PubMed Health⌕ Search

Biomedical subjects

A Kahn

Publications and source records attributed to A Kahn.

At least 19 recordsLinked to original sources
PubMed1992

Combinatorial crosstalk of transacting factors binding to the L-type pyruvate kinase promoter elements analysed in vitro.

Five Dnase 1 footprints, termed boxes L1 to L5, have been characterized in the 280 bp upstream from the cap site of the L-type pyruvate kinase (L-PK) gene. They bind from 3' to 5', the factors HNF1, HNF4, MLTF, and a non-identified protein referred to as L5 binding factor (L5-BF). These elements, individually or variably combined, were tested by cell-free transcription. The L1 box stimulates both L-PK and TK promoters, but in the context of L-PK promoter, it cooperates with the L3 element to reach a high level of transcriptional activation. The L3 and L4 elements exhibit weaker influences, increased by homologous or heterologous interactions. The L5 box behaves as a promoter-dependent negative regulatory element.

Animals↗
PubMed1992

In vitro and in vivo protein--DNA interactions on the rat erythroid-specific L' pyruvate kinase gene promoter.

The rat L-type pyruvate kinase gene possesses two alternative tissue-specific promoters, located 472 bp apart; the upstream L' promoter is erythroid-specific and the downstream L promoter is hepatocyte-specific. The erythroid-specific L' promoter is strongly active in fetal liver at day 17 of gestation, while its activity rapidly decreases thereafter. A L' promoter fragment spanning from nucleotide -320 to +10 with respect to the cap site is able to direct a weak but erythroid-specific transcription in a cell-free system. We have used DNAse I footprinting and gel mobility shift assays to characterize and identify the binding of nuclear factors from both 17-day-old fetal liver and adult liver nuclear extracts to a 320 bp fragment of the 5' flanking region of the gene in vitro. Two clusters of erythroid-specific interactions were found. The proximal cluster consists of two GATA-1 binding sites at -50 bp and -65 bp from the transcription initiation site, immediately downstream of a CACC motif and two G/C-rich elements. The distal cluster of cis-elements, located 130 bp upstream, corresponds to two GATA-1 sequences. These two sequences overlap NF1 motifs interacting with ubiquitous NF1 transcriptional factors in presence of adult hepatic extracts. Furthermore, we have examined in vivo protein-DNA interactions by DMS footprinting in livers of 17-day-old rat fetuses and adult rats. We found that the sites characterized in vitro are occupied in vivo. Therefore, in adult liver the L' promoter, although inactive, nevertheless interacts with ubiquitous factors.

Aging↗
PubMed1992

Distal transcript of the dystrophin gene initiated from an alternative first exon and encoding a 75-kDa protein widely distributed in nonmuscle tissues.

A transcript generated by the distal part of the Duchenne Muscular Dystrophy (DMD) gene was initially detected in cells where the full size 14-kilobase (kb) messenger RNA is not found at a significant level. This transcript, approximately 4.5 kb long, corresponds to the cysteine-rich and carboxyl-terminal domains of dystrophin. It begins with a novel 80- to 100-nucleotide exon containing an ATG start site for a new coding sequence of 17 nucleotides in-frame with the consecutive dystrophin cDNA sequence from exon 63. This result suggests the existence of a third promoter that would be localized about 8 kilobases upstream from exon 63 of the DMD gene. The distal transcript is widely distributed but is absent in adult skeletal and myometrial muscle. It is much more abundant in fetal tissues. With an antibody directed against the dystrophin carboxyl terminus, the protein corresponding to this transcript was detected as a 70- to 75-kDa entity on Western blots. It was found in all tissues analyzed except in skeletal muscle. It was not found in lymphoblastoid cells from a Duchenne patient with a complete deletion of the dystrophin gene. The role and subcellular localization of this protein is not known. It may explain extramuscular symptoms exhibited by some Duchenne patients.

Animals↗
PubMed1992

DNase-I hypersensitivity analysis of the L-type pyruvate kinase gene in rats and transgenic mice.

