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Biomedical subjects

F Costantini

Publications and source records attributed to F Costantini.

At least 55 records · Page 3Linked to original sources
PubMed1997

High-density lipoprotein cholesterol strongly discriminates between healthy free-living and disabled octo-nonagenarians: a cross sectional study. Associazione Medica Sabin.

Aging is frequently associated with a deterioration in health and functional status, which often induces important modifications in several biological parameters, including plasma lipids; as a consequence, the real "meaning" of lipoprotein parameters in old individuals is complex. A cross sectional study was carried out in order to investigate the lipoprotein profile in very old individuals with or without disability, and evaluate the possible influence of other biological variables on plasma lipids. One hundred selected healthy free-living (FL) and 62 disabled (DIS) subjects aged over 80 were enrolled; 91 healthy adults matched for origin were included as controls. Lipoprotein profile [total cholesterol, triglycerides, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol, apoprotein A-I and B], anthropometric parameters, and ADL were measured. The FL octo-nonagenarians featured higher HDL-cholesterol levels than adult controls. DIS octo-nonagenarians showed lower total and HDL-C levels than FL. Discriminant analysis indicated that HDL-cholesterol and apoprotein A-I, but not total cholesterol, strongly discriminated between FL and DIS octo-nonagenarians. Multivariate analysis demonstrated that the waist/hip ratio, an index of visceral adiposity, was negatively associated with HDL-C levels in FL, but not in DIS elderly. We conclude that: 1) in very old individuals, the absence or presence of disability is strongly associated with high or low HDL-cholesterol values, respectively; 2) HDL-C and apo A-I are the parameters which better discriminate between FL and DIS octo-nonagenarians; and 3) the differences in HDL-C levels between FL and DIS are not due to modifications in anthropometric parameters. Prospective studies are needed to better understand the relationship between high-density lipoprotein levels, disability and aging.

Activities of Daily Living↗
PubMed1997

Arterial disease in dipper and nondipper hypertensive patients.

Carotid artery and small vessel structural changes were evaluated in 45 dipper and 45 nondipper hypertensive subjects matched for sex, age, body mass index, smoking habit, and serum lipids. Patients underwent carotid ultrasonography to evaluate lumen diameter, intima-media thickness, cross-sectional area, and atherosclerotic plaques, and plethysmographic examination to record minimum forearm vascular resistance. In the whole study population, carotid cross-sectional area and prevalence of atherosclerotic plaques tended to be higher in nondippers than in dippers (19.9 +/- 6.0 v 17.5 +/- 6.1 mm2, P = .07, and 40% v 26%, P = .18), whereas intima-media thickness and minimum forearm vascular resistance were significantly higher in nondippers than in dippers (0.89 +/- 0.12 v 0.8 +/- 0.15 mm, P < .02, and 2.53 +/- 0.3 v 2.34 +/- 0.3 arbitrary resistance units (RU), P < .02). When gender was taken into account, different results were obtained in men and women. In men, vascular characteristics were not significantly different between dippers and nondippers. In women, carotid cross-sectional area and prevalence of atherosclerotic plaques tended to be higher in nondippers than in dippers (18.3 +/- 4.9 v 15.5 +/- 4.5 mm2, P = .06, and 40% v 15%, P = .15), and intima-media thickness and minimum forearm vascular resistance were significantly higher in nondippers than in dippers both before (0.84 +/- 0.1 v 0.73 +/- 0.1 mm, P < .01, and 2.38 +/- 0.3 v 2.16 +/- 0.2 RU, P < .01) and after adjustment for 24-h blood pressure. In conclusion, our data suggest that the progression of vascular disease may be differently affected in the two sexes by a different circadian blood pressure profile.

Blood Pressure Monitoring, Ambulatory↗
PubMed1997

White-coat resistant hypertension.

