Chemistry at the Al- and Au-ZnSe(100) interfaces.
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Journal Article↗
Biomedical subjects
A Kahn
Publications and source records attributed to A Kahn.
Four new mutations in the NADH-cytochrome b5 reductase gene from patients with recessive congenital methemoglobinemia type II.
Recessive congenital methemoglobinemia (RCM) due to NADH-cytochrome b5 reductase (cytb5r) deficiency leads to two different types of diseases. In the type I form, cyanosis is the only symptom, and the soluble enzyme is defective in red blood cells. In the type II form, cyanosis is associated with severe mental retardation and neurologic impairment; the enzymatic defect is systemic, involving both soluble and membrane-bound isoforms. We characterized mutations responsible for cytb5r deficiency in three unrelated patients with severe RCM type II. The first patient presented a homozygous exon 5 skipping. The only mutation detected was a homozygous G to C transversion at position +8, downstream from the 5' splice site of exon 5. We suggest that this unusual mutation might be responsible for the abnormal splicing of the primary transcripts, resulting in frameshift with premature STOP codon. The second mutation found corresponds to a homozygous C to T transition changing the Arg-218 codon to a premature STOP codon in exon 8. The third case was a compound heterozygote, carrying two different mutant alleles in the cyb5r gene. One allele presented a missense mutation with replacement of Cys-203 (TGC) by Arg (CGC) in exon 7. The second allele carried a 3-bp deletion (TGA) of nucleotides 815 to 817, modifying two contiguous codons in exon 9 of the cDNA with loss of Met-272. These results confirm the genetic polymorphism of cytb5r gene mutations identified in RCM type II, as observed for the mutations described in the RCM type I, and shed light on the molecular bases of the two different diseases associated with cytb5r deficiency.
Amino Acid Sequence↗
Upstream stimulatory factor proteins are major components of the glucose response complex of the L-type pyruvate kinase gene promoter.
L-type pyruvate kinase (L-PK) gene transcription is induced by glucose through its glucose response element (GlRE) composed of two degenerated E boxes able to bind in vitro ubiquitous upstream stimulator factor (USF) proteins. Here we demonstrate in vivo, by transient transfections in hepatoma cells, that (i) native USF proteins synthesized from expression vectors can act as transactivators of the L-PK promoter via the GlRE, stimulating transcription without glucose and, therefore, decreasing the glucose responsiveness of the promoter; (ii) expression of the truncated USF proteins, able to bind the GlRE but devoid of the NH2-terminal activation domain, represses the activation of the L-PK promoter by glucose; and (iii) a similar repression of the glucose effect is observed upon expression of mutant USF proteins devoid of the basic DNA binding domain, able to dimerize with endogenous USF but not to bind the GlRE. We conclude that USF proteins are components of the transcriptional glucose response complex assembled on the L-PK gene promoter.
Base Sequence↗
Structure, sequence, and chromosomal location of the gene for USF2 transcription factors in mouse.
The ubiquitously expressed upstream stimulatory factor (USF) involved in the transcription of a wide variety of cellular genes is defined as dimers of c-myc-related proteins, composed of a basic helix-loop-helix/leucine zipper region. The USF family consists of different members that split into two groups: MLTF or USF1 and USF2 or FIP. We present here evidence that USF1 and USF2 are distinct closely related genes in human, rat, and mouse. Based on the recent cloning of rat and human new cDNAs, we have isolated genomic clones encompassing the murine USF2 gene, which consists of at least 10 exons spanning a minimum of 10 kb of genomic DNA. Unexpectedly, the organization of USF2 appears very split up by introns (0.08 to over 6 kb in size), compared to the myc gene structure. The entire gene (but the larger intron) and 1.6 kb of the 5' flanking region were sequenced. This 5' flanking region is GC-rich, contains several putative transcription binding sites, and has no apparent TATA box. Gene mapping of murine USF2 and USF1 has been determined by in situ hybridization, indicating the localization of USF2 on chromosome 7 and of USF1 on chromosomes 1 and 11.
Amino Acid Sequence↗
Why should infants with sleep apneas and apparent life-threatening events be recorded polygraphically?
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Humans↗
Transcription factors and aldolase B gene expression in microdissected renal proximal tubules and derived cell lines.
