Transgenic mice with expression of elevated levels of copper-zinc superoxide dismutase in the lungs are resistant to pulmonary oxygen toxicity.

To test the hypothesis that increases in lung superoxide dismutase can cause tolerance to pulmonary oxygen toxicity, we studied transgenic mice which constitutively express elevated levels of the human copper-zinc SOD (CuZnSOD). Upon exposure to hyperoxia (greater than 99% O2, 630 torr) the transgenic CuZnSOD mice showed increased survival, decreased morphologic evidence of lung damage such as edema and hyaline membrane formation, and reduction in the number of lung neutrophils. During continuous exposure to oxygen, both control and transgenic animals who successfully adapted to hyperoxia showed increased activity of lung antioxidant enzymes such as glutathione peroxidase (GPX), glutathione reductase (GR), and glucose-6-phosphate dehydrogenase (G6PD), whereas superoxide dismutase activity remained unchanged. The results show that expression of elevated levels of CuZnSOD decreases pulmonary oxygen toxicity and associated histologic damage and mortality.

Transcription-coupled translation control of AML1/RUNX1 is mediated by cap- and internal ribosome entry site-dependent mechanisms.

AML1/RUNX1 belongs to the runt domain transcription factors that are important regulators of hematopoiesis and osteogenesis. Expression of AML1 is regulated at the level of transcription by two promoters, distal (D) and proximal (P), that give rise to mRNAs bearing two distinct 5' untranslated regions (5'UTRs) (D-UTR and P-UTR). Here we show that these 5'UTRs act as translation regulators in vivo. AML1 mRNAs bearing the uncommonly long (1,631-bp) P-UTR are poorly translated, whereas those with the shorter (452-bp) D-UTR are readily translated. The low translational efficiency of the P-UTR is attributed to its length and the cis-acting elements along it. Transfections and in vitro assays with bicistronic constructs demonstrate that the D-UTR mediates cap-dependent translation whereas the P-UTR mediates cap-independent translation and contains a functional internal ribosome entry site (IRES). The IRES-containing bicistronic constructs are more active in hematopoietic cell lines that normally express the P-UTR-containing mRNAs. Furthermore, we show that the IRES-dependent translation increases during megakaryocytic differentiation but not during erythroid differentiation, of K562 cells. These results strongly suggest that the function of the P-UTR IRES-dependent translation in vivo is to tightly regulate the translation of AML1 mRNAs. The data show that AML1 expression is regulated through usage of alternative promoters coupled with IRES-mediated translation control. This IRES-mediated translation regulation adds an important new dimension to the fine-tuned control of AML1 expression.

Upregulation of GABA neurotransmission suppresses hippocampal excitability and prevents long-term potentiation in transgenic superoxide dismutase-overexpressing mice.

Cu/Zn superoxide dismutase (SOD-1) is a key enzyme in oxygen metabolism in the brain. Overexpression of SOD-1 in transgenic (Tg) mice has been used to study the functional roles of this enzyme in oxidative stress, lipid peroxidation, and neurotoxicity. We found that Tg-SOD-1 mice are strikingly less sensitive to kainic acid-induced behavioral seizures than control mice. Furthermore, the hippocampus of Tg-SOD-1 mice was far less sensitive to local application of bicuculline, a GABA-A antagonist, than the hippocampus of control mice. GABAergic functions, expressed in extracellular paired-pulse depression, and in IPSCs recorded in dentate granular cells were enhanced in Tg-SOD-1 mice. Finally, long-term potentiation (LTP), not found in the dentate gyrus of Tg-SOD-1 mice, could be restored by local blockade of inhibition and could be blocked in control mice by injection of diazepam, which amplifies inhibition. These results indicate that constitutive elevation of SOD-1 activity exerts a major effect on neuronal excitability in the hippocampus, which, in turn, controls hippocampal ability to express LTP.

Docosahexaenoic acid-deficient phosphatidyl serine and high alpha-tocopherol in a fetal mouse brain over-expressing Cu/Zn-superoxide dismutase.

