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A Kahn

Publications and source records attributed to A Kahn.

At least 253 records · Page 14Linked to original sources
PubMed1993

Functional characterization of the L-type pyruvate kinase gene glucose response complex.

L-type pyruvate kinase (L-PK) gene expression is modulated by hormonal and nutritional conditions. We have previously shown that the glucose/insulin response element (GlRE) of the L-PK gene is built around two noncanonical E boxes (element L4) that cooperate closely with a contiguous binding site (element L3). We present in this report the identification of proteins that interact with both elements. The L3 site binds hepatocyte nuclear factor 4 (HNF4)- and COUP/TF-related proteins. In fibroblasts, the overexpression of HNF4 transactivates the L-PK promoter. On the contrary, COUP/TF strongly inhibits the active promoter in hepatocytes. The L4 site binds the major late transcription factor (MLTF) in vitro and ex vivo; mutations that suppress this binding activity also inactivated the GlRE function. Mutations transforming one or two noncanonical E boxes of element L4 into consensus MLTF/USF binding sites strongly increase the affinity for MLTF/USF and do not impair the glucose responsiveness. However, merely the ability to bind MLTF/USF does not seem to be sufficient to confer a GlRE activity: those elements in which one E box has been destroyed and the other has been transformed into a consensus MLTF/USF sequence bind MLTF/USF efficiently but do not confer a high glucose responsiveness on the L-PK gene promoter. Consequently, the full activity of the L-PK GlRE seems to require the cooperation between two putative MLTF/USF binding sites located in the vicinity of an HNF4 binding site.

Animals↗
PubMed1993

Apportioning illness management authority: how diabetic individuals evaluate and respond to spousal help.

This study explores spousal helping behavior related to diabetes management as seen from the perspective of the person with diabetes. Individuals with diabetes described spousal behaviors considered helpful in one context that were considered intrusive or unhelpful in other situations. When the helpful and unhelpful situations were compared, two factors emerged that were fundamental influences in subjects' acceptance or rejection of help and the manner in which it was accepted or refused. These factors were perceived need for help and subjects' perception of spousal motivation for action. Combinations of these factors gave rise to four distinct response patterns: grudging acceptance of help, grateful acceptance of help, respectful rejection of help, and punitive rejection of help. The impact of these response patterns on diabetes management is discussed.

Adult↗
PubMed1993

Establishment of renal proximal tubule cell lines by targeted oncogenesis in transgenic mice using the L-pyruvate kinase-SV40 (T) antigen hybrid gene.

Targeted oncogenesis allowed us to obtain two cell lines which have been derived from the proximal tubule of kidney from transgenic mice harbouring the simian virus (SV40) large T and small t antigens placed under the control of the 5' regulatory sequence from the rat L-type pyruvate kinase (L-PK) gene. The cell lines (PKSV-PCT and PKSV-PR cells) were derived from early (PCT) and late (Pars Recta, PR) microdissected proximal tubules grown in D-glucose-enriched medium. In such conditions of culture, both cell lines exhibited L-PK transcripts, a stable expression of SV40-encoded nuclear large T antigen, a prolonged life span but failed to induce tumors when injected sub-cutaneously into athymic (nu-nu) mice. Confluent cells, grown on plastic support or porous filters, were organized as monolayers of polarized cuboid cells with well developed apical microvilli and formed domes. Both cell lines exhibited morphological features of proximal tubule cells with villin located in the apical brush-border and substantial amounts of hydrolase activity. By immunofluorescence studies using specific antibodies, aminopeptidase N appeared restricted to the apical microvillar domain, whereas the H2 histocompatibility antigen was distributed in the cytoplasm and lateral membranes. These results demonstrate that the proximal morphological phenotype has been fully preserved in these cultured cells derived from tissue-specific targeted oncogenesis in transgenic mice.

Aminopeptidases↗
PubMed1993

Functional properties of proximal tubule cell lines derived from transgenic mice harboring L-pyruvate kinase-SV40 (T) antigen hybrid gene.

