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Biomedical subjects

G Grimber

Publications and source records attributed to G Grimber.

At least 37 records · Page 2Linked to original sources
PubMed1992

Inherited haemolytic anaemia created by insertional inactivation of the alpha-spectrin gene.

In the process of generating transgenic mice, inserted foreign DNA can cause insertional inactivation of the flanking genetic locus and simultaneously provide a molecular tag for localizing and cloning the inactivated gene. We describe the case of an insertional mutation leading, in animals homozygous for the insertion, to severe anaemia that was lethal within a few days after birth. The haemolytic anaemia and microspherocytosis of the red cells strongly suggested membrane abnormalities of the erythrocytes. By in situ localization of the integration site, protein analysis of the red cell membranes, northern and Southern blot analyses, we were able to demonstrate that the integrated transgene had affected the alpha-spectrin gene locus.

Anemia, Hemolytic↗
PubMed1992

Tissue expression pattern directed in transgenic mice by the LTR of an HTLV-I provirus isolated from a case of tropical spastic paraparesis.

Human T lymphotropic virus type I (HTLV-I) causes adult T cell leukemia/lymphoma and a chronic neurological disease called either tropical spastic paraparesis (TSP) or HTLV-I-associated myelopathy. The different outcomes of this infection could be due to both host and viral factors and it has been proposed that genetic differences could make some HTLV-I strains neurotropic. In this paper, we examined the pattern of tissue-specific expression determined by a long terminal repeat (LTR) obtained from a case of TSP. We constructed transgenic mice in which this LTR controlled the expression of the nlslacZ reporter gene. We observed that in three independent lines of transgenic mice, the reporter gene was expressed predominantly in the central nervous system (CNS), in choroid plexus, and in cells of the hippocampus and cerebellum. Our observations indicate the existence of CNS cells permissive for the expression of HTLV-I and which may be of importance in the pathogenesis of TSP.

Animals↗
PubMed1992

Diet-dependent carcinogenesis of pancreatic islets and liver in transgenic mice expressing oncogenes under the control of the L-type pyruvate kinase gene promoter.

The liver-type pyruvate kinase (L-PK) gene is controlled positively by insulin and carbohydrates, negatively by glucagon and fasting. Diet-inducible models of carcinogenesis were obtained using the L-PK gene promoter and regulatory sequences to control the expression of c-myc and SV40 T oncogenes in transgenic mice. L-PK/c-myc and L-PK/Tag animals fed a carbohydrate-rich diet developed hepatocarcinomas. In addition, L-PK/Tag animals developed diet-dependent, aggressive endocrine pancreatic tumors, preceded by islet hyperplasia involving the different analysed cell populations (alpha, beta and delta). Expression of the L-PK gene was demonstrated in pancreatic tumors, in rat isolated islets and in rat insulinoma-derived cells (RIN line), revealing a new tissue specificity of the L-PK gene. Our results suggest that this gene may be expressed in islet progenitor cells from which the different mature endocrine cells derive.

Animals↗
PubMed1991

Neuronal-specific expression of human copper-zinc superoxide dismutase gene in transgenic mice: animal model of gene dosage effects in Down's syndrome.

It has been suggested that copper-zinc superoxide dismutase (CuZn SOD) increment, by accelerating hydrogen peroxide formation, might promote oxidative damage within trisomy 21 cells and might be involved in the various neurobiological abnormalities found in Down's syndrome such as premature aging and Alzheimer-type neurological lesions. In order to test this hypothesis, we have developed strains of transgenic mice carrying the human CuZn SOD gene. The human transgene expression resulted in increased CuZn SOD activity predominantly in the brain (1.93 fold). Immunohistochemical and in situ hybridization analysis of brain sections revealed that human CuZn SOD protein and mRNA was preferentially expressed in neurons, particularly in pyramidal cells of Ammon's horn and granule cells of gyrus dentate. The amount of thiobarbituric acid (TBA)-reactive material was significantly higher in transgenic brains compared to controls, strongly suggesting an increased level of peroxidation in vivo. These results support the notion that CuZn SOD gene dosage effect could play a role in the pathogenesis of rapid aging features in the brain of Down's syndrome patients.

Aging↗
PubMed1991

Time-course development of differentiated hepatocarcinoma and lung metastasis in transgenic mice.

