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Biomedical subjects

E S Robinson

Publications and source records attributed to E S Robinson.

At least 19 recordsLinked to original sources
PubMed2001

Characterization of the CDKN2A and ARF genes in UV-induced melanocytic hyperplasias and melanomas of an opossum (Monodelphis domestica).

We examined the involvement of the cyclin-dependent kinase inhibitor 2A (CDKN2A) locus in the pathogenesis of ultraviolet (UV) radiation-induced melanomas in an opossum (Monodelphis domestica) melanoma model in which suckling young were exposed to UVB to produce melanocytic lesions. Monodelphis CDKN2A and alternated reading frame (ARF) cDNAs were cloned and sequenced, and the expression patterns of these genes were determined by reverse transcription-polymerase chain reaction in normal tissues, 39 primary melanocytic skin lesions, and two tumor-derived cell lines, one nonmetastatic and one metastatic. Primary melanocytic lesions, including hyperplasias, benign melanomas, melanomas metastatic to lymph nodes, and melanomas metastatic to nodes and additional visceral organs, were categorized accordingly as types I-IV. Levels of CDKN2A transcripts were most abundant in type III tumor samples and the metastatic cell line but absent in the nonmetastatic cell line. ARF transcripts were expressed in all tumors and cell lines. A UV-signature mutation was detected with the wild-type allele at the CDKN2A locus in type II and III primary tumor samples and in the nonmetastatic cell line. Interestingly, in the metastatic cell line, only the mutant allele was present and expressed. These data suggest dynamic changes in the expression and/or structure of the CDKN2A and ARF genes represent one molecular defect associated with the etiology of melanoma formation and progression in the Monodelphis model system.

Animals↗
PubMed2001

Ultraviolet B-induced melanoma in Monodelphis domestica occurs in the absence of alterations in the structure or expression of the p53 gene.

Monodelphis domestica, a South American opossum, has been established as a mammalian model for sporadic ultraviolet radiation (UVR)-induced melanoma. Using this model system, we investigated the role of changes in the p53 gene in the development of cutaneous melanocyte-derived lesions. Cutaneous melanocytic hyperplasias, benign melanomas and metastatic primary melanomas, plus affected lymph nodes and visceral organs, were screened for mutations in the Monodelphis p53 gene by single-strand conformation polymorphism analysis and direct sequencing. With the exception of a silent point mutation found in a single benign melanocytic hyperplasia sample, no p53 mutations were detected. Furthermore, a relative quantitative reverse transcriptase-polymerase chain reaction approach was used to analyse p53 gene expression at different stages of primary melanoma progression and revealed no substantial changes in p53 mRNA levels. These results suggest that, as in humans, UVR-induced melanoma in the Monodelphis model is initiated and progresses on the basis of predominantly p53-independent molecular pathways.

Animals↗
PubMed2000

Cross-matching marsupial proteins with eutherian mammal databases: proteome analysis of cells from UV-induced skin tumours of an opossum (Monodelphis domestica).

The identification and characterisation of Monodelphis proteins has required cross-species analysis. Protein expression was investigated in normal, nonirradiated adult fibroblasts and also in fibroblastic cells from a benign cutaneous tumour after chronic ultraviolet (UVB) exposure and a metastatic cutaneous tumour after intermittent exposure. Proteins were separated and visualised by two-dimensional gel electrophoresis (2-D PAGE) and a peptide mass fingerprint (PMF) was obtained for protein spots using matrix assisted laser desorption/ionisation-time of flight-mass spectrometry (MALDITOF-MS). Cross-species PMF database analysis facilitated the identification of 120 proteins, constituting 46.5% of the proteins analysed. The identification of two proteins was confirmed by internal amino acid sequencing using tandem MS. Differential protein expression was observed between normal fibroblasts and those in tumours chronically or intermittently exposed. A number of tropomyosin and vimentin isoforms were expressed only in cells from the metastatic tumour induced by intermittent exposure to UV radiation. These results highlight the value of cross-species PMF analysis for the rapid characterisation of proteins from a poorly defined species and also show how proteomics can be used to detect changes in protein expression in differentially treated cells.

Animals↗
PubMed2000

Single nucleotide primer extension (SNuPE) analysis of the G6PD gene in somatic cells and oocytes of a kangaroo (Macropus robustus).

cDNA sequence analysis of the X-linked glucose-6-phosphate dehydrogenase (G6PD) gene has shown a base difference between two subspecies of the kangaroo, Macropus robustus robustus (wallaroo) and M. r. erubescens (euro). A thymine residue in the wallaroo at position 358 in exon 5 has been replaced by a cytosine residue in the euro, which accounts for the previously reported electrophoretic difference between the two subspecies. This base difference allowed use of the Single Nucleotide Primer Extension (SNuPE) technique to study allele-specific expression of G6PD at the transcriptional level. We began by examining G6PD expression in somatic cells and observed complete paternal X inactivation in all somatic tissues of adult female heterozygotes, whereas we found partial paternal allele activity in cultured fibroblasts, thus confirming previous allozyme electrophoresis studies. In late dictyate oocytes from an adult heterozygote, the assay also detected expression of both the maternal and paternal alleles at the G6PD locus, with the maternal allele showing preferential expression. Thus reactivation of the inactive paternally derived X chromosome occurs during oogenesis in M. robustus, although the exact timing of reactivation remains to be determined.

