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A Kahn

Publications and source records attributed to A Kahn.

At least 181 records · Page 10Linked to original sources
PubMed1996

Growth and differentiation of C2 myogenic cells are dependent on serum response factor.

In order to study to what extent and at which stage serum response factor (SRF) is indispensable for myogenesis, we stably transfected C2 myogenic cells with, successively, a glucocorticoid receptor expression vector and a construct allowing for the expression of an SRF antisense RNA under the direction of the mouse mammary tumor virus long terminal repeat. In the clones obtained, SRF synthesis is reversibly down-regulated by induction of SRF antisense RNA expression by dexamethasone, whose effect is antagonized by the anti-hormone RU486. Two kinds of proliferation and differentiation patterns have been obtained in the resulting clones. Some clones with a high level of constitutive SRF antisense RNA expression are unable to differentiate into myotubes; their growth can be blocked by further induction of SRF antisense RNA expression by dexamethasone. Other clones are able to differentiate and are able to synthesize SRF, MyoD, myogenin, and myosin heavy chain at confluency. When SRF antisense RNA expression is induced in proliferating myoblasts by dexamethasone treatment, cell growth is blocked and cyclin A concentration drops. When SRF antisense RNA synthesis is induced in arrested confluent myoblasts cultured in a differentiation medium, cell fusion is blocked and synthesis of not only SRF but also MyoD, myogenin, and myosin heavy chain is inhibited. Our results show, therefore, that SRF synthesis is indispensable for both myoblast proliferation and myogenic differentiation.

Animals↗
PubMed1996

Transimmortalized mouse intestinal cells (m-ICc12) that maintain a crypt phenotype.

This study describes the properties of a clone of immortalized cells (m-ICc12 cells) derived from the bases of small intestinal villi from 20-day-old fetuses of L-type pyruvate kinase (L-PK)/ TAg1 transgenic mice. The mice harbor the simian virus 40 large T antigen under the control of the 5' regulatory sequence from the L-PK gene. m-ICc12 cells expressed nuclear large T antigen, had a prolonged life span, and were nontumorigenic when injected into nude mice. They formed confluent monolayers of cuboid cells separated by tight junctions, developed dense, short apical microvilli, and formed domes. They also possessed cytokeratins, villin, aminopeptidase N, dipeptidyl-peptidase IV, and glucoamylase and retained crypt cell features, including intracellular sucrase isomaltase and alpha-L-fucose glycoconjugates accumulation and expression of the polymeric immunoglobulin receptor and the cystic fibrosis transmembrane conductance regulator gene. Thus the m-ICc12 cell line obtained by targeted oncogenesis in transgenic mice maintained in culture several important properties and differentiated functions of intestinal crypt cells.

Animals↗
PubMed1996

Functional and growth properties of a myometrial cell line derived from transgenic mice: effects of estradiol and antiestrogens.

This study describes the properties of a myometrial cell line, m-M116, that was derived from a leiomyoma developed in an adult female transgenic mouse harboring the simian virus 40 large T antigen (Tag) under the control of the 5'-regulatory sequence of the calbindin D9k (CaBP9k) gene. As the expression of this transgene is governed by the CaBP9k estrogen-responsive element, m-M116 cells were grown in medium supplemented with 17 beta-estradiol. The cells were long lived, had Tag-positive nuclei, and were nontumorigenic when injected into nude mice. They formed irregular layers of elongated cells with typical features of uterine, smooth muscle cells, as assessed by the presence of alpha-smooth muscle actin and desmin filaments, estradiol and progesterone receptors, and expression of the CaBP9k gene. The rate of cell doublings and the expression of the Tag gene in early passaged cells depended on the presence of 17 beta-estradiol. Tamoxifen, a mixed estrogen agonist-antagonist, also stimulated the growth of m-M116 cells, whereas ICI 182 780, a pure antiestrogen, blocked cell growth. Later passages of m-M116 cells still had a smooth muscle phenotype, but proliferated even in the absence of 17 beta-estradiol. These mouse uterine smooth muscle cells obtained by targeted oncogenesis provide a useful model for studies of the progression of steroid-independent carcinomas.

Animals↗
PubMed1996

Decreased cardiac responses to auditory stimulation during prone sleep.

