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M L Hooper

Publications and source records attributed to M L Hooper.

At least 55 records · Page 3Linked to original sources
PubMed1992

A relationship between gap junction-mediated intercellular communication and the in vitro developmental capacity of murine embryonic stem cells.

Two metabolic cooperation-deficient variants, 1P9 and 2P2[1], have been isolated from the embryonic stem cell line B2B2. Characterization of these cell lines has shown that both variants are severely restricted with respect to embryoid body differentiation capacity while a revertant isolated from 1P9, designated 2H4, has its differentiation phenotype restored to a level comparable with that of the parent line. These results are interpreted as indicating that the metabolic cooperation deficiency and the restricted differentiation phenotype of 1P9 are causally related. A revertant isolated from 2P2[1], designated H19, remains severely restricted with regard to embryoid body differentiation, suggesting that in these lines a secondary event deleterious to differentiation, but unrelated to metabolic cooperation, has taken place.

Animals↗
PubMed1992

Failure of ondansetron to block the discriminative or reinforcing stimulus effects of cocaine in the rat.

Ondansetron (GR38032F), a serotonin 5HT3 antagonist, is active in numerous behavioral paradigms and neurochemical systems. Since 5HT3 antagonists have been suggested as therapeutic agents for the treatment of drug abuse, the action of ondansetron on cocaine drug discrimination and self-administration paradigms in rats was investigated. Doses of ondansetron (0.001 - 1.0 mg/kg) had no effect on the discriminative stimulus properties of 10 mg/kg cocaine. In contrast SCH23390, a dopamine D1 antagonist known to block cocaine discrimination, acted as previously reported. Ondansetron did not augment the effects of SCH23390, but at higher doses, combinations of ondansetron and SCH23390 produced disruption of lever pressing in the presence of cocaine. Ondansetron (0.001-1.0 mg/kg) had no effect on the self-administration of various doses of cocaine, nor did it have any effect on reacquisition of cocaine self-administration in animals with a history of active administration followed by a period of abstinence. As before, SCH23390, known to block cocaine self-administration, acted as previously reported. Although other 5HT antagonists may prove to be efficacious in cocaine abuse, ondansetron appears unlikely to alter the subjective or rewarding stimulus properties of cocaine.

Animals↗
PubMed1991

Creation of a large genomic deletion at the T-cell antigen receptor beta-subunit locus in mouse embryonic stem cells by gene targeting.

Recently it has become possible to introduce predesigned mutations into a given gene in the mouse germ line by homologous recombination in embryonic stem cells. The mutations are usually introduced by inserting the neomycin phosphotransferase gene into an exon of a particular gene. Here we describe an extension of this method that can result in at least a 15-kilobase-long deletion. The deletion created in the present work encompasses one of the two diversity gene segments of the mouse T-cell receptor beta-subunit locus, 10 out of the 12 joining gene segments, and both constant gene segments. This strategy is a valuable alternative to sequential targeting of multiple genes forming a gene cluster, could simplify the construction of plasmids to be used for targeting, and could be the solution for inactivating small genes that have eluded conventional targeting approaches.

Animals↗
PubMed1991

Hypoxanthine phosphoribosyl transferase deficiency, haematopoiesis and fertility in the mouse.

We have looked for effects of deficiency in hypoxanthine phosphoribosyl transferase (HPRT) in the mouse comparable to non-behavioural consequences of HPRT-deficiency in humans. HPRT-deficient humans show abnormalities in haematopoiesis and, in heterozygotes, there is strong selection in haematopoietic tissues against HPRT-deficient cells arising as a result of X-chromosome inactivation. We have examined two situations in mice in which HPRT- and HPRT+ cells occur in the same individual. First, in chimaeras resulting from the injection of HPRT- embryonal stem cells into HPRT+ blastocysts the fate of HPRT- and HPRT+ cell populations was monitored by their expression of different isozymes of glucose phosphate isomerase and also, in those chimaeras that resulted from injecting the male ES cells into female blastocysts, by in situ hybridisation using a Y-chromosome-specific repetitive DNA probe. There was a small statistically significant selection against the HPRT- population in haematopoietic tissues in both XX in equilibrium with XY and XY in equilibrium with XY chimaeras. Second, in female mice doubly heterozygous for HPRT-deficiency and for an electrophoretic variant of the X-linked enzyme phosphoglycerate kinase, there was a similar small statistically significant selection against the HPRT- population in haematopoietic tissues. While further work is required to establish whether this selection is a consequence of the HPRT mutation, it is clear that any selection against cells in the haematopoietic system as a consequence of HPRT-deficiency is at most small compared with the effect seen in humans. In HPRT-deficient human males surviving beyond the normal age of puberty, there is testicular atrophy. However, we find no effect of HPRT-deficiency on the fertility of either male or female mice. Thus, as with effects on behaviour, the consequences of HPRT-deficiency for haematopoiesis and testis development in the mouse are at most small compared with those in the human. We conclude that the reason for the difference in effects between the two species lies in a difference in purine-related intermediary metabolism per se, rather than in its interaction with brain amine biochemistry.

