by DMI treatment. 7 These results show that DMI treatment blunted the sensitivity of post‐synaptic α2‐adrenoceptors, accompanied by complex interactions manifested as increased responsiveness of α1‐adrenoceptors and 5‐HT1A receptors. Sertraline had no significant neurendocrine effects at a dose which significantly reduced gross activity.

Loss of plakophilin 2 disrupts heart development in zebrafish

The desmosomal armadillo protein plakophilin 2 is the only plakophilin expressed in the heart, and mutations in the human plakophilin 2 gene result in arrhythmogenic right ventricular cardiomyopathy. To investigate loss of function, we knocked down plakophilin 2 by morpholino microinjection in zebrafish. This resulted in decreased heart rate, cardiac oedema, blood pooling, a failure of the heart to pattern correctly and a twisted tail. Co-injection of plakophilin 2 mRNA rescued the morphant phenotype, indicating the specificity of the knockdown. Desmosome numbers were decreased in morphant hearts and the plaque and midline structures of the desmosomes in the intercalated discs were disrupted when examined by electron microscopy. cmlc2 and vmhc expression at 48 hours post-fertilization (hpf) showed incomplete looping of the heart in morphant embryos by whole mount in situ hybridization, and bmp4 expression was expanded into the ventricle. The domain of expression of the heart marker nkx2.5 at 24 hpf was expanded. At the 18 somite stage, expression of the cardiogenic gene lefty2 was abolished in the left cardiac field, with concomitant increases in bmp4, spaw and lefty1 expression, likely resulting in the looping defects. These results indicate that plakophilin 2 has both structural and signalling roles in zebrafish heart development.

Plakoglobin expression and localization in zebrafish embryo development.

Plakoglobin (gamma-catenin) and beta-catenin are major components of the adherens junctions and can be localized to the nucleus by activation of the Wnt signalling pathway. In addition, plakoglobin is also found in desmosomes, a vertebrate-specific cell-cell adhesion structure. Plakoglobin expression and localization were examined at the protein level during zebrafish embryonic development by Western blotting and confocal microscopy. Plakoglobin was expressed throughout embryo development at the protein level. Western blotting revealed that embryonic plakoglobin protein content increased between 12- and 24-h post-fertilization (hpf). Confocal microscopy showed that at stages up to 12 hpf, plakoglobin and beta-catenin were co-localized and expressed in both the nucleus and in cell-cell junctions. At 24- and 72-hpf, separate patterns were seen for plakoglobin and beta-catenin. These data indicate that plakoglobin localization in the heart region shifts from adherens junctions to desmosomes during heart chamber development.