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A Kahn

Publications and source records attributed to A Kahn.

At least 37 records · Page 2Linked to original sources
PubMed1991

Interplay of an original combination of factors: C/EBP, NFY, HNF3, and HNF1 in the rat aldolase B gene promoter.

The rat aldolase B 5' flanking region (nucleotides - 194 to +41) contains sufficient information for liver-specific expression. A detailed investigation of factors binding to the rat aldolase B 5' flanking region has allowed us to identify three distinct factors that filled different sites of this region (A, B, C). The liver-enriched C/EBP or related factors bind to box C, as demonstrated by the specific interaction with bacterially expressed C/EBP protein. Box B bearing the CCAAT sequence binds the ubiquitous factor NFY. Surprisingly, Box A is able to bind two liver enriched factors, namely HNF1 and HNF3. However, in the context of the intact promoter, as shown by footprinting competition experiments, HNF3 binds solely to this sequence. HNF3, but not HNF1 is a transcriptional activator as demonstrated in the in vitro transcription assay.

Animals↗
PubMed1991

A ubiquitous enhancer shared by two promoters in the human aldolase A gene.

The human aldolase A gene is transcribed from three different promoters, which are all clustered within a 1.6 kbp DNA domain. Two of these, PN and PH, are ubiquitous and seem to be co-regulated in most tissues while the third one, PM, is specific to adult skeletal muscle. We investigated the sequences involved in the ubiquitous activity of the PN and PH promoters of the human aldolase A gene. Deletion analysis, performed by transient expression assays of chloramphenicol acetyltransferase reporter genes in human HepG2 hepatoma cells, indicated that PH activity results from the interaction of an upstream activating region with two distinct core promoters. The upstream activating region was able to stimulate transcription from the HSV tk promoter as efficiently as the SV40 enhancer in all cell types tested. It appears, therefore, to be a strong ubiquitous enhancer. DNAsel footprinting revealed protections covering sequences scattered along the enhancer, including Sp1 and AP1 motifs. Importantly, we found that this enhancer was also necessary to activity of the other ubiquitous promoter of the aldolase A gene, PN. These studies demonstrate that expression of the human aldolase A gene is mediated by a complex interplay of enhancer and promoter elements.

Base Sequence↗
PubMed1991

Illegitimate (or ectopic) transcription proceeds through the usual promoters.

Illegitimate transcription corresponds to the low level presence of specific transcripts in nonspecific cells. This phenomenon allows to analyse any tissue-specific disease transcript in any easily accessible cell. We demonstrate here that the start sites of transcription are the same in specific and non-specific cells, which indicates that illegitimate transcription is due to a low level activity of the normal promoter. In addition, it is possible to increase about 10 fold the abundance of illegitimate transcripts through the use of cycloheximide. This treatment should, therefore, facilitate detection and qualitative analysis of illegitimate transcripts.

Base Sequence↗
PubMed1991

Anti-Müllerian hormone Bruxelles: a nonsense mutation associated with the persistent Müllerian duct syndrome.

The persistent Müllerian duct syndrome (PMDS) is characterized by the persistence of Müllerian derivatives, uterus and tubes, in otherwise normally virilized males. In a previous study, we showed that this syndrome is heterogeneous, with lack of production of anti-Müllerian hormone (AMH) by testicular tissue accounting for only some, AMH-negative, cases of this disorder. We have characterized the point mutation responsible for an AMH-negative PMDS in three siblings: a guanine to thymine transversion at position 2096 in the fifth exon changes a GAA triplet, coding for glutamic acid, to a TAA stop codon. The mutation could also be recognized, using the polymerase chain reaction, on RNA produced in trace amounts by a lymphoblastic cell line. The translation product, although undetectable in testicular tissue, could be visualized in culture medium of cells transfected with the mutant gene.

Anti-Mullerian Hormone↗
PubMed1991

Expression of the rat aldolase B gene: a liver-specific proximal promoter and an intronic activator.