The aim of this study was to evaluate whether sustained hypertensives with high clinic blood pressure, despite multiple drug treatment, show a true resistant hypertension or a "white-coat effect," and whether the pretreatment white-coat effect is maintained despite pharmacological therapy. The occurrence of resistant hypertension was determined in 250 consecutive essential hypertensives who had had an ambulatory blood pressure monitoring before treatment assignment. Twenty-seven of 250 hypertensives with persistently high clinic blood pressure despite 3 months of adequate pharmacological therapy underwent further ambulatory blood pressure monitoring. Using our internal standards, seven patients had a true resistant hypertension whereas 20 subjects showed a large white-coat effect (white-coat resistant hypertension), ie, high clinic blood pressure (> 140/90) but "normal" ambulatory daytime (< 139/90 mm Hg) and 24 h (135/85 mm Hg) blood pressure. Using other cutoff points for ambulatory blood pressure, 134/90 and 135/85 mm Hg for daytime blood pressure, 10 and 13 patients, respectively, were reclassified as true resistant hypertensives and 17 and 14, respectively, were white-coat resistant hypertensives. Interestingly, in white-coat resistant hypertensives the large differences between clinic and ambulatory daytime blood pressure (white-coat effect), recorded before treatment assignment, were not affected by drugs and remained constant over time. Left ventricular mass index in white-coat resistant hypertensives was significantly lower than in truly resistant hypertensives, suggesting that prognosis could differ between these groups. In this study, using either our internal standards or some other cutoffs reported in the literature, the white-coat phenomenon was an important cause of resistant hypertension. The use of ambulatory blood pressure monitoring in these patients may avoid misdiagnosis of resistant hypertension, unnecessary overtreatment, and expensive procedures to look for possible secondary hypertension.

Antihypertensive Agents↗
PubMed1997

Two dominant mutations in the mouse fused gene are the result of transposon insertions.

The mouse Fused locus encodes a protein that has been implicated in the regulation of embryonic axis formation. The protein, which has been named Axin to distinguish it from the product of the unrelated Drosophila melanogaster gene fused, contains regions of similarity to the RGS (regulators of G-protein signaling) family of proteins as well as to dishevelled, a protein that acts downstream of Wingless in D. melanogaster. Loss-of-function mutations at Fused lead to lethality between days 8 and 10 of gestation. Three dominant mutations result in a kinked tail in heterozygotes. Two of the dominant mutations, Fused and Knobbly, result from insertion of intracisternal A particle retrotransposons into the gene. The insertion in Fused, within the sixth intron, creates a gene that produces wild-type transcripts as well as mutant transcripts that initiate at both the authentic promoter and the 3'-most long terminal repeat of the insertion. Knobbly, an insertion of the retrotransposon into exon 7, precludes the production of wild-type protein. Thus the Fused homozygote is viable whereas Knobbly is a recessive embryonic lethal. In both mutants the dominant kink-tailed phenotype is likely to result from the synthesis of similar amino-terminal fragments of Axin protein that would contain the RGS domain, but lack the dishevelled domain.

Animals↗
PubMed1997

In vivo formation of 8-Epi-prostaglandin F2 alpha is increased in hypercholesterolemia.