Animals↗
PubMed1991

Sex chimaerism, fertility and sex determination in the mouse.

Adult intraspecific mouse chimaeras, derived by introducing male embryonal stem cells into unsexed host blastocysts, were examined to determine whether gonadal sex was correlated with the sex chromosome composition of particular cell lineages. The fertility of XX in equilibrium XY and XY in equilibrium XY male chimaeras was also compared. The distribution of XX and XY cells in 34 XX in equilibrium XY ovaries, testes and ovotestes was determined by in situ hybridisation using a Y-chromosome-specific probe. Both XX and XY cells were found in all gonadal somatic tissues but Sertoli cells were predominantly XY and granulosa cells predominantly XX. The sex chromosome composition of the tunica albuginea and testicular surface epithelium could not, in general, be fully resolved, owing to diminished hybridisation efficiency in these tissues, but the ovarian surface epithelium (which like the testicular surface epithelium derives from the coelomic epithelium) was predominantly XX. These findings show that the claim that Sertoli cells were exclusively XY, on which some previous models of gonadal sex determination were based, was incorrect, and indicate instead that in the mechanism of Sertoli cell determination there is a step in which XX cells can be recruited. However, it remains to be established whether the sex chromosome constitution of the coelomic epithelium lineage plays a causal role in gonadal sex determination. Male chimaeras with XX in equilibrium XY testes were either sterile or less fertile than chimaeras with testes composed entirely of XY cells. This impaired fertility was associated with the loss of XY germ cells in atrophic seminiferous tubules. Since this progressive lesion was correlated with a high proportion of XX Leydig cells, we suggest that XX Leydig cells are functionally defective, and unable to support spermatogenesis.

Animals↗
PubMed1990

Extragonadal teratocarcinoma derived from embryonal stem cells in chimaeric mice.

Three tumours which arose in two (one male and one hermaphrodite) out of 63 chimaeric mice resulting from injection of E14TG2a embryo stem (ES) cells into host blastocysts have been investigated. All of the tumours appeared within the first 3 weeks after birth. The tumour in the male chimaera and one of the tumours in the hermaphrodite were in the perigenital region but were extragonadal. The third, smaller tumour in the hermaphrodite was on the caecum. The perigenital tumour in the male chimaera was a teratocarcinoma with a wide variety of differentiated tissues, including non-pigmented retina, as well as nests of undifferentiated embryonal carcinoma (EC) cells with high levels of alkaline phosphatase activity. The perigenital tumour in the hermaphrodite was a teratoma, less differentiated and with no evidence of EC cells. Glucose phosphate isomerase isozyme analysis indicated that both perigenital tumours were predominantly of the injected ES cell rather than the host blastocyst type. The possible origins of these tumours, which are the first reported to have arisen from ES cells in chimaeric mice, are discussed.

Abdominal Neoplasms↗
PubMed1989

Germ line transmission and expression of a corrected HPRT gene produced by gene targeting in embryonic stem cells.

The deletion mutation in the HPRT-deficient mouse embryonic stem (ES) cell line E14TG2a has been corrected by gene targeting. The presence of plasmid sequences in the correcting vector DNA did not affect the frequency of correction. We have characterized three different HPRT gene structures in correctants. Cells from one corrected clone have been introduced into mouse blastocysts, and germ line transmission of the ES cell-derived corrected gene has been achieved. The corrected gene has the same pattern of expression as the wild-type gene, with the characteristic elevated level of expression in brain tissue. Hence, we have demonstrated the feasibility of introducing targeted modifications into the mouse germ line by homologous recombination in ES cells.