A precise targeting of the SV40 T early region expression in the liver of transgenic mice was obtained using 700 bp of the antithrombin III regulatory sequences to control oncogene expression. In the strain expressing the highest level of large T antigen (Tag), the incidence of hepatocarcinoma was 100%. The evolution was reproducible and characterized by a marked cytolysis occurring as early as 4 weeks, when no morphological and histological modifications were visible, a preneoplastic state marked by a progression from hyperplasia to proliferative nodules composed of highly differentiated cells exhibiting a high Tag expression, which elicited tumor formation in nude mice and could proliferate in vitro, and hepatocellular carcinoma associated, in 10% of the cases, with lung metastasis. These transgenic mice constituted a useful model for therapeutic assays and fundamental studies on carcinogenesis.

Animals↗
PubMed1991

Transcriptional selectivity in early mouse embryos: a qualitative study.

The mouse zygotic genome is activated at the 2-cell stage. At this stage, microinjected DNA can be expressed and its transcription, analysed qualitatively with LacZ reporter genes, has the following characteristics (i) Sp1-sensitive promoters are active; (ii) the SV40 early promoter does not require upstream enhancers; (iii) genes driven by the -447, +563 region of murine leukemia virus (M-MuLV) are repressed and; (iv) activation of promoters is possible as shown for the promoter of acetylcholine receptor alpha-subunit by MyoD. This transactivation can occur before the formation of the zygotic genome. The transcriptional selectivity of 2-cell embryos also characterizes oocytes and 4-cell embryos. Therefore the elements involved are present in the oocytes and they persist after fertilization. This transcriptional selectivity has numerous common characteristics with that in EC cells, and may be indicative of a genetic control program specific for multipotential cells.

Animals↗
PubMed1991

Expression of human Cu-Zn superoxide dismutase gene in transgenic mice: model for gene dosage effect in Down syndrome.

It was suggested that increased Cu-Zn superoxide dismutase (SOD-1) might be involved in the various biological abnormalities found in Down's syndrome (DS) such as premature aging and Alzheimer-type neurological lesions. As a model system for testing this hypothesis we have developed two strains of transgenic mice carrying only one copy of the human SOD-1 gene. In the first strain (TG1), no expression has been found by northern blot analysis. The second strain (TG2) exhibited human SOD-1 mRNA and increased SOD-1 activity in the brain (1.93 fold), in the heart (1.69 fold), thymus (1.49 fold) and to a lesser extent in muscle (1.25 fold), liver (1.19 fold), kidney (1.18 fold), spleen (1.35 fold), lung (1.26 fold) and erythrocytes (1.09 fold). In this strain, increased SOD-1 activity in the brain did not induce modifications in the seleno-dependent glutathione peroxidase, glutathione reductase and glutathione S-transferase activities. In brain homogenates, we have focused our studies on Tau proteins which are known to be the major antigenic components of paired helical filaments (PHF), both in DS and Alzheimer's disease. Our results suggested that, in our experimental conditions, the overexpression of SOD-1 did not induce the modifications of Tau proteins similar to those seen during neurofibrillary degeneration.

Animals↗
PubMed1990

Patterns of expression of position-dependent integrated transgenes in mouse embryo.

The abilities to introduce foreign DNA into the genome of mice and to visualize gene expression at the single-cell level underlie a method for defining individual elements of a genetic program. We describe the use of an Escherichia coli lacZ reporter gene fused to the promoter of the gene for hypoxanthine phosphoribosyl transferase that is expressed in all tissues. Most transgenic mice (six of seven) obtained with this construct express the lacZ gene from the hypoxanthine phosphoribosyltransferase promoter. Unexpectedly, however, the expression is temporally and spatially regulated. Each transgenic line is characterized by a specific, highly reproducible pattern of lacZ expression. These results show that, for expression, the integrated construct must be complemented by elements of the genome. These elements exert dominant developmental control on the hypoxanthine phosphoribosyltransferase promoter. The expression patterns in some transgenic mice conform to a typological marker and in others to a subtle combination of typology and topography. These observations define discrete heterogeneities of cell types and of certain structures, particularly in the nervous system and in the mesoderm. This system opens opportunities for developmental studies by providing cellular, molecular, and genetic markers of cell types, cell states, and cells from developmental compartments. Finally this method illustrates that genes transduced or transposed to a different position in the genome acquire different spatiotemporal specificities, a result that has implications for evolution.

Animals↗
PubMed1990

In vivo activation by ultraviolet rays of the human immunodeficiency virus type 1 long terminal repeat.

