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Biomedical subjects

G Bain

Publications and source records attributed to G Bain.

At least 19 recordsLinked to original sources
PubMed1995

Neuronal differentiation of P19 embryonal carcinoma cells in defined media.

The P19 embryonal carcinoma cell line is a useful model system for analyzing the factors that regulate neuronal differentiation. In order to analyze the extrinsic factors that are involved in differentiation, it is necessary to carry out experiments in fully defined media. Here we have investigated the neuronal differentiation of P19 cells in two defined media. Cells that are propagated and induced with retinoic acid in standard serum-containing medium are capable of differentiating into neuron-like cells in N2 medium. Dividing fibroblast-like cells also appeared in these cultures. After about 10 days in culture in N2 medium, the great majority of neuron-like cells died. On the other hand, culturing induced cells in N2 medium for 5 days and then switching to a defined medium consisting of Neurobasal medium plus B27 supplement allowed the neuron-like cells to survive for prolonged periods of time. This defined medium thus provides a suitable system for analyzing extrinsic factors that affect the survival and differentiation of P19 neurons. P19 cells induced with retinoic acid and plated in N2 were exposed to bFGF and EGF, which are known to be mitogens for neuronal precursor cells. Both growth factors were mitogenic for a subpopulation of the induced cells. In separate experiments, cells cultured in N2 in the presence of RA were induced to differentiate into neuron-like cells.

Animals↗
PubMed1995

Embryonic stem cells express neuronal properties in vitro.

Mouse embryonic stem (ES) cells cultured as aggregates and exposed to retinoic acid are induced to express multiple phenotypes normally associated with neurons. A large percentage of treated aggregates produce a rich neuritic outgrowth. Dissociating the induced aggregates with trypsin and plating the cells as a monolayer results in cultures in which a sizable percentage of the cells have a neuronal appearance. These neuron-like cells express class III beta-tubulin and the neurofilament M subunit. Induced cultures express transcripts for neural-associated genes including the neurofilament L subunit, glutamate receptor subunits, the transcription factor Brn-3, and GFAP. Levels of neurofilament L and GAD67 and GAD65 transcripts rise dramatically upon induction. Physiological studies show that the neuron-like cells generate action potentials and express TTX-sensitive sodium channels, as well as voltage-gated potassium channels and calcium channels. We conclude that a complex system of neuronal gene expression can be activated in cultured ES cells. This system should be favorable for investigating some of the mechanisms that regulate neuronal differentiation.

Animals↗
PubMed1995

A comparison of the chest radiograph and computerised tomography in assessing lung changes in acute spinal injuries--an assessment of their prevalence and the accuracy of the chest X-ray compared with CT in their assessment.

Lung function in patients following an acute spinal injury is frequently much more compromised than may be expected from the level of injury and the chest radiograph appearance. Experimental evidence in anaesthetised patients and subsequently our own experience with patients with acute spinal injuries suggested that in paralysed patients lung changes were frequent and that in many spinal patients large pleural effusions and lung consolidation could be present without the usual associated chest radiograph changes being recognised. This study was performed to assess the prevalence of chest pathology and the sensitivity of the chest radiograph in portraying it. Sixty patients (50 males, 10 females, 31 cervical, 29 thoracic or thoracolumbar; 15 incomplete, 45 complete; ages 17-66, mode 22 years) with spinal injuries from a variety of causes were assessed with a supine chest radiograph and three computerised tomography axial cuts at standardised locations through the thorax. The chest radiograph agreed with the computerised tomography in only 12 patients (six normal, six abnormal). The chest radiograph suggested that there were 19 normals but computerised tomography only showed 12. In a total of 35 patients, the chest radiograph significantly underestimated the degree of change and in 13 the chest radiograph suggested greater pathology than was shown on computerised tomography. Thirteen of the 20 patients with cervical lesions but no chest trauma had lung changes.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1994

E2A proteins are required for proper B cell development and initiation of immunoglobulin gene rearrangements.