F2-isoprostanes are bioactive prostaglandin (PG)-like compounds that are produced from arachidonic acid through a nonenzymatic process of lipid peroxidation catalyzed by oxygen free-radicals. 8-Epi-PGF2 alpha may amplify the platelet response to agonists, circulates in plasma, and is excreted in urine. We examined the hypothesis that the formation of 8-epi-PGF2 alpha is altered in patients with hypercholesterolemia and contributes to platelet activation in this setting. Urine samples were obtained from 40 hypercholesterolemic patients and 40 age- and sex-matched control subjects for measurement of immunoreactive 8-epi-PGF2 alpha. Urinary excretion of 11-dehydro-thromboxane (TX) B2, a major metabolite of TXA2, was measured as an in vivo index of platelet activation. Low-dose aspirin, indobufen, and vitamin E were used to investigate the mechanism of formation and effects of 8-epi-PGF2 alpha on platelet activation. Urinary 8-epi-PGF2 alpha was significantly (P = .0001) higher in hypercholesterolemic patients than in control subjects: 473 +/- 305 versus 205 +/- 95 pg/mg creatinine. Its rate of excretion was inversely related to the vitamin E content of LDL and showed a positive correlation with urinary 11-dehydro-TXB2. Urinary 8-epi-PGF2 alpha was unchanged after 2-week dosing with aspirin and indobufen despite complete suppression of TX metabolite excretion. Vitamin E supplementation was associated with dose-dependent reductions in both urinary 8-epi-PGF2 alpha and 11-dehydro-TXB2 by 34% to 36% and 47% to 58% at 100 and 600 mg daily, respectively. We conclude that the in vivo formation of the F2-isoprostane 8-epi-PGF2 alpha is enhanced in the vast majority of patients with hypercholesterolemia. This provides an aspirin-insensitive mechanism possibly linking lipid peroxidation to amplification of platelet activation in the setting of hypercholesterolemia. Dose-dependent suppression of enhanced 8-epi-PGF2 alpha formation by vitamin E supplementation may contribute to the beneficial effects of antioxidant treatment.

Antioxidants↗
PubMed1997

Serum amyloid P component enhances induction of murine amyloidosis.

Serum amyloid P component (SAP), a common component of all known types of amyloid fibrils, protects amyloid fibrils from proteolysis in vitro. It is therefore speculated to contribute to the deposition of amyloid fibrils in various types of amyloidoses. However, a role for SAP in amyloid deposition is not yet known. To investigate the relationship between SAP and amyloid deposition, we used gene targeting techniques to generate a unique strain of mice carrying a null mutation at the sap locus. The resultant SAP-deficient mice displayed no obvious phenotypic abnormalities. We asked whether experimental amyloid A (AA) amyloidosis could be induced in the SAP-deficient mice. The wild-type and SAP-deficient mice did not differ in their synthesis of serum amyloid A, the precursor protein of AA amyloid fibril, in response to acute inflammation. The induction of AA amyloidosis, however, was significantly retarded in the SAP-deficient mice relative to wild-type mice. Our experiments present, for the first time, compelling evidence that, although not essential in the deposition of AA amyloid, SAP significantly accelerates this reaction. Thus, SAP enhances the induction of murine amyloidosis and may play an important role in the pathogenesis of human amyloidoses, including Alzheimer's disease.

Acute-Phase Reaction↗
PubMed1996

Magnetic resonance evidence of hypoxia in a homozygous alpha-knockout of a transgenic mouse model for sickle cell disease.

All transgenic mouse models for sickle cell disease express residual levels of mouse globins which complicate the interpretation of experimental results. We now report on a mouse expressing high levels of human betaS and 100% human alpha-globin. These mice were created by breeding the alpha-knockout and the mouse beta(major)-deletion to homozygosity in mice expressing human alpha- and betaS-transgenes. These betaS-alpha-knockout mice have accelerated red cell destruction, altered hematological indices, ongoing organ damage, and pathology under ambient conditions which are comparable with those found in alphaH betaS-Ant[betaMDD] mice without introduction of additional mutations which convert betaS into a "super-betaS" such as the doubly mutated betaS-Antilles. This is of particular importance for testing strategies for gene therapy of sickle cell disease. Spin echo magnetic resonance imaging at room air and 100% oxygen demonstrated the presence of blood hypoxia (high levels of deoxygenated hemoglobin) in the liver and kidneys that was absent in control mice. We demonstrate here that transgenic mice can be useful to test new noninvasive diagnostic procedures, since the magnetic resonance imaging technique described here potentially can be applied to patients with sickle cell disease.

Anemia, Sickle Cell↗
PubMed1996

Increased expression of laminin-1 and collagen (IV) subunits in the aganglionic bowel of ls/ls, but not c-ret -/- mice.