The nature and location of the cis-acting DNA sequences regulating expression of the rat aldolase B gene has been investigated. Two liver-specific DNAse I hypersensitive sites were detected, one located just upstream from the cap site, the second in the middle of the first, 4.8-kbp-long, intron. A fragment of 190 bp 5' to the cap site behaved as a tissue-specific but weak core promoter: it directed a detectable reporter gene expression in the Hep G2 cells and hepatocytes, but not in fibroblasts. The tissue-specific expression was stimulated at least 16 fold when constructs contained the entire first intron. The intronic activating sequences could be ascribed to an inner 2 kbp fragment in which the downstream liver-specific DNAse I hypersensitive site was located.

Chloramphenicol O-Acetyltransferase↗
PubMed1991

cis-acting DNA elements regulating expression of the liver pyruvate kinase gene in hepatocytes and hepatoma cells. Evidence for tissue-specific activators and extinguisher.

To identify the DNA sequences that cis-regulate the expression of the rat liver pyruvate kinase (L-PK) genes, a series of constructs in which the chloramphenicol acetyltransferase reporter genes is driven by various deleted fragments of the 3200 base pairs (bp) upstream of the L-PK gene cap site have been assayed for transient expression after introduction into hepatoma HepG2 cells, rat hepatocytes in primary culture, fibroblast LTK- cells, myogenic C2C12 cells, and CHO cells. Four distinct regulatory domains have been characterized. A proximal promoter region containing a binding site for the hepatocyte nuclear factor 1 (HNF1) which is sufficient to confer liver specificity, even in the presence of a ubiquitous enhancer. A distal promoter region (-96 to -283 bp) containing binding sites for the liver-specific factor A1 (LFA1), the ubiquitous nuclear factor 1 (NF1), the major late transcriptional factor (MLTF), and so far unidentified proteins binding to the L5-PK region which is essential to maximally activate expression of the construct in HepG2 cells. An extinguisher region, located between positions -2082 and -1170 bp, which decreases efficiency of the L-PK promoter in HepG2 cells, but not in hepatocytes in primary culture. Finally, a far upstream region (-2900 to -2500 bp) which seems to correspond to a liver-specific DNase I hypersensitive site and which behaves in HepG2 cells as an activating sequence efficient in the absence of the extinguisher.

Animals↗
PubMed1991

Regulation of the multiple promoters of the human aldolase A gene: response of its two ubiquitous promoters to agents promoting cell proliferation.

The human aldolase A gene is transcribed from three distinct promoters, the two ubiquitous promoters PN and PH and the muscle specific promoter PM. In the present study, we investigate further aldolase A mRNA structure and expression. We demonstrate that the upstream N-type exon is, in fact, extremely heterogeneous. RNAse H mapping experiments permit quantification of relative abundance of N, M, and H type mRNAs and show that the level of transcripts containing the downstream H-type exon is at least 30 times higher than that of those containing N exon, in all tissues tested. Aldolase A level is up-regulated in proliferating cells. Here we show that both N and H type mRNAs, although barely detectable in normal liver, are highly expressed in human hepatomas biopsies. Furthermore, in human lymphocytes, N-type mRNA level is enhanced by serum treatment, while in cultured Hep G2 cells, both N-type and H-type mRNA levels are increased by serum and by the tumor promoting agent PMA. Using CAT constructs in transfection experiments, we demonstrate that the H exon plus its upstream region can function autonomously: the 420 base pairs upstream of the H exon are sufficient to confer to promoter PH an efficiency comparable that of the complete SV40 early promoter and enhancer in two cell lines.

Base Sequence↗
PubMed1991

Glucose-dependent and -independent effect of insulin on gene expression.