Animals↗
PubMed1989

Dominance and independent segregation of metabolic cooperation-competence and pluripotency in an embryonal carcinoma cell hybrid.

We report the isolation of a fusion hybrid, PR3, from a pluripotent embryonal carcinoma (EC) cell line, PSA4, which is metabolic cooperation-competent, and an EC line R5/3OA which has a reduced capacity for metabolic cooperation and a restricted developmental capacity. PR3 resembles its pluripotent parent PSA4 in its capacity for gap-junction-mediated transfer of uridine nucleotides and in its pluripotency both in embryoid bodies in vitro and in tumors in vivo. This enabled the relationship between pluripotency and metabolic cooperation to be examined by the selection of segregant lines. Cooperation-deficient lines were isolated from a thioguanine-resistant intermediate line (PR3Tg12) using "Kiss of Death" selection. A novel method was devised for the selection of differentiation-deficient segregants using feeder cell-conditioned medium which partially inhibits in vitro differentiation. It was found that communication-competence and in vitro pluripotency segregated independently, demonstrating that the loss of developmental capacity in R5/3OA cannot be attributed to its communication-deficiency.

Animals↗
PubMed1989

Factors influencing the differentiation of embryonal carcinoma and embryo-derived stem cells.

The effects of aggregation, retinoic acid, and medium conditioned by Buffalo rat liver (BRL) cells, alone and in combination, on the differentiation of PSA4TG12 embryonal carcinoma and E14 embryonal stem cells are reported. The observations indicate that BRL-conditioned medium has more than one effect on the differentiation process, that retinoic acid has at least two effects which operate in different concentration ranges, and that both agents influence the choice of differentiation pathway as well as the extent of differentiation.

Animals↗
PubMed1989

A close relationship between microvilli and metabolic cooperation deficiency in embryonal carcinoma cells.

A revertant clone has been isolated from the metabolic cooperation-deficient embryonal carcinoma clone PT2md1. In PT2md1, its cooperation-competent parent, and its cooperation-competent revertant an inverse correlation has been demonstrated between cooperation competence and the incidence of microvilli. This demonstration, together with a similar correlation previously reported in an independently isolated series of cell lines, establishes a close causal relationship between the incidence of microvilli and communication deficiency.

Animals↗
PubMed1989

Differentiation in medulloblastomas and other primitive neuroectodermal tumours.

Fifty-four cases of primitive neuroectodermal tumour (PNET) including 47 medulloblastomas, were examined for evidence of neuronal and glial differentiation, using antibodies to neuron-specific enolase (NSE), neurofilament protein (NF), and glial fibrillary acidic protein (GFAP). In 30 of the cases, antibodies to vimentin, alphafetoprotein, cytokeratin, epithelial membrane antigen and lymphoid markers were also used. Most of the 47 medulloblastomas in the group were NSE positive but NF negative; about half were GFAP positive and three of them were positive for both neuronal markers and for GFAP. Vimentin was demonstrated in four cases and was not always co-expressed with GFAP. Medulloblastomas were negative for all the other markers. Supratentorial PNETs were sometimes positive with neuronal markers but were GFAP negative. The cell specificity of these markers and the interpretation of immunocytochemical findings are discussed in relation to differentiation potential in primitive neuroectodermal tumours.

Adolescent↗
PubMed1987

Buffalo rat liver cells produce a diffusible activity which inhibits the differentiation of murine embryonal carcinoma and embryonic stem cells.

Many pluripotent embryonal carcinoma (EC) cell lines and all embryonic stem (ES) cell lines have hitherto been maintained in the undifferentiated state only by culture on feeder layers of mitomycin C-treated embryonic fibroblasts. We now demonstrate that medium conditioned by incubation with Buffalo rat liver (BRL) cells prevents the spontaneous differentiation of such cells which occurs when they are plated in the absence of feeders. This effect is not mediated via cell selection but represents a fully reversible inhibitory action ascribed to a differentiation-inhibiting activity (DIA). BRL-conditioned medium can therefore replace feeders in the propagation of homogeneous stem cell populations. Such medium also restricts differentiation in embryoid bodies formed via aggregation of EC cells and partially inhibits retinoic acid-induced differentiation. The PSA4 EC line gives rise only to extraembryonic endoderm-like cells when aggregated or exposed to retinoic acid in BRL-conditioned medium. This suggests that DIA may be lineage-specific. DIA is a dialysable, acid-stable entity of apparent molecular weight 20,000-35,000. Its actions are reproduced neither by insulin-like growth factor-II nor by transforming growth factor-beta. DIA thus appears to be a novel factor exerting a negative control over embryonic stem cell differentiation.