The rat L-type pyruvate kinase gene possesses two promoters located 500 bp apart. The L' promoter is specific to erythroid cells. The L promoter is specific to liver and is regulated by diet and hormones; positively by glucose and insulin and negatively by glucagon via cAMP. The DNA elements involved in this tissue-specific and hormone-regulated gene expression are located within 3.2 kbp of 5' flanking region as previously demonstrated by transgenic mice analysis [Tremp, G. L., Boquet, D., Ripoche, M. A., Cognet, M., Yu-Chun, L., Jami, J., Kahn, A. and Daegelen, D. (1989) J. Biol. Chem. 264, 19,904-19,910]. Moreover, we have observed in these mice that gene expression was dependent on the transgene copy number and independent of the integration site. We present here DNase-I-hypersensitivity analysis of the endogenous rat L-type pyruvate kinase gene and of two transgene constructs in relation to development, tissue differentiation, nutritional and hormonal status. In rats, two groups of proximal sites were detected on the endogenous gene; hypersensitive site (HSS) HSS-1 in adult liver and HSS-A in fetal liver (a major erythropoietic tissue). Both groups are probably related to the transcriptional initiation complexes at either the L or L' promoter. Two other distal groups were detected; HSS-2 at -3 kbp (with respect to the liver-specific cap site) in adult liver and HSS-B around -4 kbp in fetal liver. These sites are thought to correspond to activating sequences; in adult liver, deletion of a fragment encompassing HSS-2 provokes a dramatic reduction of transcription starting at the L promoter of the transgene. In adult liver, HSS-1 appears to be a transcription-associated site, being greatly weakened in fasted rats, while HSS-2 is transcription independent. The pattern of DNase-I hypersensitivity is similar for the rat endogenous gene and for the complete rat transgene; the liver-specific HSS-1 and HSS-2 are present and the intensity of the sites is correlated to the number of integrated copies. Interestingly, HSS-1 is still detectable and its intensity remains proportional to the number of integrated copies in a truncated transgene with HSS-2 deletion, while this transgene is very weakly (but nevertheless tissue-specifically) expressed. These results strongly suggest that each transgene copy possesses a complete set of specific nucleoprotein complexes and that, with or without HSS-2, the DNA is in a potentially active configuration.

Animals↗
PubMed1992

Positive and negative regulatory DNA elements including a CCArGG box are involved in the cell type-specific expression of the human muscle dystrophin gene.

The muscle-specific promoter of the dystrophin gene is active in skeletal, cardiac, and smooth muscles and is specifically stimulated during differentiation of myoblasts into multinucleated myotubes. An 850-base pair (bp) DNA fragment upstream from the cap site is able to confer a partial muscle specificity to a reporter gene. The region between -850 and -140 bp includes nonspecific negative and positive regulatory sequences. A continuous stretch of 140 bp upstream from the cap site exhibits a striking conservation between rodents and human (93% homology) and still retains muscle preference of expression. It contains two putative binding sites for factors involved in regulation of other muscle-specific genes, a CCArGG box and an E box. This latter element, however, is unable to confer the ability to be transactivated by MyoD1 to the dystrophin promoter. The -140-bp promoter fragment exhibits antagonist effects contributed by one inhibiting sequence (nucleotide -140/-96), active in all cell types, and one activating region, from nucleotide -96 to the cap site, sufficient to confer a muscle preference of expression, in which the CCArGG box seems to play a major role.

Animals↗
PubMed1992

Cis-regulation of the L-type pyruvate kinase gene promoter by glucose, insulin and cyclic AMP.

The glucose/insulin response element of the L-pyruvate kinase gene is a perfect palindrome located from nt -168 to -144 with respect to the cap site. This element (L4) is partially homologous to MLTF binding sites. Its full efficiency requires cooperation with a contiguous binding site for HNF4, termed L3 and located from nt -145 to -125. In the presence of the L4 element contiguous to L3, cyclic AMP inhibits activity of the L-PK promoter while in its absence, or when the normal L4-L3 contiguity is modified, cyclic AMP behaves as a transcriptional activator that does not seem to be sequence-specific. Therefore, we propose that the mechanism of inhibition of the L-PK gene by cyclic AMP requires precise interactions between the nucleoprotein complex built up at sites L4 and L3 and other components of the L-PK transcription initiation complex.