OBJECTIVE: To evaluate the relationship between body position during sleep and the infants' cardiac responses to auditory stimulation. METHODS: Thirty healthy infants with a median age of 11 weeks were studied polygraphically for one night, while sleeping successively prone and supine, or vice versa. Their behavioral and cardiac responses were recorded during rapid eye movement (REM) sleep, both before and after exposure to 90 dB (A) of white-noise. RESULTS: Ten infants were excluded from the study, because they woke up during the challenge. For the 20 infants included in the analysis, no significant difference was seen between the prone and the supine position for total sleep time, sleep efficiency, percent of REM and nonrapid eye movement sleep, number of gross body movements, transcutaneous oxygen saturation levels, mean cardiac rate, heart rate variability, number of heart rate drops; mean respiratory rate, and number or duration of central or obstructive apneas. Auditory challenges induced significantly less overall changes in heart rate, less heart rate drops, less heart rate variability, as well as fewer and shorter central apneas in the prone than in the supine position. Autoregressive power spectral analysis of the heart rate was consistent with a possible increase in orthosympathetic tone in the prone position. CONCLUSION: Prone sleeping was associated with a decrease in cardiac responses to auditory stimulation and a possible increase in orthosympathetic activity. Prone positioning could favor a reduced reactivity to danger-signaling stimuli during REM sleep.

Acoustic Stimulation↗
PubMed1996

Technical results from the eversion technique of carotid endarterectomy.

A total of 167 carotid endarterectomies by the eversion technique were completed in 158 patients at a teaching hospital during the 6-year period ending July 1995. The average patient age was 66 years with a range of 39 to 89 years, and 99 (63%) were male. General anesthesia was employed routinely, and temporary indwelling shunts, were not used. Indications for endarterectomy included hemispheric transient ischemic attack (43), amaurosis fugax (20), stroke (41), and asymptomatic stenosis (63). Associated patient risk factors were not significantly different for men and women, and included diabetes mellitus (22%), tobacco abuse (72%), hypertension (69%), hypercholesterolemia (76%), cardiac disease (54%), and renal disease (21%). One (0.6%) permanent operative stroke and two (1%) 30-day hospital deaths occurred. Vascular laboratory follow-up was accomplished by duplex scanning with a documented sensitivity of 98 per cent in detecting a > or = 40 per cent stenosis. Eighty-nine per cent (148) of the 167 endarterectomies were tested at least once postoperatively. Overall laboratory follow-up averaged 17 months and ranged from one to 69 months. Residual stenosis, included perioperative thrombosis, occurred in 8 (5%) arteries. Recurrent stenosis was detected in four (2%) cases at 9, 24, 54, and 66 months after endarterectomy. Statistical analyses failed to implicate any specific patient risk factor, age, sex, or operative indication relevant to recurrent stenosis. Residual stenosis was correlated with younger patient age (P = 0.002), female gender (P = 0.12), and endarterectomy on the right side (P = 0.008). Carotid eversion endarterectomy appears to be a universally applicable, safe, and durable operative technique.

Adult↗
PubMed1996

Tissue specificity of L-pyruvate kinase transgenes results from the combinatorial effect of proximal promoter and distal activator regions.

The L-type pyruvate kinase (L-PK) gene is regulated by diet and hormones and expressed at high levels in the hepatocytes, enterocytes, and proximal tubular cells of the kidney and at low levels in the endocrine pancreatic cells. Two regulatory regions have been shown to be important in transgenic mice to confer on a reporter gene a similar tissue-specific and diet-responsive expression: a proximal promoter fragment, with binding sites for the tissue-specific hepatocyte nuclear factors 1 and 4, and presence of the glucose-response element (GIRE) and a distal activator corresponding to a liver-specific hypersensitive site at -3000 bp with respect to the cap site. Although the proximal promoter is able to confer by itself tissue-specific expression on a reporter gene, its activity in vivo is strongly stimulated by the distal activator. To determine the possible role of the distal region on diet responsiveness and tissue specificity of the L-PK gene expression, we have created lines of transgenic mice in which the gene for SV40 T antigen (Tag) was directed by composite regulatory sequences consisting of the L-PK promoter and different enhancers: either the SV40 early enhancer (SV) or the H enhancer of the aldolase A gene (H). The induction of the composite H-PK/Tag and SV-PK/Tag transgenes by a carbohydrate-rich diet in the liver was similar to that of the endogenous L-PK gene. This suggests that in fasted mice the L-PK promoter, and especially the GIRE, is able to silence the activating influence of a strong viral enhancer such as the SV40 enhancer. The H-PK/Tag mice expressed the transgene similarly to the endogenous gene, except in the pancreas, where expression was practically undetectable. Consistently, whereas L-PK/Tag mice develop insulinomas, H-PK/Tag mice develop only hepatomas. In contrast, the transgene expression was partly aberrant in SV-PK/Tag mice. In addition to a normal activation of the transgene in the liver, a strong expression was also detected in the kidney medulla, whereas the transgene was practically silent in enterocytes. Finally, the effect of the distal region (-2070 to -3200) on an ubiquitous promoter was tested by ligating the distal L-PK gene fragment in front of a thymidine kinase/CAT transgene. Such a transgene was constantly expressed in the pancreas and, strikingly, in the brain. It appears, therefore, that the L-PK distal activator exhibits, by itself, a certain neuropancreatic specificity required in combination with the proximal promoter for L-PK gene expression in pancreas endocrine cells.