E12 and E47 are two helix-loop-helix transcription factors that arise by alternative splicing of the E2A gene. Both have been implicated in the regulation of immunoglobulin gene expression. We have now generated E2A (-/-) mice by gene targeting. E2A-null mutant mice fail to generate mature B cells. The arrest of B cell development occurs at an early stage, since no immunoglobulin DJ rearrangements can be detected in homozygous mutant mice. While immunoglobulin germline I mu RAG-1, mb-1, CD19, and lambda 5 transcripts are dramatically reduced in fetal livers of E2A (-/-) mice, B29 and mu degrees transcripts are present, but at lower levels. In addition, we show that Pax-5 transcripts are significantly reduced in fetal livers of E2A (-/-) mice. These data suggest a crucial role for E2A products as central regulators in early B cell differentiation.

Animals↗
PubMed1994

Expression of retinoid X receptors in P19 embryonal carcinoma cells and embryonic stem cells.

The pluripotent mouse embryonal carcinoma cell line P19 provides an excellent model system to study the mechanisms by which retinoic acid (RA) exerts its biological effects. When aggregated and exposed to low concentrations of RA, P19 cells differentiate into neuron- and glial-like cells. The diverse biological effects of RA are mediated by two families of receptors localized in the cell nucleus, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). Each family consists of three members designated alpha, beta and gamma. While the patterns of expression of the RARs have been studied in P19 cells, similar data for the RXRs have not been available. We demonstrate here that these receptors are expressed in P19 and are regulated during RA-induced differentiation. We also show that pluripotent mouse embryonic stem cells express the RXRs as well.

Animals↗
PubMed1994

From embryonal carcinoma cells to neurons: the P19 pathway.

The differentiation of mammalian neurons during development is a highly complex process involving regulation and coordination of gene expression at multiple steps. The P19 mouse embryonal carcinoma cell line is a suitable model system with which to analyze regulation of neuronal differentiation. These multipotential cells can be maintained and propagated in tissue culture in an undifferentiated state. Exposure of aggregated P19 cells to retinoic acid results in the differentiation of cells with many fundamental phenotypes of mammalian neurons. Undifferentiated P19 cells are amenable to genetic manipulations such as transfection and establishment of stable clonal cell lines expressing introduced genes. Proteins that play a key role in the neuronal differentiation of P19 cells are beginning to be identified. These include retinoic acid receptors, the epidermal growth factor receptor and the transcription factors Oct-3 and Brn-2. The biological and technical advantages of this system should facilitate deeper analysis of the activities of proteins that play a role in neuronal differentiation.

Animals↗
PubMed1993

Expression of the genes coding for glutamic acid decarboxylase in pluripotent cell lines.

The expression of glutamic acid decarboxylase (GAD) is a basic characteristic of a wide array of inhibitory neurons the use gamma-aminobutyric acid as a neurotransmitter. Clonal cell models will be essential for investigating the mechanisms which are responsible for the selective expression of GAD. P19 embryonal carcinoma cells are an important model for the analysis of neuronal gene expression. Depending on culture conditions, undifferentiated cells can be induced to form cells as widely divergent as cardiac muscle-like cells and neuron-like and glial-like cells. P19 cells are amendable to a number of powerful genetic manipulations including transformation with foreign DNA and selection of mutants. In this study we used nuclease protection assays and Northern blot analysis to determine if P19 cells express the GAD1 and GAD2 genes. The results show that uninduced P19 cells express these genes at very low but easily detectable levels. When the cells are induced to differentiate along the neuronal pathway with retinoic acid, the levels of transcripts for both GAD genes rise dramatically. At least some RNA transcripts of both genes from induced cells comigrate with the corresponding mRNA from the brain and thus probably represent processed mRNA. The expression of GAD genes in undifferentiated cultures of embryonal stem (ES) cells was also investigated. These cultures express levels of GAD1 transcripts that are higher than uninduced P19 cells. In contrast, expression of the GAD2 gene was barely detectable. These results indicate that P19 EC cells and ES cells will be useful for the investigation of the mechanisms that regulate the expression of the GAD1 and GAD2 genes.