Renal expression of the aldolase B isoenzyme and transcription factors previously shown to regulate the aldolase B gene promoter in the liver were analyzed in whole kidney, microdissected tubules, and the two PKSV-PCT and PKSV-PR proximal tubule cell lines derived from transgenic mice. Aldolase B gene expression appeared restricted to the proximal tubule, the site where HNF1 alpha, HNF1 beta, C/EBP alpha, and DBP transcripts were also abundant. Compared to the liver, another organ synthesizing aldolase B, proximal tubules from the kidney were characterized by the absence of HNF3 and the presence of higher ratio of HNF1 beta/HNF1 alpha transcripts. The same features were conserved in both PKSV-PCT and PKSV-PR proximal tubule cell lines. Transactivation experiments in PKSV-PCT cultured cells showed that HNF1 alpha, C/EBP alpha, and DBP behave as transactivators of the 190-bp aldolase B gene promoter, and that HNF1 beta had a low transactivating efficiency. HNF1 beta, as well as HNF3, antagonized the HNF1 alpha-dependent transactivation of the aldolase B promoter. The fact that both HNF1 beta and HNF3 factors play similar negative roles by competitively binding close to or on the HNF1 site could suggest that, in proximal tubule renal cells, HNF1 beta has the same attenuator effect on the aldolase B gene promoter as HNF3 in hepatocytes. Thus, these results indicate that such models of established renal tubule cell lines, which have conserved the same features of parental cells, represent valuable tools for studies of the regulation of genes expressed in proximal tubules of the kidney.
Animals↗
Targeted oncogenesis: A powerful method to derive renal cell lines.
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Animals↗
Transient expression of genes transferred in vivo into heart using first-generation adenoviral vectors: role of the immune response.
Gene therapy for heart diseases requires availability of an efficient vector for gene transfer into myocardium. Recombinant adenovirus expressing the Escherichia coli beta-galactosidase (beta-Gal) gene was shown to infect rat cardiocytes efficiently in vivo. However, a time course of gene expression showed that transgene expression was maximal during the first week following injection, then declined and disappeared by day 21. An immunosuppressive treatment prolonged beta-Gal expression for at least 21 days. On the contrary, a preimmunization of the animals by two intraperitoneal injections of the vector led to a decreased transgene expression 48 hr after intramyocardial injection and to a barely detectable expression at the sixth day. Appearance of adenovirus neutralizing antibodies in preimmunized animals could have contributed to such a refractoriness to further adenoviral infection. Finally, a neonatal intrathymic injection of the vector was able to induce long-term LacZ expression for more than 2 months after heart injection, although neutralizing as well as anti-beta-Gal antibodies were detected in sera of the animals. These results indicate that an immune response against first-generation replication-defective adenoviral vectors is a major cause of transient transgene expression, a cellular response being most probably responsible for ablation of transgene expression in immunocompetent animals.
Adenoviridae↗
Overproduction of a truncated hepatocyte nuclear factor 3 protein inhibits expression of liver-specific genes in hepatoma cells.
Transcription of hepatocyte-specific genes requires the interaction of their regulatory regions with several nuclear factors. Among them is the hepatocyte nuclear factor 3 (HNF3) family, composed of the HNF3 alpha, HNF3 beta, and HNF3 gamma proteins, which are expressed in the liver and have very similar fork head DNA binding domains. The regulatory regions of numerous hepatocyte-specific genes contain HNF3 binding sites. We examined the role of HNF3 proteins in the liver-specific phenotype by turning off the HNF3 activity in well-differentiated mhAT3F hepatoma cells. Cells were stably transfected with a vector allowing the synthesis of an HNF3 beta fragment consisting of the fork head DNA binding domain without the transactivating amino- and carboxy-terminal domains. The truncated protein was located in the nuclei of cultured hepatoma cells and competed with endogenous HNF3 proteins for binding to cognate DNA sites. Overproduction of this truncated protein, lacking any transactivating activity, induced a dramatic decrease in the expression of liver-specific genes, including those for albumin, transthyretin, transferrin, phosphoenolpyruvate carboxykinase, and aldolase B, whereas the expression of the L-type pyruvate kinase gene, containing no HNF3 binding sites, was unaltered. Neither were the concentrations of various liver-specific transcription factors (HNF3, HNF1, HNF4, and C/EBP alpha) affected. In partial revertants, with a lower ratio of truncated to full-length endogenous HNF3 proteins, previously extinguished genes were re-expressed. Thus, the transactivating domains of HNF3 proteins are needed for the proper expression of a set of liver-specific genes but not for expression of the genes encoding transcription factors found in differentiated hepatocytes.