Animals↗
PubMed1992

Illegitimate transcription: its use in the study of inherited disease.

In 1988, by using the powerful and accurate cDNA/PCR technique, it was demonstrated that there are very low levels of dystrophin mRNA in a variety of non-muscle tissues, including cultured fibroblasts and lymphoblastoid cell lines. The phenomenon was also shown for a number of other tissue-specific genes, including beta-globin, factors VIIIc and IX, anti-Müllerian hormone, L-pyruvate kinase, retinal blue pigment, phenylalanine hydroxylase. The level of transcript in inappropriate cells is exceedingly low, perhaps one mRNA per 100-1000 cells. This low-level ubiquitous transcription of tissue-specific genes was called "illegitimate" or "ectopic" transcription, and has been proven to occur for 17 gene transcripts to date. The mechanism and biological significance of illegitimate transcription are still obscure, but, since illegitimate transcripts exhibit the same pathology as legitimate transcripts, they have been useful tool in the study of already 9 inherited diseases. This strategy will be applied widely for diseases where samples from the appropriate tissue for study is difficult to obtain, or where an mRNA is easier or more informative to study than a genomic DNA (as for large genes, or where alternative splicing is involved).

Animals↗
PubMed1992

Histone H4-related osteogenic growth peptide (OGP): a novel circulating stimulator of osteoblastic activity.

It has been established that regenerating marrow induces an osteogenic response in distant skeletal sites and that this activity is mediated by factors released into the circulation by the healing tissue. In the present study we have characterized one of these factors, a 14 amino acid peptide named osteogenic growth peptide (OGP). Synthetic OGP, identical in structure to the native molecule, stimulates the proliferation and alkaline phosphatase activity of osteoblastic cells in vitro and increases bone mass in rats when injected in vivo. Immunoreactive OGP in high abundance is present physiologically in the serum, mainly in the form of an OGP-OGP binding protein complex. A marked increase in serum bound and unbound OGP accompanies the osteogenic phase of post-ablation marrow regeneration and associated systemic osteogenic response. Authentic OGP is identical to the C-terminus of histone H4 and shares a five residue motif with a T-cell receptor beta-chain V-region and the Bacillus subtilis outB locus. Since these latter proteins have not been implicated previously in the control of cell proliferation or differentiation, OGP may belong to a novel, heretofore unrecognized family of regulatory peptides. Perhaps more importantly, OGP appears to represent a new class of molecules involved in the systemic control of osteoblast proliferation and differentiation.

Amino Acid Sequence↗
PubMed1992

CFTR illegitimate transcription in lymphoid cells: quantification and applications to the investigation of pathological transcripts.

Since the isolation of the cystic fibrosis transmembrane conductance regulator gene (CFTR) and the characterization of the main mutation (delta F508) in 1989, a large number of rare mutations has been found. Full screening of the CFTR gene is difficult because it is split into 27 exons covering 250 kb of genomic DNA. This gene is essentially expressed in the lung and intestinal tract, neither of which are easily accessible for routine investigations. The recent description of a faint transcription of highly tissue-specific genes in any cell, a phenomenon known as illegitimate transcription, would facilitate the research of mutations and the characterization of truncated m-RNA caused by splicing mutations. Using the polymerase chain reaction on cDNA (cDNA-PCR), we detected transcripts of the CFTR gene in lymphocytes and lymphoblast cells at a very low level (about 300 times less than in lung or intestine). This strategy allowed us to obtain a sufficient amount of cDNA-PCR product compatible with further molecular analyses. We have, therefore, analyzed a cDNA fragment overlapping exons 10 and 11 by polyacrylamide gel electrophoresis and direct sequencing, and detected the delta F508 mutation at this level. Our protocol can be generalized to the investigation of the total 4.5-kb CFTR coding sequence.