Animals↗
PubMed1986

Clones defective in metabolic cooperation selected from a pluripotent feeder-dependent mouse embryonal carcinoma cell line.

Starting from embryonal carcinoma (e.c.) cells capable of extensive differentiation in culture, the technique of thioguanine 'kiss of death' has been used to select four independent metabolic cooperation-defective variants. The communication ability of these variant cell lines has been quantified by autoradiographic measurement of the transfer of uridine nucleotides, and also by an assay of the extent of junction-mediated rescue from ouabain toxicity by resistant fibroblasts. The cell lines which are defective in ability to transfer nucleotides, as measured by the uridine nucleotide transfer assay, are also defective in their ability to differentiate into endoderm and to form the cavitated 'embryoid bodies' which are produced by the parental cell line when grown in suspension culture. However, it is not clear whether this is related to the defects in metabolic cooperation, since clones which had been subjected to the same selective conditions but which cooperate normally have also lost some of the capacity to undergo this differentiation. Endoderm differentiation was classified into two categories, one being visceral endoderm and the other, primary plus parietal endoderm, on the basis of morphology, immunocytochemical staining for alpha-fetoprotein, and basement membrane formation. With the exception of correlations arising from variations between experiments and differences between cell lines, there is no statistical association between these two categories of differentiation. The formation of cavities was observed only in embryoid bodies with endoderm differentiation: the present of either category was a sufficient condition for cavitation to occur.

Animals↗
PubMed1986

Selection of a mouse embryonal carcinoma clone resistant to the inhibition of metabolic cooperation by retinoic acid.

A novel selection for cell lines resistant to the blocking of metabolic cooperation by chemical inhibitors is described. The selection has been used to isolate a pluripotent embryonal carcinoma clone which cooperates in retinoic acid (RA), an inhibitor of junctional communication in all other cell lines tested to date. The selected cells require higher levels of RA to induce differentiation, but show little or no increase in resistance to toxic effects of RA.

Animals↗
PubMed1985

Gap-junctional communication in a communication-defective and in a communication-competent teratocarcinoma cell line.

We examined the gap-junctional communication properties of a communication-defective cell line R5/3 and its communication-competent revertant H2T12. For these studies, we carried out microelectrode impalements to monitor ionic coupling and dye coupling. Our dye-injection experiments revealed that the H2T12 cells are much more efficient in dye coupling than the R5/3 cells. This latter observation is in agreement with the previous finding that the H2T12 cells are much better metabolically coupled than the R5/3 cells. With ionic coupling measurements, however, both cell lines exhibited similar levels of cell-cell coupling. The R5/3 cells demonstrated an ionic coupling coefficient of 0.19 +/- 0.011 (S.E.M.) and H2T12 a coupling coefficient of 0.25 +/- 0.009 (S.E.M.). These results in conjunction with observations from other studies indicate that the different experimental approaches for monitoring gap-junctional communication may have different levels of sensitivity for detecting as opposed to measuring the level of cell-cell coupling.

Cell Communication↗
PubMed1984

The mode of cell death associated with cavitation in teratocarcinoma-derived embryoid bodies.

Cell death occurring in embryoid bodies derived from the embryonal carcinoma cell line, PSA4, which undergo cavitation, and in those from the related cell line S2, which do not undergo cavitation, was classified as apoptosis or necrosis by ultrastructural criteria. Both modes of cell death were seen in PSA4 embryoid bodies while apoptosis alone was seen in S2 embryoid bodies. No significant difference was found between PSA4 and S2 embryoid bodies either in apoptotic incidence score or in the spatial distribution of apoptotic events. We therefore conclude that although apoptosis and tissue modelling coexist in PSA4 embryoid bodies, necrosis rather than apoptosis is causally related to formation of the cavity.

Animals↗