The over-expressed Cu/Zn-superoxide dismutase (Cu/Zn-SOD) gene has been found in some circumstances phenotypically deleterious and associated with oxidative injury-mediated aberrations while in other studies it was considered neuroprotective. In this work we examine a number of biochemical markers in fetal and adult brain from transgenic (tg) mice expressing the human Cu/Zn-SOD gene, which may determine this dual characteristic. These markers include the polyunsaturated fatty acid (PUFA) profile in discrete phospholipid species, the alpha-tocopherol levels, a marker for lipid anti-oxidant status, and thiobarbituric acid reactive substance (TBARS), a marker for the tissue oxidative status. The PUFA profile in choline- and ethanolamine-phosphoglycerides was similar in tg and nontransgenic (ntg) animals of either fetal or adult brain. Serine-phosphoglycerides, however, showed a marked decrease from 20. 07+/-0.53 to 14.92+/-0.87 wt% and 14.52+/-1.15 wt% in docosahexaenoic acid (DHA; 22:6 n3), in the tg 51 and tg 69 fetal brains, respectively, but not in the comparable adult tissues. The alpha-tocopherol levels were significantly higher in the fetal compared to the adult brain. There were no differences in the anti-oxidant levels between the ntg and tg fetal brains, but there were differences in the adult animals; the tg mice were higher by at least two-fold than the control animals. The basal TBARS in the tg 51 fetal brain was 35% lower than that of ntg mouse and in the presence of Fe(2+), brain slices from the former released less TBARS (57% reduction) into the medium than the latter. These results suggest that higher dosages of Cu/Zn-SOD gene are compatible with increased alpha-tocopherol levels, reduced basal TBARS levels and a DHA deficiency in the fetal, but not the adult, tg brain.

Expression of AML1-d, a short human AML1 isoform, in embryonic stem cells suppresses in vivo tumor growth and differentiation.

The human AML1 gene encodes a heterodimeric transcription factor which plays an important role in mammalian hematopoiesis. Several alternatively spliced AML1 mRNA species were identified, some of which encode short protein products that lack the transactivation domain. When transfected into cells these short isoforms dominantly suppress transactivation mediated by the full length AML1 protein. However, their biological function remains obscure. To investigate the role of these short species in cell proliferation and differentiation we generated embryonic stem (ES) cells overexpressing one of the short isoforms, AML1-d, as well as cells expressing the full length isoforms AML1-b and AML2. The in vitro growth rate and differentiation of the transfected ES cells were unchanged. However, overexpression of AML1-d significantly affected the ES cells' ability to form teratocarcinomas in vivo in syngeneic mice, while a similar overexpression of AML1-b and AML2 had no effect on tumor formation. Histological analysis revealed that the AML1-d derived tumors were poorly differentiated and contained numerous apoptotic cells. These data highlight the pleiotropic effects of AML1 gene products and demonstrate for the first time an in vivo growth regulation function for the short isoform AML1-d.

Impaired glucose-induced insulin response in transgenic mice overexpressing the L-phosphofructokinase gene.

The selective impairment of glucose-induced insulin secretion in NIDDM can be attributed to defects in the glucose-signaling system. An alteration in the activity of phosphofructokinase (PFK), a key enzyme in the glycolytic pathway, may play a role in the abnormal glucose-induced insulin secretion. In this study, we evaluated insulin secretion in transgenic (Tg) mice overexpressing the liver-type subunit of phosphofructokinase (PFKL). Three independently derived Tg-PFKL lines showed random and postprandial hyperglycemia with diminished acute insulin response following intravenous glucose tolerance load. Isolated islets of Tg-PFKL mice exhibited a shift to the right of the glucose insulin dose curve. However, the maximal insulin secretory capacity, as well as the potentiation effect by arginine, were retained. PFK activity in Tg-PFKL islets was increased by 30-70%, because of the overexpression of PFKL. Conceivably, a selective overexpression of the PFKL isoform in Tg-PFKL mice altered the enzymatic properties of the tetrameric PFK and thereby affected glucose metabolism. A similar phenomenon was previously observed in transfected PC12-PFKL cells. The data show that overexpression of PFKL in transgenic mice was associated with diminished glucose-induced insulin response and suggest a mechanism to explain the role of beta-cell PFK activity in glucose-induced insulin secretion.