Animals↗
PubMed1996

Restoration of hexosaminidase A activity in human Tay-Sachs fibroblasts via adenoviral vector-mediated gene transfer.

Tay-Sachs disease (TSD) is a lysosomal storage disease due to hexosaminidase A deficiency caused by mutations in the gene for alpha-chain (Hex alpha). A human Hex alpha cDNA was subcloned into the adenoviral plasmid pAdRSV. Hex alpha. Replication-deficient adenovirus was generated by homologous recombination in 293 cells. Human fibroblasts from a patient suffering from TSD were infected with the recombinant adenovirus. TSD fibroblasts expressing the recombinant alpha-chain had an enzyme activity on the natural substrate ranging from 40 to 84% of the normal. The corrected cells secreted up to 25 times more Hex alpha than control fibroblasts. The Hex alpha encoded by the adenovirus was shown to be correctly transported into the lysosomes and to normalize the impaired degradation of GM2 ganglioside in TSD fibroblasts.

Adenoviridae↗
PubMed1996

[Gene therapy of neurological diseases].

In hereditary neurological diseases, gene transfer into neurons is made difficult by: the nature of the cells (postmitotic cells, that cannot be cultured, genetically modified ex vivo, then retransplanted), sometimes, their widespread localization, the blood-brain barrier. However, three viral vectors derived from adenovirus, Herpes simplex virus and adeno-associated virus have been shown to be very efficient in transferring DNA into brain cells. All of these vectors can infect resting cells, especially neurons, and are efficient in vivo. Retroviral vectors which can infect dividing cells only are mainly used for ex vivo genetic modification of cells (neural progenitor cells, myoblasts, fibroblasts) followed by intracerebral transplantation. Alternatively, genetically modified cells can be transplanted in a peripheral site if the transgene product is able to cross the blood-brain barrier or to be transported retrogradely from the nerve terminals. We have especially investigated the potential interest of adenoviral vectors to transfer foreign genes into brain cells and to treat animal models of neurological diseases. These vectors allowed us to transfer the lacZ gene into any neural cell type, including neurons, glia, photoreceptors and olfactory receptors, ex vivo, in cell culture, and in vivo, by stereotactic administration. In addition, axonal transport of adenoviral vectors has been demonstrated, e.g. in the substantia nigra after injection into the striatum, in the olfactory bulb after intranasal instillation and in spinal motor neurons after intramuscular injection. After intracerebroventricular injection, ependymal cells are massively infected and express the transgene for several months, as this is also observed in neurons. Through the spinal canal and cerebrospinal fluid, the vector can diffuse to a considerable distance from the injection point, e.g. to the lumbar spinal cord after injection in the suboccipital region. To test the biological function of transgenes transferred through adenoviral vectors, we have constructed vectors with cDNAs or genes for various neutrophic factors: CNTF, NT3, BDNF and GDNF. These vectors were biologically active on target cells, ex vivo and in vivo. In the pmn mouse model of progressive motor neuronal degeneration, some of these vectors, alone or combined, allowed for prolongation of life of homozygous animals by more than two fold, and for decrease in the demyelination of phrenic nerve axons. Finally, we have also constructed an adenoviral vector carrying the alpha-hexosaminidase cDNA, encoding the enzyme subunit deficient in Tay Sachs patients. This vector permitted to normalize ganglioside metabolism in Tay Sachs fibroblasts and is currently tested in knock out mice deficient in hexosaminidase A. In spite of all these encouraging results, we are nevertheless aware that progress in vector design and delivery strategies will be needed before gene therapy can become a realistic therapeutical strategy in humans.