Amino Acid Substitution↗
PubMed2000

Behavioural and physiological effects induced by an infusion of antisense to alpha(2D)-adrenoceptors in the rat.

1. The aim of this study was to investigate the behavioural and physiological effects of an i.c.v. infusion of antisense oligonucleotide to the alpha(2D)-adrenoceptor subtype. Behavioural and physiological parameters were monitored for 2 days before the infusion, throughout the 3-day infusion period and for 3 days following the end of the infusion. 2. The antisense infusion resulted in a significant increase in behavioural activity characterized by increased locomotion and grooming scores. Behavioural activity scores of rats treated with antisense to alpha(2D)-adrenoceptors were significantly higher than those of rats treated with vehicle (H(2)O) or the mismatch toxicity control on day 4 and day 5 and, significantly higher than vehicle controls on day 6. 3. Body weight gain was significantly reduced in the antisense-treated rats at the end of the study compared to the vehicle (34%) and mismatch groups (30%), although daily food and water intakes were not significantly different at any time point. 4. Pupil diameters of rats infused with antisense to alpha(2D)-adrenoceptors were significantly greater than those of animals treated either with vehicle or mismatch oligonucleotide on day 5 of the study. On day 6, the pupil diameters of these animals were still significantly greater than the mismatch group. 5. In conclusion, an i.c.v. infusion of antisense to the alpha(2D)-adrenoceptor induced behavioural activation in rats, increased pupil diameter and reduced total weight gain. These effects were specific to the antisense-treated group and were fully reversed post-infusion.

Animals↗
PubMed2000

The Monodelphis melanoma model: initial report on large ultraviolet A exposures of suckling young.

The objective of this study was to determine whether exposure of early suckling young of the opossum Monodelphis domestica to ultraviolet A (UVA) radiation (320-400 nm) can lead to the development of melanocytic lesions similar to those induced after exposure to ultraviolet B (UVB) radiation (280-320 nm) to total doses as low as 380 J/m2. A total of 576 sucklings received nine exposures of 0.6, 2.6 or 15.5 kJ/m2 per dose (total doses approximately 6, 23 and 140 kJ/m2, respectively) from a Blak Ray lamp source with a narrow range emission at 365 nm. A further 280 sucklings were exposed in the same way to doses of 2.6 kJ/m2 per dose (total approximately 23 kJ/m2) broad-band UVA with visible wavelengths from a Dermalight lamp. Frequency of litter loss following all of the UVA-exposure protocols was similar to that within the same stocks in the colony at large. Only one of the 856 UVA-exposed individuals possessed a melanocytic lesion at the 5 month assessment point. No radiation-induced lesions of any type were evident on the skin of the other animals exposed as sucklings. The affected male was from a group of 70 individuals exposed to the highest total dose (140 kJ/m2) from the Blak Ray light source. The melanocytic hyperplasia was provisionally identified as a potential melanoma but it slowly regressed as the animal aged. We conclude that in the opossum suckling exposure system, the potency of UVA for melanoma induction is extremely low compared with that of UVB. Possible explanations, amenable to further investigations, are given for the low UVA sensitivity of the suckling model compared to the adult exposure model of Ley (Ley, R. D. [1997] Cancer Res. 57, 3682-3684).

Animals↗
PubMed1999

Autoradiographical and behavioural effects of a chronic infusion of antisense to the alpha2D-adrenoceptor in the rat.

1. The aims of this study were, firstly to use receptor autoradiography to investigate the effect of antisense oligonucleotides to the alpha2D-adrenoceptor on receptor binding and, secondly to measure behavioural and physiological parameters to determine whether the chronic antisense infusion had any effect on alpha2-adrenoceptor function in vivo. 2. A 3 day infusion of antisense to the alpha2D-adrenoceptor significantly reduced specific [3H]-RX821002 binding in the septum (20 - 30%) and anterior hypothalamic area (20 - 30%). beta-Adrenoceptor expression was unaffected in those brain areas examined, indicating the antisense knockdown was specific to the alpha2-adrenoceptors. 3. On the second day of the infusion, the hypothermic response to UK 14,304 was significantly attenuated in the antisense-treated group compared with both vehicle and mismatch controls. The effect was fully reversible and a similar decrease in body temperature was observed in all the treatment groups 4 days after the end of infusion. 4. During the second day of the infusion, the effects of UK 14,304 on behaviour were reduced in the antisense-treated rats, but were not significantly lower than those of the vehicle and mismatch, UK 14, 304 controls. These trends were not observed 4 days after the end of the infusion. 5. In conclusion, antisense has been shown to selectively knockdown alpha2-adrenoceptor expression in specific brain areas. The consequence of this knockdown is a significant attenuation of UK 14,304-induced hypothermia and a reduction in its sedative actions. These changes were fully reversed 4 days after the end of the infusion.