Extracellular matrix molecules, including laminin, affect the development of enteric neurons and accumulate in the aganglionic colon of ls/ls mice. Quantitative Northern analysis revealed that mRNAs encoding the beta 1 and gamma 1 subunits of laminin and collagens alpha 1(IV) and alpha 2(IV) are increased in the colons of ls/ls mice. Transcripts of laminin alpha 1 were evaluated quantitatively with reverse transcription and the competitive polymerase chain reaction (RT-cPCR). The abundance of laminin alpha 1 transcripts was developmentally regulated, but greater in the ls/ls than the wild-type colon at each age examined. In situ hybridization revealed that transcripts in the colon encoding laminin alpha 1 and beta 1 and collagen alpha 2(IV) were initially expressed in the endoderm, but by E15, expression shifted to cells of the colonic mesenchyme (ls/ls > wild type) where crest-derived cells migrate. The expression of laminin alpha 1 was examined in the totally aganglionic intestine of E15 and newborn c-ret -/- mice, to determine whether an increase occurs when neurogenesis fails independently of the ls/ls defect. RT-cPCR revealed no difference from control in mRNA encoding laminin alpha 1 in the c-ret -/- colon in either E15 or newborn animals. The accumulation of immunohistochemically demonstrable laminin that is prominent in the newborn ls/ls colon could not be detected in that of c-ret -/- animals. These observations suggest that transcripts encoding laminin-1 and collagen (IV) are increased in the colon and surrounding pelvic mesenchyme of ls/ls mice because of an intrinsic lesion, rather than a secondary consequence of aganglionosis. The data are compatible with the hypothesis that the increased expression of laminin-1 contributes to the failure of crest-derived cells to complete their colonization of the ls/ls colon.

Animals↗
PubMed1996

GDNF signalling through the Ret receptor tyrosine kinase.

Mutational analysis in humans and mice has demonstrated that the Ret, the product of the c-ret proto-oncogene, a member of the receptor tyrosine kinase (RTK) superfamily, is essential for development of the enteric nervous system and kidney. Despite the established role of Ret in mammalian embryogenesis, its cognate ligand(s) is currently unknown. Here we demonstrate, by using a Xenopus embryo bioassay, that glial-cell-line-derived neurotrophic factor (GDNF), a distant member of the transforming growth factor (TGF)-beta superfamily, signals through the Ret RTK. Furthermore, using explant cultures from wild-type and Ret-deficient mouse embryos, we show that normal c-ret function is necessary for GDNF signalling in the peripheral nervous system. Our data strongly suggest that Ret is a functional receptor for GDNF, and that GDNF, in addition to its potential role in the differentiation and survival of central nervous system neurons, has profound effects on kidney organogenesis and the development of the peripheral nervous system.

Animals↗
PubMed1996

Protection from lethal malaria in transgenic mice expressing sickle hemoglobin.

Previous studies from our laboratories have shown that transgenic mice expressing high levels of beta S globin are well-protected from Plasmodium chabaudi adami and partially protected against P berghei (Shear et al, Blood 81:222, 1993). We have now infected transgenic mice expressing low (39%), intermediate (57%), and high (75%) levels of beta S with the virulent strain of P yoelii (17XL) that appears to cause cerebral malaria. We find that the level of protection in these three groups of mice correlates positively with the level of beta S chain expression in the mice. Seven of nine mice expressing the high level of beta S recovered from infection, as did 7 of 9 mice expressing the intermediate level of beta S. Control mice and mice expressing the lower level of beta S all succumbed to infection. In mice expressing high and intermediate levels of beta S, parasites were found almost exclusively in reticulocytes during recovery, suggesting that mature red blood cells expressing beta S are more resistant than reticulocytes. These studies confirm epidemiologic data and offer insight into the mechanism of protection of sickle trait individuals against falciparum malaria.

Animals↗
PubMed1996

Differential effects of an erythropoietin receptor gene disruption on primitive and definitive erythropoiesis.