Animals↗
PubMed1992

Brain-stimulation reward: a model for the study of the rewarding effects of abused drugs.

The robustness of the findings of the BSR effects of abused substances indicates that almost any technique used to measure these effects will be useful. The simplest procedure, however, of selecting a single intensity of stimulation and determining the effects of drugs on the rate of response for that selected intensity is fraught with difficulty in interpretation. More than 25 years ago the interpretation of changes in response rate as a reflection of changes in the reward value of the stimulation was challenged (Hodos and Valenstein 1962). Today it is rare to see a single stimulation intensity used in a published manuscript. However, most studies still use procedures of which rate of response is an integral part. Because animals will press a lever more than 80 times per minute and--on a continuous reinforcement schedule--receive as many as 80 stimulations a minute, conclusions about the specificity of the effects of drugs are difficult. The results of BSR experiments on mechanisms of action of abused substances clearly indicate that the reinforcing effect of most, if not all, such substances is probably the result of activation of a reward system that originates in the cell bodies of the ventral tegmental area and courses rostrally to the limbic and frontal projection sites of the mesocortical system. Thus, we believe, the technique has clearly fulfilled its promise as a "window on the brain" (Olds 1977).

Animals↗
PubMed1991

Disruption of the gene encoding the p34/31 polypeptides affects growth and development of Dictyostelium discoideum.

We have used homologous recombination to disrupt the gene which codes for p34 and p31, two polypeptides related to a cAMP-binding protein (CABP1) in Dictyostelium discoideum. By screening a total of 80 independent transformants by Southern blotting, four mutants have been isolated. Two of these mutants were analyzed in detail. Our results indicate that, while a null allele has not been obtained, both mutants express drastically reduced levels of truncated p34 and p31. Phenotypic analysis has demonstrated that both of them grow significantly more slowly than wild-type controls when bacteria are used as a food source. Interestingly, this growth defect is not seen when the cells are cultured axenically. In addition, the mutants possess an altered developmental profile. They complete development approximately 3 h later than wild-type controls. These results indicate that p34 and p31 play roles in both growth and development in this organism.

Alleles↗
PubMed1991

Isolation and characterization of cDNA clones encoding polypeptides related to a Dictyostelium discoideum cyclic AMP binding protein.

By screening a cDNA library with a cDNA encoding the Dictyostelium discoideum cAMP-binding protein CABP1, under conditions of reduced stringency, we have isolated clones which code for two closely related molecules. Hybrid selection experiments indicated that these cDNAs encoded polypeptides with molecular masses of 34 (p34) and 31 (p31) kDa, both of which were recognized by anti-CABP1 monoclonal antibodies. Sequence analysis revealed that the clones were identical except for the presence of a 102 nucleotide segment inserted in-frame in the p34 cDNAs, just downstream of the translation initiation codon. DNA blot analysis suggested that p34 and p31 were encoded by the same gene. This hypothesis was strongly supported by the observation that both polypeptides were generated when a single cDNA was expressed under the control of the actin 15 promoter in D. discoideum cells. RNA blot analysis indicated that the cDNAs were complementary to three developmentally regulated transcripts of sizes 1.15 kb, 1.25 kb and 1.4 kb. Comparison of the derived amino acid sequences of p34 and p31 with those of the two subunits of CABP1 indicated that these polypeptides were very closely related, and that the corresponding genes probably arose by duplication followed by sequence divergence. Finally, the carboxy termini of these four polypeptides demonstrated 50% similarity to two polypeptides encoded by a bacterial plasmid which confers resistance to tellurium anions.

Actins↗
PubMed1990

The molecular basis for alternative splicing of the CABP1 transcripts in Dictyostelium discoideum.