Animals↗
Glucose, growth hormone, cortisol, and insulin responses to glucagon injection in normal infants, aged 0.5-12 months.
We performed glucagon stimulation tests in 59 normally growing siblings of children who died from sudden infant death syndrome. These investigations were performed to exclude a possible metabolic disorder (found in 4 siblings) as an underlying cause of sudden infant death syndrome. The remaining 55 siblings (32 boys and 23 girls) provide control data for this age range. Testing was performed at 0800 h after a 15-h fast. The median age was 98 days (range, 13-349 days). Plasma glucose and serum cortisol, insulin, and GH were determined before and 30, 60, 90, 120, 150, and 180 min after im injection of 0.1 mg/kg glucagon. No side-effects were observed during the procedure. Asymptomatic hypoglycemia was noted in 11% of the infants at least once between 120-180 min. Basal and peak GH concentrations were greater than 10 micrograms/L in 31% and 80% of the infants, respectively. There was a significant negative correlation between age and basal GH concentration [Spearman's rank correlation coefficient (rs) = -0.37; P < 0.01]. There was a significant correlation between age and glucagon-stimulated cortisol at 120, 150, and 180 min (rs) = 0.41; P < 0.005), but not between age and changes in glucose levels. There was no significant correlation between age and basal cortisol or peak GH concentrations and no difference between boys and girls for any of the variables studied. In conclusion, the glucagon stimulation test is well tolerated in very young subjects. The peak GH response to glucagon injection is independent of age between 0.5-12 months. The age-related increase in the glucagon-stimulated cortisol response despite a similar decrease in glucose suggests the existence of a postnatal maturation in the response of the pituitary-adrenal axis to stress.
Aging↗
Gene delivery into the central nervous system by nasal instillation in rats.
Replication-deficient adenoviruses have been used successfully to transfer foreign DNA into postmitotic cells. This article demonstrates that it is possible to transfer the Escherichia coli lacZ gene in vivo into the central nervous system structures of rats after nasal instillation of replication-defective adenoviral vector AdRSV beta gal. Mitral cells from the olfactory bulb, neurons from the anterior olfactory nucleus, locus coeruleus and area postrema expressed beta-galactosidase for at least 12 days. No cytopathic effect was observed in the CNS structures studied at the viral titer used (1-3 x 10(9) plaque-forming units (p.f.u.)). This method could be useful for the gene therapy of diseases affecting different CNS structures.
Adenoviridae↗
Reduction in obstructive breathing events during body rocking: a controlled polygraphic study in preterm and full-term infants.
OBJECTIVE: To investigate the effect of body rocking on infant respiratory behavior during sleep. METHODS: Eighteen infants with documented obstructive sleep apneas were studied. There were eight premature infants with persistent bradycardias and 10 infants born full-term, admitted after an idiopathic apparent life-threatening event. No cause for the obstructive apneas was found. The infants were recorded with polygraphic techniques during two successive nights. They were randomly assigned to a rocking or a nonrocking mattress. The conditions were reversed the following night, in a crossover design. RESULTS: In both groups of infants, no significant difference was seen between the two consecutive nights for most of the variables studied: total sleep time, the proportion of non-rapid-eye-movement and rapid-eye-movement sleep, the number of arousals, the number and maximal duration of central apneas, the frequency of periodic breathing, the level of oxygen saturation, and heart rate. During the nonrocking nights, all infants had repeated obstructive breathing events. In seven of the eight preterm infants and in nine of the 10 full-term subjects, body rocking was associated with a significant decrease in the frequency of obstructive events. During rocking, in the preterm infants the obstructions fell from a median of 2.5 to 1.8 episodes per hour (P = .034). In the full-term infants, rocking reduced the obstructive events from a median of 1.5 obstructions per hour to 0.7 (P = .005). No difference was seen for the duration of the obstructive episodes. CONCLUSION: In preterm and full-term infants prone to obstructive sleep apneas, gentle side-to-side body rocking is associated with a significant decrease in the frequency of upper-airway obstructions.
Beds↗
Age-related bone loss. A hypothesis and initial assessment in mice.