Although the hormone erythropoietin (Epo) and its receptor (EpoR) are known to play important roles in the regulation of erythropoiesis, several questions remain concerning the developmental role of Epo/EpoR signaling. As the functions of Epo have been defined primarily through studies of definitive erythroid cells, its importance for primitive, embryonic erythropoiesis remains uncertain, as does the significance of EpoR expression in several nonerythroid cell types. To address these questions, mouse embryonic stem cells and embryos lacking a functional EpoR gene were produced by gene targeting. The effects of the mutation were examined in embryos developing in vivo, in chimeric adult mice produced with homozygous mutant embryonic stem cells, and in hemopoietic cells cultured in vitro. No defects were apparent in nonerythroid cell lineages in which the EpoR normally is expressed, including megakaryocytes and endothelial cells. In the mutant yolk sac, primitive erythrocytes were produced in normal numbers, they underwent terminal differentiation, and expressed near normal levels of embryonic globins, although they were reduced in size and their proliferation was severely retarded after E9.5. In contrast, in the fetal liver, definitive erythropoiesis beyond the late progenitor (CFU-E) stage was drastically inhibited by the EpoR mutation, and virtually no definitive erythrocytes were produced in vivo, leading to embryonic death by E13.5. Thus, our results suggest a fundamental difference in the molecular mechanisms stimulating primitive and definitive erythropoiesis. It was also observed that a few mutant definitive erythroid cells could terminally differentiate when cultured with additional cytokines, demonstrating that although Epo/EpoR signaling is important for definitive erythroid cell survival and proliferation, it is not an obligatory step in differentiation.

Animals↗
PubMed1996

Diagnosis of rabbit haemorrhagic disease (RHD) and European brown hare syndrome (EBHS) by indirect sandwich polyclonal ELISA.

The establishment of two indirect sandwich polyclonal ELISAs for the virological diagnosis of rabbit haemorrhagic disease (RHD) and European brown hare syndrome (EBHS) is described. Each assay uses rabbit and guinea pig antisera raised to RHD or EBHS purified virus particles. The tests are sensitive and specific, but cross-reactions between rabbit haemorrhagic disease virus (RHDV) and European brown hare syndrome virus (EBHSV) are observed. Most rabbit liver homogenates identified as RHDV-positive by ELISA do not haemagglutinate human red blood cells at room temperature.

Animals↗
PubMed1996

Systemic oxidative stress and its relationship with age and illness. Associazione Medica "Sabin".

OBJECTIVE: It has recently been proposed that increased oxidative stress may play a role in the aging process and age-associated degenerative diseases. DESIGN AND MEASUREMENTS: A cross-sectional study was carried out to assess the relationship of circulating antioxidants, namely vitamins E and C, beta-carotene, proteic thiols (P-SH) and ceruloplasmin, and of lipid peroxides, with both aging and aging with disability, i.e., unsuccessful aging. PARTICIPANTS: One hundred healthy free living and 62 disabled octo-nonagenarians and 91 healthy adults were enrolled in the study. RESULTS: Free living and disabled older adults had lower antioxidant and higher lipid peroxide levels than healthy adults, as well as the disabled older adults compared with free living older persons. Using logistic regression, we observed that plasma concentrations of vitamins E and C, P-SH, and lipid peroxides were independently associated with either aging or aging with disability, apparently representing biochemical indicators of patient status. In particular, aging and unsuccessful aging were associated with higher levels of lipid peroxides independently of circulating levels of vitamins C and E, suggesting that the increased oxidative stress was not merely an effect of a lower dietary intake of antioxidants. Serum ceruloplasmin was significantly higher in free living older adults than in healthy adults, and in the disabled compared with free living octo-nonagenarians. CONCLUSIONS: Our findings are consistent with the presence of systemic oxidant load in older adults, and this phenomenon is far more evident in unsuccessful aging.