Adenoviridae↗
PubMed1996

Independence and interdependence of the three human aldolase A promoters in transgenic mice.

The human aldolase A gene is transcribed from three alternative promoters, clustered in a small 1.6-kb DNA domain. In transgenic mice, the upstream pN and the downstream pH promoters are ubiquitous, whereas the pM promoter, located between pN and pH, is activated specifically in fast skeletal muscles. A strong ubiquitous enhancer, lying upstream of the pH promoter, is necessary for both pN and pH ubiquitous activities, whereas a fast-muscle-specific enhancer, located upstream of the pM promoter, is required for pM-specific activation. In the present study, we use the transgenic mice model to further investigate the contribution of these two regulatory elements to the overall control of these three promoters. We confirm that the pM and pH promoters are activated independently of each other and, in particular, we show that the activation of pM in fast muscle is not responsible for the downregulation of the downstream pH in this tissue. By contrast, the pN promoter needs the presence of both enhancers to reproduce its correct pattern of activity and is unable to function autonomously in vivo.

Animals↗
PubMed1995

Myotube-specific activity of the human aldolase A M-promoter requires an overlapping binding site for NF1 and MEF2 factors in addition to a binding site (M1) for unknown proteins.

The human aldolase A gene is expressed in several tissues through the use of three alternative promoters. The activity of one of the promoters, pM, is restricted to skeletal muscle. We reported previously that a proximal 280 bp pM fragment confers tissue-specific expression to a CAT reporter gene in transgenic mice. This small regulatory region directs expression to muscle composed mainly of fast-twitch fibers. Here we show that a minimal promoter fragment from base-pairs -164 to +45 is sufficient to highly active pM during myoblast differentiation in cell culture and demonstrate that two DNA elements play a major role in this activation. These elements consist of a binding site (M1) for unknown ubiquitous proteins and an overlapping binding site for MEF2 and NF1 families of transcription factors. The NF1 factor constitute the main binding activity on the MEF2/NF1 site and, interestingly, some of the DNA-protein complexes that form with muscle nuclear extracts on the NF1 element differ from those that form with non-muscular extracts.

Animals↗
PubMed1995

[Gene therapy of cancer].

Therapeutic gene transfer for cancer can aim at different goals, the main of them being: 1. to increase anti-tumor cell immune response; 2. to introduce a drug-activating gene into tumor cells; 3. to normalize cell cycle by inhibiting oncogenes or transferring antioncogenes. Significant, sometimes spectacular results have been reported using models of transplantation of tumor cell lines to animals, but not yet with spontaneous animal tumors. Although most of the about 130 clinical trials of gene therapy authorized around the world are devoted to cancer, it is so far impossible to predict the future of this strategy, and when, if any, it will succeed.

Animals↗
PubMed1995

Functional dissection of the brain-specific rat aldolase C gene promoter in transgenic mice. Essential role of two GC-rich boxes and an HNF3 binding site.

The aldolase C gene product is a glycolytic isoenzyme specifically detected in brain. We have previously defined a short 115-base pair promoter fragment able to confer on a reporter chloramphenicol acetyltransferase (CAT) gene a specific expression in brain of transgenic mice. In this promoter fragment, two GC-rich regions (A/A' and B boxes) were detected by in vitro DNase1 footprinting experiments with brain, fibroblast, or liver nuclear extracts. Both A/A' and B boxes, sharing structural homology, are able to interact with Sp1, Krox20/Krox24 factors and with other proteins (Thomas, M., Makeh, I., Briand, P., Kahn, A., and Skala, H. (1993) Eur. J. Biochem. 218, 143-151). In this paper, we describe a new ubiquitous factor termed Ub able to bind the A/A' box. We also delimit a third element (box C) binding a hepatocyte-enriched protein displaced by a hepatocyte nuclear factor 3-specific oligonucleotide. The functional involvement of each binding site in brain-specific transcription of the aldolase C gene has been tested in transgenic mice carrying different mutant promoters cloned in front of the CAT gene. A promoter containing only box C was totally inactive, suggesting an essential role of the region containing A/A' and B boxes. However, mutations or deletions of either the A/A' or the B box have no significant effect on the CAT gene expression. We therefore hypothesize that the A/A' and B sites may be functionally redundant. Indeed, constructs harboring only one of these two boxes (A/A' or B) linked to the C box displayed a brain-specific CAT activity similar to that obtained with the wild-type promoter. Furthermore, a transgene with disruption of the C box, keeping intact the A/A' and B boxes, was totally inactive, suggesting a crucial role of the hepatocyte nuclear factor 3 binding site in activation of the aldolase C gene.