Animals↗
PubMed1992

Gene expression in hepatocyte-like lines established by targeted carcinogenesis in transgenic mice.

New hepatocyte-like cell lines (mhAT) were derived from the liver of a transgenic mouse expressing SV40 early genes under the direction of the liver-specific antithrombin III gene promoter (ATIII-TSV40). Their differentiated phenotypes were improved and stabilized by the use of liver-specific growth media (arginine-free, glucose-free, or low-fructose/glucose-free medium). The best differentiated lines display a very high level of albumin, transferrin, and L-type pyruvate kinase (L-PK) gene expression that is comparable to that observed in the mouse liver. Abundance of the aldolase B and phosphoenolpyruvate carboxykinase (PEPCK) transcripts varied from 5 to 35% of the in vivo concentrations while abundance of the alpha-fetoprotein and phenylalanine hydroxylase transcripts remained very low. Hormonal (cAMP and insulin) and nutritional (glucose) gene controls of PEPCK and L-PK were, at least partially, conserved. mhAT cells are readily transfectable by the calcium phosphate coprecipitation technique and exhibit a liver-specific pattern of expression of exogenous genes. Thus, mhAT cells seem suitable for the analysis of the regulatory regions involved in the tissue-specific transcription of genes. This work demonstrates, therefore, the great efficiency of targeted carcinogenesis in transgenic mice to create new differentiated cell lines. The availability of various lines of liver-specific cells with different phenotypes will constitute useful tools to establish correlations between expression of trans-acting factors and control of the phenotype.

Albumins↗
PubMed1992

Sleep and cardiorespiratory characteristics of infant victims of sudden death: a prospective case-control study.

We studied the polygraphic sleep recordings of 30 infants who eventually died of sudden infant death syndrome (SIDS) and those of 60 matched control infants. All records were extracted from 20,750 sleep studies collected prospectively in 10 sleep laboratories. Of the 30 future SIDS victims, 5 were siblings of SIDS victims and 9 were studied after an apparent life-threatening event. For each SIDS victim, two normal control infants were matched for sex, gestational age, postnatal age and weight at birth. The future SIDS infants were reported to have more frequent episodes of regurgitations after feeding (p = 0.01) and profuse sweating during sleep (p = 0.01) than the control subjects. Only two polysomnographic variables characterized the future SIDS infants. Compared to control subjects, the SIDS infants moved less during sleep (p = 0.04) and had significantly more frequent obstructed breathing events. Obstructive and mixed apneas were seen in 23 of 30 future SIDS victims, but in only 9 of 60 control subjects (p = 0.01). The obstructed and mixed apneas lasted longer in the SIDS than in the control infants (p = 0.01) but did not exceed 15 seconds. The obstructed breaths occurred mainly in rapid eye movement sleep (78% of the events) and were accompanied by drops in heart rates to 68 beats per minute and in SaO2 levels to 75%. The present report adds further indirect evidence for a possible sleep-related impairment of respiratory control in some infants who eventually died of SIDS.

Case-Control Studies↗
PubMed1992

Effects of body movement restraint on cardiac response to auditory stimulation in sleeping infants.

When exposed to an unfamiliar and sudden noise, infant rodents may show an abrupt bradycardia, as part of a "fear-paralysis response". The response is enhanced by body movements restraint. To investigate if this reaction is seen in humans, 15 normal infants with a median age of 12 weeks were studied polygraphically. They were exposed to a 100-dB (A) white noise, while sleeping in REM sleep. Each infant was studied in both "unrestrained" and "restrained" conditions. Restraint of body movement was obtained by means of sand bags and tightly-binding bed sheets. During movement restraint, the infants had a significantly greater and earlier decrease in heart rate compared with during the unrestrained condition. No infant had a minimal heart rate less than 95 beats/min. The present observation raises questions about the possible unfavorable effects of tightly wrapped bed sheets around sleeping infants.