Aged↗
PubMed1996

Common origin and developmental dependence on c-ret of subsets of enteric and sympathetic neuroblasts.

c-ret encodes a tyrosine kinase receptor that is necessary for normal development of the mammalian enteric nervous system. Germline mutations in c-ret lead to congenital megacolon in humans, while a loss-of-function allele (ret.k-) causes intestinal aganglionosis in mice. Here we examine in detail the function of c-ret during neurogenesis, as well as the lineage relationships among cell populations in the enteric nervous system and the sympathetic nervous system that are dependent on c-ret function. We report that, while the intestine of newborn ret.k- mice is devoid of enteric ganglia, the esophagus and stomach are only partially affected; furthermore, the superior cervical ganglion is absent, while more posterior sympathetic ganglia and the adrenal medulla are unaffected. Analysis of mutant embryos shows that the superior cervical ganglion anlage is present at E10.5, but absent by E12.5, suggesting that c-ret is required for the survival or proliferation of sympathetic neuroblasts. In situ hybridization studies, as well as direct labelling of cells with DiI, indicate that a common pool of neural crest cells derived from the postotic hindbrain normally gives rise to most of the enteric nervous system and the superior cervical ganglion, and is uniquely dependent on c-ret function for normal development. We term this the sympathoenteric lineage. In contrast, a distinct sympathoadrenal lineage derived from trunk neural crest forms the more posterior sympathetic ganglia, and also contributes to the foregut enteric nervous system. Overall, our studies reveal previously unknown complexities of cell lineage and genetic control mechanisms in the developing mammalian peripheral nervous system.

Animals↗
PubMed1996

Renal agenesis and hypodysplasia in ret-k- mutant mice result from defects in ureteric bud development.

The c-ret gene encodes a receptor tyrosine kinase that is expressed in the Wolffian duct and ureteric bud of the developing excretory system. Newborn mice homozygous for a mutation in c-ret displayed renal agenesis or severe hypodysplasia, suggesting a critical role for this gene in metanephric kidney development. To investigate the embryological basis of these defects, we characterized the early development of the excretory system in mutant homozygotes, and observed a range of defects in the formation, growth and branching of the ureteric bud, which account for the spectrum of renal defects seen at birth. Co-culture of isolated ureteric buds and metanephric mesenchyme show that the primary defect is intrinsic to the ureteric bud. While the mutant bud failed to respond to induction by wild-type mesenchyme, mutant mesenchyme was competent to induce the growth and branching of the wild-type bud. Furthermore, the mutant metanephric mesenchyme displayed a normal capacity to differentiate into nephric tubules when co-cultured with embryonic spinal cord. These findings suggest a model in which c-ret encodes the receptor for a (yet to be identified) factor produced by the metanephric mesenchyme, which mediates the inductive effects of this tissue upon the ureteric bud. This factor appears to stimulate the initial evagination of the ureteric bud from the Wolffian duct, as well as its subsequent growth and branching.

Animals↗
PubMed1995

A second generation transgenic mouse model expressing both hemoglobin S (HbS) and HbS-Antilles results in increased phenotypic severity.