We have determined the nucleotide sequence of the CABP1 gene from Dictyostelium discoideum. Together with previous data on cDNA sequences, we establish that alternative splicing of transcripts derived from this gene is responsible for the production of the two CABP1 subunits. RNA blot analysis suggested that alternative splicing of the CABP1 transcripts occurs during growth and throughout development. In addition, we have compiled the intron sequences of Dictyostelium pre-mRNAs and observed that the GUAAGU hexanucleotide at the 5' splice site is highly conserved. The 5' splice site of CABP1 deviates from the consensus hexanucleotide in having a sequence of GUAAUA. To assess the role of the modified 5' splice on differential splicing, we have constructed an actin-CABP1 fusion gene and transformed it into Dictyostelium cells. Analysis by immunoprecipitation, with anti-CABP1 antibody and amplification of specific cDNAs by polymerase chain reaction show that the transcripts generated by the fusion gene are alternatively spliced. When the 5' splice site of the fusion gene is mutated to conform to the consensus sequence, the resulting transcripts are constitutively spliced. These observations suggest that changes in positions 5 and 6 of the donor splice site are involved in the alternative splicing of the CABP1 transcripts.

Actins↗
PubMed1990

The distribution of changes in local cerebral energy metabolism associated with brain stimulation reward to the medial forebrain bundle of the rat.

Using the quantitative 2-[14C]deoxyglucose autoradiographic method, local rates of glucose utilization were measured in rats during brain stimulation reward to the medial forebrain bundle. Metabolic activation was observed both rostral and caudal to the site of stimulation. These sites included the nucleus accumbens, olfactory tubercle, lateral septum, and ventral tegmental area. In many cases, increases in glucose utilization occurred bilaterally. These data suggest the involvement of both ascending and descending systems in brain stimulation reward. Furthermore, despite the unilateral nature of the electrical stimulation, increases in glucose utilization were observed both ipsilateral and contralateral to the site of stimulation.

Animals↗
PubMed1988

Characterization of an unusual cAMP receptor and its related polypeptides in Dictyostelium discoideum.

Several lines of evidence indicate that cAMP modulates developmental gene activity via cell-surface receptors. We describe here a novel cAMP receptor, CABP1, whose properties are consistent with the idea that this protein is involved in gene regulation. Firstly, immunological techniques using anti-CABP1 antibodies as probes showed that this cAMP receptor can be detected on the surface of developing cells. Secondly, there is a steady migration of CABP1 to the nucleus during development. Thirdly, some genetic variants exhibiting an altered pattern of development are found to possess modified CABP1. We also showed that CABP1 co-purifies with at least seven other polypeptides which share common epitopes with CABP1. Interestingly, four of the CABP1-related polypeptides can be detected on the cell surface as well as in the nucleus.

Dictyostelium↗
PubMed1976

Naloxone blockade of morphine analgesia: a dose-effect study of duration and magnitude.

The purpose of the experiment was to determine the minimal dose of naloxone needed to block the analgesic effect of morphine as measured by the foot-shock titration procedure in the rat. Various groups of rats received, subcutaneously, various doses of naloxone hydrochloride, from 0.03 to 2.0 mg/kg, 15 minutes before receiving 10 mg/kg of morphine sulfate. The results indicate that 1.0 mg/kg of naloxone is the minimum dose necessary to provide full blockade of the measured morphine effect for up to 5 hours after the morphine administration. Partial blockade was evident at doses much smaller than 1.0 mg/kg of naloxone. A dose of 0.125 mg/kg of naloxone completely blocked the onset of a measurable effect from 10 mg/kg of morphine for 60 minutes in six out of the seven animals tested at that dose. The results provide a clear dose-effect relationship for both magnitude and duration of naloxone blockade of the analgesic effect of morphine.

Animals↗
PubMed1968

Morphine: single-dose tolerance.

Rats show a significant degree of tolerance to a second dose of morphine, with the degree of tolerance increasing the longer the delay between the two doses of morphine. To measure the morphine effect a foot-shock attenuation procedure that allowed the animal to adjust the shock intensity was used in studying delays of up to 180 days.

Animals↗