This study describes the functional characterization of two cell lines derived from the proximal convoluted (PKSV-PCT cells) and proximal straight (PKSV-PR) tubules microdissected out from kidneys of transgenic mice harboring the simian virus 40 (SV40) large T and small t antigens placed under the control of the rat L-type pyruvate kinase (L-PK) 5' regulatory sequence. Both cell lines exhibited cellular cyclic AMP stimulated by parathormone (PTH) and calcitonin (CT) and a sodium-dependent glucose transporter. Uptake of the fluid-phase marker [3H]inulin showed that both cell lines grown on filters exhibited biphasic apical and basolateral endocytic rates. Results from Northern blot analysis indicate that the expression of the T antigen gene (Tag) is dependent on the concentration of D-glucose in the medium and show that the L-PK construct has maintained its capacity for up- or down-regulation by carbohydrates. Replacement of D-glucose by neoglucogenic substrates (lactate, oxaloacetate) blunted the expression of Tag transcripts and induced arrest of cell growth. Compared to cell grown in D-glucose-enriched medium, the hormonal sensitivities to PTH and CT and the sodium-dependent glucose uptake were unchanged whereas quiescent cells exhibited increased hydrolase content. Thus the proximal function has been preserved in these cultured cells derived from tissue-specific targeted oncogenesis in transgenic mice. As the expression of Tag transcripts is controlled by D-glucose, the structural and physiological characteristics of these cell lines can be studied in either quiescent or active growth conditions.

Animals↗
PubMed1993

[Values and limits of adenoviral vectors for gene transfer in vivo].

Review of the therapeutic use of DNA transfer to treat a certain number of hereditary diseases (such as adenosine deaminase deficiency) or acquired diseases. The strategies (ex vivo manipulation or direct in vivo transfer of the corrective gene), vectors (retrovirus, adenovirus, nonviral vectors), and diseases which can benefit from gene therapy are considered and discussed together with an evaluation of the risk of gene therapy.

Adenoviridae↗
PubMed1993

Prone or supine body position and sleep characteristics in infants.

OBJECTIVE: To evaluate the potential relation between body position and sleep characteristics in normal infants. PATIENTS: Two groups of 3-month-old healthy infants were evaluated: 40 infants who usually slept supine, and 40 who usually slept prone. The two groups were matched for gender, gestational age, postnatal age, birth weight, and total recording time. RECORDING TECHNIQUES: The 80 infants were studied polygraphically during one night in the pediatric sleep laboratory. They were allowed to fall asleep in their usual sleep position, and every 3 hours were gently turned from prone to supine, or from supine to prone. RESULTS: In each group, 6 infants were excluded from the analysis, because they woke up after having been turned over. In both groups, no significant difference was seen between the prone and the supine body positions for the following variables: number of sleep state changes; number of gross body movements; percent of rapid eye movement sleep; saturation with oxygen, arterial blood levels; number and duration of acid esophageal reflux; rectal temperature; mean respiratory rates; water evaporation rates from the forehead skin; and number or duration of central or of obstructive apneas. In both groups of infants, prone body position was associated with a significant increase in sleep duration (+16%) and in non-rapid eye movement sleep (+25%) and a significant decrease in number of arousals (-40%) and in their duration (-43%). CONCLUSIONS: No explanation has been found for the sleep-promoting effect of prone body positioning. The finding could be of interest to the study of infants' sleep quality, as well as to the potential relation between body positions and sudden death during sleep.

Female↗
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

Characterization of the rat pulmonary surfactant protein A promoter.

The expression of the pulmonary surfactant protein A (SP-A) is developmentally regulated and controlled by several hormones. In an attempt to characterize cis-acting elements involved in the regulation of SP-A expression, we have cloned the 5' flanking sequence of the rat SP-A gene. The promoter region contains a TATA box but no CAAT box. The transcription start site has been identified by anchored polymerase chain reaction and S1 nuclease mapping of the mature and precursor transcripts. S1 mapping of precursor transcripts has confirmed the stimulating effect of glucocorticoids on SP-A rat gene transcription in vivo. This hormonal effect may be mediated by a putative glucocorticoid responsive element located 140 bp upstream from the initiation site and protected against DNase 1 digestion in footprinting experiments. In vitro transcription of a G-free reporter cassette linked to the 212-bp 5' flanking DNA fragment has established that this putative promoter region is functional. Efficient transcription of the G-free reporter cassette was obtained with cell-free fetal lung extracts, whereas no transcript was detectable with cell-free liver extracts. Comparative analysis of the human and rat 5' flanking sequences shows the presence of strongly conserved motifs, unrelated to previously known consensus sequences. Some of these motifs, specifically protected in DNase 1 footprinting studies, could therefore be involved in the regulation of SP-A gene expression.

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↗