We report on a second generation of transgenic mice produced by crossing a transgenic mouse line expressing high levels of human alpha and beta S chains (alpha H beta S [beta MDD]) with a line expressing human alpha and beta S-Antilles (beta SAnt). We hypothesized that mice expressing both hemoglobins (Hbs) would have a more severe phenotype because the reduced oxygen affinity and solubility of the beta S-Antilles might enhance the rate and extent of polymer formation. We obtained mice that expressed both beta S and beta S-Antilles. The doubly transgenic mice that are heterozygous for deletion of mouse beta Major (beta MD) occurred with reduced frequency and those that are homozygous for deletion of mouse beta Major (beta MDD) occurred at a much reduced frequency and suffered early mortality. Human alpha was 58% of all alpha globin for all animals, whereas beta S and beta S-Antilles were 34% and 28% of all beta globins for beta MD mice and 42% and 36% for beta MDD mice. Hematocrit, Hb, and mean corpuscular Hb were normal for all transgenic mice, but reticulocyte levels were higher for the doubly transgenic mice versus alpha H beta S [beta MDD] mice older than 30 days (10.0% +/- 1.0% v 4.3% +/- 0.4%; P < .001, mean +/- SE, n = 20 and n = 10, respectively) and control mice (3.9% +/- 0.4%). Reticulocytosis was more severe in mice less than 30 days old ( > 20% for alpha H beta S beta S-Ant[beta MDD] mice). The median mean corpuscular hemoglobin concentration of doubly transgenic mice was higher than that of alpha H beta S[beta MDD] mice with a variable number of very dense cells. Delay times for polymerization of Hb in red blood cells from alpha H beta S beta S-Ant[beta MDD] mice were shorter than those of alpha H beta S[beta MDD] mice, and there were fewer cells with delay times greater than 100 seconds. Urine-concentrating ability in control mice under ambient conditions is 2,846 +/- 294 mOsm and was reduced 30% to 1,958 +/- 240 mOsm, P < 4 x 10(-8) in all mice expressing both transgenes. We conclude that doubly transgenic mice have a more severe phenotype than either of the two parental lines. These mice may be suitable for validating therapeutic intervention in sickle cell disease.

Anemia, Sickle Cell↗
PubMed1995

Sequences located 3' to the breakpoint of the hereditary persistence of fetal hemoglobin-3 deletion exhibit enhancer activity and can modify the developmental expression of the human fetal A gamma-globin gene in transgenic mice.

Expression of fetal gamma-globin genes in individuals with the deletion forms of hereditary persistence of fetal hemoglobin (HPFH) has been attributed either to enhancement by 3' regulatory elements juxtaposed to gamma-globin genes or to deletion of gamma-gene silencers normally residing within the beta-globin gene cluster. In the present study, we tested the hypothesis of imported enhancers downstream of beta-globin gene using the HPFH-3 deletion as a model. The abnormal bridging fragment of 13.6 kilobases (kb) containing the A gamma-gene with its flanking sequences and 6.2 kb of the juxtaposed region was microinjected into fertilized mouse eggs. Twelve transgenic mice positive for the fragment were generated. Samples from 11.5-day yolk sacs, 16-day fetal liver, and adult blood were analyzed for A gamma-mRNA using RNase protection assays. Three mice lacked A gamma expression in the yolk sac indicating non-optimal integration site. Four expressed A gamma-mRNA at the embryonic stage only, while two expressed A gamma-mRNA in both embryonic and fetal liver erythroid cells. Since the A gamma-gene with its normal flanking sequences and in the absence of the locus control region is expressed only in embryonic cells of transgenic mice, these data suggest that the juxtaposed sequences have altered the developmental specificity of the fetal gamma-globin gene. These sequences were further tested for the presence of an enhancer element, by their ability to activate a fusion reporter gene consisting of the CAT gene linked to the gamma-globin gene promoter, in erythroid (K562) and non-erythroid (HeLa) cells. A 0.7-kb region located immediately 3' to the breakpoint, enhanced chloramphenicol acetyltransferase activity by 3-fold in erythroid cells. The enhancer also activated the embryonic epsilon-globin gene promoter by 2-fold but not the adult beta- or delta-globin gene promoters. The enhancer represents a region of previously known complex tandem repeats; in this study we have completed the sequencing of the region encompassing the 0.7-kb enhancer element. Multiple areas of the enhancer region exhibit homology to the core element of the simian virus 40 enhancer and to the sequences of the human 3' A gamma- and the chicken 3' beta-globin enhancers. A consensus binding site for the erythroid specific GATA-1 transcription factor and seven consensus sites for the ubiquitous CP1 transcription factor are also included within the enhancer. These data suggest that these sequences located immediately 3' to the breakpoint of the HPFH-3 deletion, exhibit both the structure and the function of an enhancer, and can modify the developmental specificity of the fetal gamma-globin genes, resulting in their continued expression during adult life.

Animals↗