It has been previously shown in vitro that the human immunodeficiency virus type 1 long terminal repeat (LTR) is activated by ultraviolet irradiation. In order to analyze if a similar effect could occur in vivo, transgenic mice carrying the lacZ gene under the control of the viral LTR were irradiated at 280-300 and 254 nm. These mice spontaneously expressed the transgene in the epidermis and the lens of both adults and embryos. Irradiations caused a significant increase in skin beta-galactosidase activity. This phenomenon might be involved in viral activation and could be of interest in regard to the skin pathology observed during an HIV infection.

Animals↗
PubMed1988

Correction of mouse ornithine transcarbamylase deficiency by gene transfer into the germ line.

The sparse fur with abnormal skin and hair (Spf-ash) mouse is a model for the human X-linked hereditary disorder, ornithine transcarbamylase (OTC) deficiency. In Spf-ash mice, both OTC mRNA and enzyme activity are 5% of control values resulting in hyperammonemia, pronounced orotic aciduria and an abnormal phenotype characterized by growth retardation and sparse fur. Using microinjection, we introduced a construction containing rat OTC cDNA linked to the SV40 early promoter into fertilized eggs of Spf-ash mice. The expression of the transgene resulted in the development of a transgenic mouse whose phenotype and orotic acid excretion are fully normalized. Thus, the possibility of correcting hereditary enzymatic defect by gene transfer of heterologous cDNA coding for the normal enzyme has been demonstrated.

Amino Acid Metabolism, Inborn Errors↗
PubMed1987

A new family affected by the syndrome of hyperornithinaemia, hyperammonaemia and homocitrullinuria.

We have reported three cases of hyperornithinaemia associated with hyperammonaemia and homocitrullinuria (HHH). They deal with two brothers and a sister from a family where the parents and four other children are healthy on clinical and biochemical examination. The biochemical findings in our patients indicate the existence of a defect in the transport of ornithine into the mitochondria. Cultured skin fibroblasts from two of these patients incorporated six times less [14C]ornithine into protein as compared to control cells. The most characteristic sign of the clinical picture is the progressive spastic paraparesis found in one of the cases. Ornithine supplementation and restricted protein intake may be useful in the treatment of this syndrome since after three years of treatment the clinical response was favourable and the patients showed no adverse clinical effects.

Adolescent↗
PubMed1987

A beta-galactosidase hybrid protein targeted to nuclei as a marker for developmental studies.

The Escherichia coli lacZ gene has been used as an indicator gene for the study of cell lineage in vivo. To adapt this marker for gene expression studies, a sequence encoding a modified beta-galactosidase and including the simian virus 40 large tumor nuclear location signal (nls-beta-Gal) has been introduced into vectors. In differentiated cells, multipotential cells, and embryos, the constructs led to the expression of an enzymatically active protein. Its location was examined by its beta-galactosidase activity or by using antibodies and electron microscopy. The results show that the nls-beta-Gal protein remains mainly located at the nuclear periphery (probably at the nuclear pores) but does not reach the nucleoplasm. It suggests that an interaction with the nuclear membrane is necessary but not sufficient for protein uptake into the nucleus. In multipotential cells, the expression of nuclear location signal LacZ (nls-LacZ) interferes neither with cell growth nor with differentiation. Using various lacZ constructs, the transcriptional activity of embryos was studied. At the two-cell stage, the promoters of the Rous sarcoma virus, simian virus 40, and the beta-actin gene are functional but the Moloney murine leukemia virus long terminal repeat is not. Thus, transcriptional specificity must already be present at the stage of activation of the embryonic genome.

Animals↗
PubMed1984

Potentiation by ammonia of the metabolic effects of pent-4-enoate in isolated rat hepatocytes.

The metabolic effects of pent-4-enoate were studied in isolated rat hepatocytes; 1 mM-pent-4-enoate did not significantly inhibit gluconeogenesis from lactate, alanine and glycerol, but significantly decreased glucose synthesis from pyruvate. The addition of 1 mM-NH4Cl led to a drastic inhibition of glucose synthesis from all these substrates. In hepatocytes incubated with 10 mM-alanine and 1 mM-oleate, pent-4-enoate at 0.05-1 mM slightly inhibited glucose synthesis and ketogenesis. The addition of ammonia resulted in a dramatic potentiation of the metabolic effects of pent-4-enoate. Half-maximum effect of ammonia was observed at 0.2 mM concentration. Concomitant cellular concentrations of ATP and acetyl-CoA were also decreased by the addition of ammonia, as were lactate/pyruvate ratio and beta-hydroxybutyrate/acetoacetate ratio. These data suggest that ammonia seriously interferes with the cellular metabolism of pent-4-enoate and leads to a dramatic potentiation of its effects.