Insulin action on gene expression can be glucose-dependent or -independent. Accumulation of aldolase B mRNA and of an unidentified 5.4-kilobase mRNA as well as accumulation of L-type pyruvate kinase mRNAs (Decaux, J.F., Antoine, B., and Kahn, A. (1989) J. Biol. Chem. 264, 11584-11590) in cultured hepatocytes isolated from fasted rats require the presence of both glucose and insulin, these agents not being effective individually. In contrast, maintaining the amount of albumin and transferrin mRNAs in these hepatocytes requires the presence of insulin alone, glucose having no effect by itself. Transcription of the albumin gene, investigated by run-on assay, is active in the presence of insulin alone, with or without glucose, whereas transcription of the aldolase B gene is stimulated by glucose and insulin together, but not by insulin or glucose alone. In addition, the stability of the albumin and aldolase B mRNAs in cultured hepatocytes is lowered in the absence of glucose and insulin together as compared to the stability in the presence of one or both agents. These results confirm that transduction of the insulin signal occurs via distinct pathways; one of these pathways could involve a secondary insulin-dependent modification of metabolite concentration, whereas other pathways could be more directly related to the activity(ies) of the occupied insulin receptor.

Albumins↗
PubMed1991

Evidence of proteoglycan/proteoglycan interactions within aggregates.

Nonaggregated proteoglycan monomers, digested fragments of the monomers, as well as link proteins have been shown to self-associate. These associations have not been shown to occur on the aggregate. However, previous reports, using the Kleinschmidt technique of monolayer electron microscopy, have noted proteoglycan subunits on the aggregate that appear to interact, either as branched proteoglycans or as proteoglycan subunits that appear to share the same attachment site on the hyaluronic acid chain. Branching and shared attachments were noted in all aggregates analyzed in this study. Increasing the average space between proteoglycan subunits on the reconstituted aggregate resulted in a significant decrease in branched proteoglycans, indicating either a weak association occurring on the aggregate, or an artifact created by a three-dimensional structure being reduced to a two-dimensional monolayer image. The shared attachments were independent of both the presence of link proteins and changes in spacing between proteoglycans, suggesting a proteoglycan-proteoglycan interaction occurring before aggregation. The interactions were not influenced by proteoglycan concentration at the time of aggregation. Link proteins, however, did increase the number of proteoglycans on the aggregate that could be cross-linked with a bifunctional reagent, suggesting that link proteins facilitate proteoglycan-proteoglycan interactions.

Animals↗
PubMed1991

Positive and negative regulation of gene expression by insulin and glucagon: the model of L-type pyruvate kinase gene.

L-type pyruvate kinase gene regulation is an excellent model of gene control by hormones and diet. In vivo and ex vivo experiments allowed us to established that thyroid hormones and glucocorticoids act on pyruvate kinase gene expression at the post-transcriptional level. In contrast, glucose and insulin together stimulate transcription of this gene while glucagon inhibits it. Insulin or glucose are individually inefficient and glucagon-dependent transcriptional inhibition seems to be dominant in insulin + glucose-dependent activation. A 14-kbp fragment encompassing the entire pyruvate kinase gene and 3.2-kbp of 5' flanking sequences is expressed in transgenic mice exactly like the endogenous gene; the 3.2-kbp upstream region is sufficient to confer this tissue-specific and hormone/diet-regulated expression to reporter genes. In vivo, DNAse I hypersensitivity analysis revealed the presence of 3 liver-specific groups of hypersensitive sites (HSS). The proximal sites, between + 1 and -183 bp with respect to the start site of transcription, were, in addition, transcription-dependent. The nature and functional role of proteins binding to this proximal upstream sequence were analyzed by in vitro binding and cell free transcription experiments. The existence of more upstream cis-acting elements was investigated by transient transfection assays using differentiated hepatoma cell lines and hepatocytes in primary culture. These experiments permitted the detection of an extinguisher active in hepatoma Hep G2 cells but not in hepatocytes, and of an activating element which could correspond to a distal HSS. Unfortunately, this investigation has not yet allowed us to determine with accuracy the DNA elements responsible for response to diet and hormones.

Animals↗
PubMed1991

Seven novel Tay-Sachs mutations detected by chemical mismatch cleavage of PCR-amplified cDNA fragments.