Acoustic Stimulation↗
PubMed1992

Elements responsible for hormonal control and tissue specificity of L-type pyruvate kinase gene expression in transgenic mice.

L-type pyruvate kinase (L-PK) is a key enzyme of the glycolytic pathway specifically expressed in the liver and, to a lesser degree, in the small intestine and kidney. One important characteristic of L-PK gene expression is its strong activation by glucose and insulin and its complete inhibition by fasting or glucagon treatment. Having previously established that the entire rat L-PK gene plus 3.2 kbp of 5'-flanking region functions in mice in a tissue-specific and hormonally regulated manner, various deletions of these 3.2 kbp of 5'-flanking regions were tested in transgenic animals to map the cis-acting elements involved in transcriptional gene regulation. Our experiments indicate that the proximal region between -183 and +11 confers tissue specificity and contains all the information necessary for dietary and hormonal control of L-PK gene expression in vivo. We found, however, that the transcriptional activity generated by this proximal promoter fragment can be modulated by distal sequences in a tissue-specific manner. (i) Sequences between bp -183 and -392 seem to play a dual role in the liver and small intestine; they induce L-PK expression in the liver but repress it in the small intestine. (ii) Sequences from bp -392 up to -1170 do not seem to have any additional effect on promoter activity. (iii) Between bp -1170 and -2080, we found a putative extinguisher whose transcriptional inhibitory effect is much more marked in the small intestine than in the liver. (iv) Finally, between bp -2080 and -3200, we identified an activating sequence required for full expression of the gene in the liver.

Animals↗
PubMed1992

[Illegitimate transcription: discovery and application to gene molecular pathology].

In 1988, by using the powerful cDNA/PCR technique, it was demonstrated that there are very low levels of dystrophin mRNA in a variety of non-muscle tissues, including cultured fibroblasts and lymphoblastoid cell lines. The phenomenon was also shown for a number of other tissue-specific gene, including beta-globin, factors VIIIc and IX, anti-müllerian hormone, L-pyruvate kinase, retinal blue pigment, phenylalanine hydroxylase. The level of transcript in inappropriate cells is exceedingly low, perhaps one mRNA per 100-1,000 cells. This low-level ubiquitous transcription of tissue-specific genes was called "illegitimate" or "ectopic" transcription, and has been proven to occur for 17 gene transcripts to date. The mechanism and biological significance of illegitimate transcription are still obscure, but, since illegitimate transcripts exhibit the same pathology as legitimate transcripts, they have been useful tool in the study of already 9 inherited diseases. This strategy will be applied widely for diseases where samples from the appropriate tissue for study is difficult to obtain, or where an mRNA is easier or more informative to study than a genomic DNA (as for large genes, or where alternative splicings is involved).

Humans↗
PubMed1979

The permeability coefficient of albumin of the isolated rat mesentery. Modification by some mediators of inflammation, cyclic AMP and calcium.

In order to study the mechanisms whereby mediators of inflammation exert their exudative effects, we used isolated rat mesentery placed as a separation membrane between the two compartments of a diffusion cell. In this experimental arrangement, the permeability coefficient of albumin (PA) can be easily computed from the equilibration rate of 125I-labelled albumin added to one compartment. Histamine, bradykinin, serotonin and prostaglandins A1, A2, E1, E2, F1 alpha and F2 alpha all increased PA to some extent, the maximal values being approx. +60%. Dibutyryl-cyclic AMP, theophylline and isoproterenol also increased PA, thus suggesting involvement of cyclic AMP. Direct measurements of this nucleotide confirmed this hypothesis; furthermore, a linear relation between cyclic AMP levels and PA could be demonstrated. In contrast, cyclic GMP is probably not involved in the control of PA. Calcium-depleted tissues had a low PA (approx. 40% below controls), and did not respond to exogenous prostaglandin E1. These results suggest that transmesenteric passage of albumin is at least partly controlled by cyclic AMP and intracellular Ca2+ levels.

Animals↗