Animals↗
PubMed1995

Identification and functional characterization of an erythroid-specific enhancer in the L-type pyruvate kinase gene.

The rat L-type pyruvate kinase gene is transcribed either from promoter L in the liver or promoter L' in erythroid cells. We have now cloned and functionally characterized an erythroid-specific enhancer, mapped in the fetal liver as hypersensitive site B (HSSB) at 3.7 kilobases upstream from the promoter L'. Protein-DNA interactions were examined in the 200-base pair core of the site by in vivo footprinting experiments. In the fetal liver, footprints were revealed at multiple GATA and CACC/GT motifs, whose association is the hallmark of erythroid-specific regulatory sequences. Functional analysis of the HSSB element in transgenic mice revealed properties of a cell-restricted enhancer. Indeed, this element was able to activate the linked ubiquitous herpes simplex virus thymidine kinase promoter in erythroid tissues. The activation was also observed in a variety of nonerythroid tissues known to synthesize GATA-binding factors. In the context of L'-PK transgenes, HSSB was not needed for an erythroid-specific activation of the L' promoter, while it was required to stimulate the L' promoter activity to a proper level. Finally, HSSB cannot be replaced by strong ubiquitous viral or cellular enhancers, suggesting a preferential interaction of the HSSB region with the L' promoter.

Animals↗
PubMed1995

Glucose responsiveness of a reporter gene transduced into hepatocytic cells using a retroviral vector.

An MMLV-based retroviral vector containing the chloramphenicol acetyl transferase reporter gene under the control of a glucose-dependent internal promoter derived from the L-type pyruvate kinase gene was constructed. After transfection into psi-CRIP packaging cells, clones producing recombinant retrovirus were selected. These retroviruses were used to infect cultured established hepatocytic cells whose endogenous L-type pyruvate kinase gene is transcriptionally regulated by glucose. In the infected cells, the reporter gene was as responsive to glucose as the endogenous L-type pyruvate kinase gene, and the glucose gene activation was time- and concentration-dependent. The possibility to confer a glucose responsiveness on a transgene carried by a retroviral vector provides a powerful tool in the prospect of gene therapy for diabetes mellitus.

Animals↗
PubMed1995

Pulmonary surfactant protein A (SP-A) is expressed by epithelial cells of small and large intestine.

Surfactant protein A (SP-A) is the most abundant protein associated with phospholipids in pulmonary surfactant. There are several lines of evidence that pulmonary and gastrointestinal epithelium produce closely related surface-active materials, although the presence of SP-A in gastrointestinal tract has so far not been reported. Indirect immunofluorescence experiments using different antibodies raised against rat pulmonary SP-A showed that some jejunal and colonic but not gastric epithelial cells positively stained for SP-A. Analysis of the proteins in cell lysates from rat small intestine and colon studied by Western blot revealed several immuno-reactive bands, including the characteristic triplet of 26-, 32-, and 38-kDa monomeric proteins, less strongly labeled than in lung cells, and higher molecular mass forms of 66 and 120 kDa also present in lung cells. The 66- and 120-kDa bands displayed the expected isoelectric pH of SP-A after two-dimensional electrophoresis. Alkylation induced conversion of the 120-kDa form (almost completely) and the 66-kDa form (partly) into the 26-38-kDa monomeric species. The presence of SP-A mRNA in rat stomach, small intestine, and colon was then searched for by conventional cDNA/reverse transcriptase-polymerase chain reaction. Products of appropriate size (372 base pairs) identical to that of pulmonary tissue were amplified in small intestine and colon but not in stomach or in other tissues used as controls. Cloning and sequencing of rat colon SP-A cDNA revealed the same sequence as the one reported for rat lung SP-A. Furthermore, analysis of the transcriptional initiation site of SP-A gene in colon by anchored-polymerase chain reaction showed that transcription was initiated at the same site in both colon and lung. These data, which demonstrate that small intestine and colon express SP-A constitutively and that this protein is present in some epithelial cells, extend the concept of intestinal surfactant and underline its close relationships to pulmonary surfactant.

Animals↗