Total RNA was isolated from cultured fibroblasts from 12 unrelated patients with Tay-Sachs disease, an autosomal recessive disorder due to beta-hexosaminidase A deficiency. beta-Hexosaminidase mRNA was amplified by cDNA-PCR in four overlapping segments spanning the entire coding sequence. In two patients, abnormal size cDNA-PCR fragments in which exons were removed resulted from splicing mutations that were characterized at the genomic DNA level: both were G to A transitions, at the first position of intron 2 and at the fifth position of intron 4. Five other mutations have been identified by cDNA-PCR chemical mismatch analysis and direct sequencing of an amplified fragment containing the mismatch site. One missense mutation alters the codon for Ser210 to Phe in exon 6 and the other one alters the codon for Arg504 to Cys in exon 13. A 3-bp deletion results in the deletion of a phenylalanine residue in exon 8. Two nonsense mutations in exon 3 (Arg137 to stop) and in exon 11 (Arg393 to stop) are associated with a marked decrease of mRNA abundance, probably because they result in mRNA instability. Three of the six single base mutations involve the conversion of a CpG dinucleotide in the sense strand to TpG. These results demonstrate the extreme molecular heterogeneity of mutations causing Tay-Sachs disease. The procedure described in this paper allows the rapid detection of any type of mutation, except those impairing the promoter function. Applicable even to patients with splicing or nonsense mutations and very low mRNA abundance, it has therefore a potentially broad application in human genetics, for both diagnostic and fundamental purposes.

Base Sequence↗
PubMed1991

Prevention of airway obstructions during sleep in infants with breath-holding spells by means of oral belladonna: a prospective double-blind crossover evaluation.

We investigated whether the brief airway obstructions seen during sleep in infants with breath-holding spells were controlled by the autonomic nervous system. We studied 20 infants, with a history of breath-holding spells and a median age of 12 wk (range 4-46 wk). During sleep they had a median of 6 airway obstructions per 10-hr recording (range 3-16 events), with a median duration of 8 sec (range 4-12 sec). No explanation was found for the airway obstructions. In every infant, a double-blind crossover challenge was conducted. It included oral administration of tincture of belladonna, equivalent to 0.01 mg/kg weight of atropine, and placebo syrup containing no belladonna. The belladonna, or the placebo, was administered at bedtime for 7 days, followed by a 7-day washout period. Another 7-day series of syrup administration was then undertaken. A nighttime polygraphic recording was made after each 7-day series. It was the belladonna, and not the placebo, that induced the disappearance of the obstructions in 10 infants; these were called "drug responsive". In 5 children no effect was observed after either the placebo or belladonna; these infants were defined as "drug unresponsive". In 4 subjects the obstructions disappeared after both belladonna and the placebo; the children were considered to have an "inconclusive response". One infant was excluded from the study because he developed an airway infection. It is concluded that in some breath-holding infants, obstructed breathing episodes during sleep disappear after the administration of an atropinic drug. The observation could indicate a role of the autonomic nervous system in the control of the upper airways during sleep in infants.

Airway Obstruction↗
PubMed1991

Illegitimate transcription. Application to the analysis of truncated transcripts of the dystrophin gene in nonmuscle cultured cells from Duchenne and Becker patients.

We have previously demonstrated that there is a low level of transcription of tissue-specific genes in every cell type. In this study, we have taken advantage of this phenomenon, called illegitimate transcription, to analyze the muscle-type dystrophin mRNA in easily accessible cells such as lymphoid cells, fibroblasts, and peripheral blood cells from Duchenne and Becker muscular dystrophies with known internal gene deletion. The results showed that, in the studied regions surrounding the deletions, processing of truncated transcripts is identical in specific (muscle tissue) and in nonspecific cells (lymphoid cells). In Becker cases with out-of-frame deletions, the already described alternatively spliced species found in muscle samples were also found in nonspecific cells. These results demonstrate that illegitimate transcripts are a bona fide version of tissue-specific mRNA, and that they represent a useful material to investigate the qualitative consequences of gene defects at the mRNA level.

Base Sequence↗
PubMed1991

Proopiomelanocortin gene expression in normal and tumoral human lung.