Animals↗
PubMed1999

In vitro and in vivo approaches to the characterization of the alpha2-adrenoceptor.

1. In order to more fully understand the role of the alpha2-adrenoceptor in brain function, a combination of in vitro and in vivo techniques were utilized including radioligand binding, autoradiography, brain microdialysis and antisense oligonucleotides. 2. Binding studies showed the tritiated form of the selective alpha2-adrenoceptor antagonist, RX821002 (methoxy-idazoxan) labelled an apparent single population of sites in rat brain membranes with high affinity (1 nM), for which prazosin had low affinity (1107 nM). Similar studies in rabbit brain membranes found that prazosin and oxymetazoline were able to displace [3H]-RX821002 in a biphasic manner indicating the presence of subtypes of alpha2-adrenoceptors. 3. Receptor autoradiography revealed a distribution of [3H]-RX821002 binding in rat brain consistent with the labelling of all alpha2-adrenoceptor subtypes, namely alpha(2A/D-), alpha2B and alpha2C. 4. In rat, in vivo brain dialysis experiments demonstrated peripherally administered RX821002 elevated basal noradrenaline in frontal cortex and also, although to a lesser extent, in ventral hippocampus. RX821002 was also able to elevate extracellular dopamine in the striatum. 5. A 7-day i.c.v. infusion of an antisense oligonucleotide targeting the alpha(2A/D)-adrenoceptor, resulted in a significant reduction in the autoradiographic density of [3H]-RX821002 binding in specific brain areas, notably the lateral septal nuclei and anterior hypothalamic area. 6. Several years of research by our group has extended our knowledge of the pharmacology and function of the alpha2-adrenoceptor and has provided evidence of the roles of this receptor in the control of monoamine turnover. The successful use of antisense technology to knockdown expression of the alpha(2A/D) subtype provides future opportunities to explore the physiology of this receptor subtype.

Adrenergic alpha-Antagonists↗
PubMed1999

Genetic analysis of susceptibility to infection with Ascaris lumbricoides.

Epidemiologic studies of helminthic infections have shown that susceptibility to these parasites frequently aggregates in families, suggesting the possible involvement of genetic factors. This paper presents a genetic epidemiologic analysis of Ascaris lumbricoides infection in the Jirel population of eastern Nepal. A total of 1,261 individuals belonging to a single pedigree were assessed for intensity of Ascaris infection at two time points. Following an initial assessment in which all individuals were treated with albendazole, a follow-up examination was performed one year later to evaluate reinfection patterns. Three measures of worm burden were analyzed, including eggs per gram of feces, direct worm counts, and worm biomass (weight). For all traits, variance component analysis of the familial data provided unequivocal evidence for a strong genetic component accounting for between 30% and 50% of the variation in worm burden. Shared environmental (i.e., common household) effects account for between 3% and 13% of the total phenotypic variance.

Adolescent↗
PubMed1998

Attitudes towards helminthic infection in the Jirel population of eastern Nepal.

Intestinal worm infections, including roundworm, hookworm, and whipworm, are major international health concerns, affecting approximately one fourth of the world's population. Many intervention schemes have been attempted to control these infections in heavily exposed populations, but success has been limited because individuals are readily reinfected upon renewed exposure. Few data are available concerning people's health beliefs about soil-transmitted helminthic infections in such populations. The purpose of this study was to assess health beliefs about common helminthiasis in a population experiencing moderate to high rates of infection. The focal population for the study was the Jirel population, a tribal group distributed across nine villages in the Jiri Region of Dolakha District, eastern Nepal. The results indicate that beliefs about the types, causes, and treatments of helminthic infections have been developed and reinforced by experience and empirical evidence. People's frequent inability to confirm the efficacy of drug therapy by observing worms in stools has led to dissatisfaction with biomedical approaches. Carefully planned education programs are required to alter prevailing attitudes and improve control of soil-transmitted helminthiasis in the region.

Adolescent↗
PubMed1998

Low-dose ultraviolet exposure early in development can lead to widespread melanoma in the opossum model.