Acetyl Coenzyme A↗
PubMed1984

Potentiation by piridoxilate of the synthesis of hippurate from benzoate in isolated rat hepatocytes. An approach to the determination of new pathways of nitrogen excretion in inborn errors of urea synthesis.

The synthesis of hippurate from benzoate as compared to ureagenesis has been investigated in isolated rat hepatocytes. Half-maximal synthesis of hippurate was observed at 0.3 mmol/l benzoate. In the presence of 1 mmol/l benzoate, hippurate synthesis proceeded linearly with time at a rate of 40 +/- 10 mumol X h-1 X g-1 dry weight. This provided less than 10% of nitrogen epuration supported by concomitant urea synthesis (350 +/- 82 mumol X h-1 X g-1 dry weight). The incorporation of benzoate to hippurate was markedly limited by the availability of glycine. Half-maximal hippurate synthesis was observed at 2 mmol/l glycine. In the absence of glycine, piridoxilate, a glyoxylate derivative, markedly potentiated hippurate synthesis. Half-maximal stimulation was observed at 10 mmol/l piridoxilate. In the presence of 10 mmol/l piridoxilate, hippurate synthesis (420 +/- 35 mumol X h-1 X g-1 dry weight) provided more than 50% of nitrogen epuration supported by urea synthesis. It is concluded that supplementation with nitrogen-free analogues of glycine such as piridoxilate are required to potentiate hippurate synthesis in an attempt to replace ureagenesis as an alternative pathway of waste nitrogen excretion in inborn errors of urea synthesis.

Alanine↗
PubMed1984

Inhibition of urea synthesis by pent-4-enoic acid: potentiation by ammonia.

Pent-4-enoic acid inhibited ureagenesis approximatively 90% in rat hepatocytes incubated with pyruvate, ammonia and ornithine. Inhibition of ureagenesis was much less with alanine as substrate (approximatively 10%). The addition of ammonia led to a drastic dose-dependent inhibition of ureagenesis by pent-4-enoate. Half-maximum effect of ammonia was observed at 0.2 mM concentration. Concomitant cellular concentrations of N-acetylglutamate were also drastically modified by the addition of ammonia as was the accumulation of citrulline. These data suggest that ammonia may seriously interfere with the metabolism of pent-4-enoic acid and leads to a dramatic potentiation of its toxicity.

Ammonium Chloride↗
PubMed1983

Role of N-acetylglutamate and acetyl-CoA in the inhibition of ureagenesis by isovaleric acid in isolated rat hepatocytes.

1 and 10 mmol/l isovalerate strongly inhibited urea synthesis in isolated rat hepatocytes incubated with 10 mmol/l alanine and 3 mmol/l ornithine. Isovalerate also markedly decreased N-acetylglutamate levels, and the decrease correlated with the inhibition of urea synthesis by isovalerate. This compound also lowered cellular levels of acetyl-CoA, a substrate of N-acetylglutamate synthase (EC 2.3.1.1). Isovalerate did not significantly affect the cellular levels of ATP and had no direct effect on N-acetylglutamate synthase activity. These results suggest that the inhibition of urea synthesis by isovalerate is due to decrease in N-acetylglutamate levels.

Acetyl Coenzyme A↗
PubMed1983

Inhibition of ureagenesis by valproate in rat hepatocytes. Role of N-acetylglutamate and acetyl-CoA.

Valproate (0.5-5 mM) strongly inhibited urea synthesis in isolated rat hepatocytes incubated with 10 mM-alanine and 3 mM-ornithine. Valproate at the same concentrations markedly decreased concentrations of N-acetylglutamate, an essential activator of carbamoyl-phosphate synthetase I (EC 6.3.4.16), in parallel with the inhibition of urea synthesis by valproate. This compound also lowered the cellular concentration of acetyl-CoA, a substrate of N-acetylglutamate synthase (EC 2.3.1.1); glutamate, aspartate and citrulline were similarly decreased. Valproate in a dose up to 2 mM did not significantly affect the cellular concentration of ATP and had no direct effect on N-acetylglutamate synthesis, carbamoyl-phosphate synthetase I and ornithine transcarbamoylase (EC 2.1.3.3) activities.

Acetyl Coenzyme A↗