Proopiomelanocortin (POMC) gene expression is not restricted to the pituitary corticotroph cell, but also takes place in many normal and tumoral nonpituitary tissues. In contrast, the ectopic ACTH syndrome is a rare event. Because it is most often associated with lung tumors, we specifically studied this tissue, analyzing the different forms of POMC RNAs in normal specimens as well as in various types of tumors. The endocrine nature of the tumors was assessed by both histological examination and measurements of secretogranin-I fragments in the tissue extracts. POMC RNA was first detected by Northern blot analysis; its absolute amounts and its various molecular forms were more precisely quantified and discriminated by S1 mapping studies using a single stranded DNA probe located at the 5' end of exon 3. In five bronchial carcinoid tumors associated with the ectopic ACTH syndrome, a highly predominant, if not single, POMC RNA identical to the 1200-nucleotide (nt) pituitary message was present, the high amounts of which were correlated with those of POMC peptides in the same tissues. In five bronchial carcinoid tumors not associated with the ectopic ACTH syndrome, the same message was detected (four of five), with a second, often predominant, short RNA of about 800 nt (five of five), and the overall amounts of POMC RNAs were low. Similar patterns of POMC RNAs were observed in squamous cell tumors, adenocarcinomas, and normal lung, where the short 800-nt RNA tended to be predominant. These results show that POMC gene expression can be demonstrated in normal lung tissue and in all types of lung tumors. The ectopic ACTH syndrome only occurs with tumors capable of generating high amounts of the pituitary-like message, a phenomenon that seems to be restricted to an occasional tumor with features of neuroendocrine differentiation.

Carcinoid Tumor↗
PubMed1991

Vanadate induction of L-type pyruvate kinase mRNA in adult rat hepatocytes in primary culture.

In primary culture of adult rat hepatocytes, vanadate in the presence of glucose stimulates the expression of the liver (L-type) pyruvate kinase gene. Glucose by itself was inactive, and vanadate, like insulin, was also inefficient in the absence of glucose. Similar results were obtained on glucokinase gene expression. An analogue of cAMP, 8-(4-chlorophenylthio)-cAMP, inhibited the production of L-type pyruvate kinase and glucokinase mRNAs in the presence of glucose plus vanadate.

Animals↗
PubMed1991

[Dystrophin expression in skeletal muscles in endurance-trained hypo-hyperthyroid and growth-hormone treated rats].

The aim of this study was to determine the effect of chronic muscular exercise, thyroid status and growth hormone administration on skeletal muscle dystrophin expression. Relative abundance of dystrophin (quantity in arbitrary units/50 micrograms of protein) was measured by immunoblotting and densimometry. Our results indicate that relative abundance of dystrophin in slow-or fast-twicht muscle was not modified by chronic muscular exercise (5 weeks), thyroxine administration (4 weeks), antithyroid drug treatment (6 weeks) or growth hormone administration (6 weeks).

Actinin↗
PubMed1991

Arousals induced by proximal esophageal reflux in infants.

We investigated whether acid reflux in the proximal esophagus can induce arousal from sleep in infants. Fifty normal infants with occasional regurgitations were studied at the age of 8 weeks (range 4-26 weeks). In each child a pH probe was placed in the proximal portion of the thoracic esophagus, in front of the third vertebra, under radiologic control. Polygraphic monitoring of state of alertness and of proximal esophageal pH changes was recorded continuously during one night. The data were analyzed blind. For 41 of the 50 infants, a total of 97 drops below pH 4 was computed. Reflux was more frequently associated with wakefulness (41% of the episodes), or rapid eye movement (REM) sleep (39%), than with nonREM (19%) or indeterminate sleep (1%). Comparing the fifth and the last minutes preceding the pH drops, a significant increase in the number of behavioral arousals was observed (p = 0.003). In comparison with the minute before the drop in esophageal pH, a further significant increase in the number of arousals occurred during the first minutes following the pH drop (p = 0.001). Although the first minutes following the 97 episodes of reflux represented only 0.4% of the total sleep time, 76% of all arousals (74 out of 97) took place during this time period (p = 0.001). Five minutes after the pH drops, the behavioral changes tended to return to prereflux values. It is concluded that in infants, during sleep, proximal gastroesophageal reflux can act as a strong arousal stimulus.

Arousal↗