Suckling young of opossums (Monodelphis domestica) were exposed to ultraviolet radiation (UVR, predominantly UVB: 290-320 nm) in part to determine an optimal protocol for induction and progression of melanoma in this species. In all, 620 litters were introduced to one of seven protocols. The lowest dose (175 J/m2) administered three times a week for almost three weeks led to the highest incidence of melanotic lesions with melanoma potential (8.1%) among young (5-month-old) adults. Among 101 much older animals (> 17 months at necropsy), 43% showed metastatic melanoma to the lymph nodes and almost one-third of these had progressed to widespread dissemination. Three of the latter animals, from a total of 13 obtained so far, were selected for detailed histological examination of disseminated disease. At necropsy, all three showed widespread metastases beyond the lymph nodes to the spleen, lungs, and other distant sites. Histological changes typical of malignant melanoma included junctional activity, mitotic figures, and nerve and vessel invasion. This novel finding leads us to conclude that UVR can act as a complete carcinogen for progression to widely disseminated disease and that exposure of sucklings can lead, in old age, to widespread metastatic melanoma in this model. The results are thus not inconsistent with the view that, in humans, early exposure to sunlight might act as an initiating factor in a later progression to malignant melanoma.

Age Factors↗
PubMed1997

Antisense oligonucleotides in psychopharmacology and behaviour: promises and pitfalls.

Antisense oligonucleotides are used to study the expression and function of a diverse range of proteins. Areas for which antisense has been used for pharmacological investigation include receptors, neuropeptides and immediate early genes, particularly when specific ligands or markers are not yet available. Antisense oligonucleotides target a specific mRNA and block the expression of the protein by sequence specific hybridization. This technique has not only been shown to be a valuable pharmacological tool but also to have potential therapeutic applications. In this review we discuss the technology behind the technique including developments in methodology employed in antisense experiments. Although antisense provides a novel and highly specific tool, the reliability of the technique and many of the problems associated with antisense experiments are discussed. The main focus of this article is the use of antisense in psychopharmacology to investigate behavioural changes following antisense-mediated inhibition of the expression of specific brain proteins and receptors.

Animals↗
PubMed1997

Development in vitro of Marsupials: a comparative review of species and a timetable of cleavage and early blastocyst stages of development in Monodelphis domestica.

The development of marsupial oocytes and embryos in vitro is reviewed. Most stages of development have been cultured successfully, usually in a complex medium with added fetal calf serum. Simpler media without added serum have been developed for fertilization and cleavage in vitro. Culture systems have been established for oocyte maturation and fertilization in the grey short-tailed opossum and for cleavage from the zygote to the early expanding unilaminar blastocyst in a number of other marsupials. Survival in vitro of the unilaminar and early bilaminar blastocyst stages is limited in all species examined. In contrast, late bilaminar, trilaminar, embryonic and fetal stages develop at rates approximating those in vivo. More stages have been cultured successfully in Sminthopsis macroura than in any other species. It has been cultured from the late bilaminar blastocyst to within 18 h of birth. Stages of cleavage and unilaminar blastocyst formation of Monodelphis domestica timed by videotaping mating animals, proceeded at similar rates in vivo and in vitro. As in other marsupials, cleavage in this opossum is characterized by a polarized conceptus. This polarity is expressed in the distribution of organelles in the zygote and the localization of secretion of the extracellular matrix material into the cleavage cavity and of the initial cell-zona attachment. Because cell-cell adhesion follows cell-zona adhesion, a unilaminar blastocyst forms without the development of an intervening morula stage.

Animals↗
PubMed1996

Intersexual phenotypes and sex chromosome complements of five South American opossums (Monodelphis domestica).

Intersexual opossums (Monodelphis domestica) from a large captive colony are described. These are the first naturally existing New World (didelphoid) intersexual marsupials for which reproductive phenotype and sex chromosome constitution are reported. One animal was XX, two were XY, and two were XO; all had lower body weight than normal males or females and the overall appearance of females. They were first recognized as abnormal by the presence of a small flaccid, nonstalked scrotum, markedly smaller than the scrotum of a normal male but in an equivalent position cranial to the cloacal aperture. Each scrotum contained a core of fatty connective tissue, but none contained testicular tissue. Teat patterns, seen only after close shaving of the hair over the area of the teat field, varied within and between the various sex genotypes, with one XY and one XO having the paired rudiments typical of normal males. All individuals had gonads, with no transabdominal migration. In the XX intersex there were mature ovaries with Graffian follicles, but in the XY and XO intersexes there was gonadal dysgenesis. The urogenital tract of all was female in appearance but was immature except in the XX intersex. Development of the scrotum and of the teat primordia can be explained on the basis of regulatory gene influences on the X chromosome. Intersex incidence in the colony is probably much higher than that observed because of ascertainment bias.

Animals↗