The osteopenia associated with advanced age appears to be a universal phenomenon in humans and animals, but the mechanisms by which it occurs are understood incompletely. However, the explanation must lie in an absolute or relative diminution in the level of osteoblastic bone-forming activity when compared with osteoclastic bone-resorbing activity. The authors postulated that with old age there would be a reduction in the number or function or both of osteoblastic stem cells that could account for part of the diminution in bone formation. They further postulated that there would be either no change or an increase in osteoclastic potential and bone resorption. To test these concepts, bone marrow cells were isolated from 4- to 6-month-old or 24-month-old mice and cultured in vitro under a variety of circumstances that permitted an assessment of the stromal osteogenic cells and marrow hemopoietic progenitor cells belonging to the monocyte and osteoclast series. These data show a marked reduction in the number and in vitro activity of stromal osteogenic cells from old animals. There is an increase in old mice in the number of marrow cells capable of forming osteoclasts in coculture and responsive to the growth factors believed operational in the monocyte and osteoclast series. The authors now are exploring the hypothesis that an age-related diminution in transforming growth factor-beta levels is responsible for these changes in progenitor cell levels in marrow and their functional status as expressed in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)
Aging↗
[Gene therapy. Use of genes as drugs].
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Animals↗
[Genetics and cancer: advances and perspectives].
Cancer genes can be ranged in three families: oncogenes, stimulating cell divisions; antioncogenes, blocking cell division; anti-apoptotic genes, blocking programmed cell death. Tumor progression usually results from cooperation between modifications of genes belonging to these different families. The familial predispositions to cancer are most frequently due to mutations of antioncogenes or of repair genes; in this later case, the original mutation results in abnormal DNA repair, then in accumulation of somatic mutations which could involve cancer genes. Recently, genes predisposing to familial forms of breast and colon cancers have been identified. Knowledge of such genes leads to possible presymptomatic prediction of the disease, but the preventive possibilities seem to be limited, today, to a preventive surgical exeresis. In the future, it would be desirable that elucidation of the mechanisms of cancer predisposition leads to less aggressive preventive or curative treatments.
Breast Neoplasms↗
In vivo transfer of a marker gene to study motoneuronal development.
Adenovirus vectors containing a marker gene (lacZ from Escherichia coli) are potent for transferring the gene to neurones after intraparenchymal injections. Expression of the marker gene may lead to the synthesis of an enormous amount of beta-galactosidase which diffuses throughout the entire neurone, providing a 'Golgi-like' staining. This suggested that the technique may be used to study the morphology of specific neuronal populations. We have validated this hypothesis by analysing the postnatal development of motoneurones in the rat cervical cord. Injections of the viral suspension into one ventral horn were performed at different ages after birth. Histochemical staining using X-Gal revealed morphological changes occurring within the first 3 weeks with enlargement of the perikaryon and increased dendritic complexity. Immunoreactivity for CGRP was visualized in double-staining experiments. In vivo transfer of a marker gene therefore provides a new way to analyse neuronal morphology which allows selection of the cells to be studied and double-labelling with immunohistochemical markers.
Adenoviridae↗
ZnSe(100) surface: Atomic configurations, composition, and surface dipole.
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Journal Article↗
Activity of the rat liver-specific aldolase B promoter is restrained by HNF3.
Although it contains binding sites for HNF1, NFY and C/EBP/DBP, the proximal promoter of the aldolase B gene is surprisingly weak when tested by transient transfection in differentiated hepatoma cells. This low activity could be due to overlapping between HNF1 and HNF3 binding sites in element PAB, from -127 to -103 bp with respect to the cap site. Replacement of the PAB region by a consensus HNF1 binding site unable to bind HNF3, results in a 30 fold activation of the promoter, in accordance with the hypothesis that activity of the wild-type promoter is normally restrained by HNF3 binding to PAB competitively with HNF1. Consistently, transactivation of the wild-type promoter by excess HNF1 is very high, most likely due to the displacement of HNF3, while the construct with the exclusive HNF1 binding site is weakly transactivated by HNF1. The inhibitory effect of HNF3 on HNF1-dependent transactivation is clearly due to competition between these two factors for binding to mutually exclusive, overlapping sites; indeed, when HNF1 and HNF3 sites are contiguous and not overlapping, the resulting promoter is as active as the one containing an exclusive HNF1 binding site. A construct in which PAB has been replaced by an exclusive HNF3 binding site is weakly expressed and is insensitive to HNF3 hyperexpression. DBP-dependent transactivation, finally, is independent of the nature of the element present in the PAB region.
Animals↗