Site-directed mutagenesis supports a three-dimensional model of the runt domain.

The "runt domain" (RD) is a 128 amino acid region of the Drosophila pair-rule gene runt. This highly conserved region delineates the DNA-binding domain of a new family of transcription factors; the RD proteins. The family includes genes from Drosophila, chicken and mammals that are involved in a wide range of developmental processes, from sex determination and neurogenesis in Drosophila to hematopoiesis and osteoblast differentiation in mouse and human. The RD confers DNA binding ability and mediates the interaction of mammalian RD proteins with the beta-subunit (CBFbeta), which enhances the DNA binding. The primary sequence of RD shows no similarity to other known DNA-binding motifs and its three-dimensional (3D) structure is not known. We employed molecular modeling-based mutagenesis to generate a 3D model of RD. Fold recognition programs identified the palm subdomain of rat DNA polymerase beta as the most likely fold for RD. In the predicted model, the RD region which interacts with DNA contains two arginine residues, R130 and R135, which appear to be in close contact with the major groove of the DNA and to interact with the three essential guanine bases of the core DNA motif PyGPyGGT. We mutated these two R residues and demonstrated that mutations markedly reduced the binding of RD to DNA with no effect on RD interaction with CBFbeta. The data provide important clues about the possible 3D structure of the RD and its interaction with the core DNA motif.

Spatial and temporal expression pattern of Runx3 (Aml2) and Runx1 (Aml1) indicates non-redundant functions during mouse embryogenesis.

The human RUNX3/AML2 gene belongs to the 'runt domain' family of transcription factors that act as gene expression regulators in major developmental pathways. Here, we describe the expression pattern of Runx3 during mouse embryogenesis compared to the expression pattern of Runx1. E10.5 and E14.5-E16.5 embryos were analyzed using both immunohistochemistry and beta-galactosidase activity of targeted Runx3 and Runx1 loci. We found that Runx3 expression overlapped with that of Runx1 in the hematopoietic system, whereas in sensory ganglia, epidermal appendages, and developing skeletal elements, their expression was confined to different compartments. These data provide new insights into the function of Runx3 and Runx1 in organogenesis and support the possibility that cross-regulation between them plays a role in embryogenesis.

Runx2 (Cbfa1) inhibits Shh signaling in the lower but not upper molars of mouse embryos and prevents the budding of putative successional teeth.

Heterozygous mutations in the RUNX2 (CBFA1) gene cause cleidocranial dysplasia, characterized by multiple supernumerary teeth. This suggests that Runx2 inhibits successional tooth formation. However, in Runx2 knockout mice, molar development arrests at the late bud stage, and lower molars are more severely affected than upper ones. We have proposed that compensation by Runx3 may be involved. We compared the molar phenotypes of Runx2/Runx3 double-knockouts with those of Runx2 knockouts, but found no indication of such compensation. Shh and its mediators Ptc1, Ptc2, and Gli1 were down-regulated only in the lower but not the upper molars of Runx2 and Runx2/Runx3 knockouts. Interestingly, in front of the mutant upper molar, a prominent epithelial bud protruded lingually with active Shh signaling. Similar buds were also present in Runx2 heterozygotes, and they may represent the extension of dental lamina for successional teeth. The results suggest that Runx2 prevents the formation of Shh-expressing buds for successional teeth.

Radiation sensitivity of Down's syndrome fibroblasts might be due to overexpressed Cu/Zn-superoxide dismutase (EC 1.15.1.1).

Trisomy 21 (Down's syndrome, DS) is the most frequent chromosomal aberration. Triplication of a small region of chromosome 21, the fragment 21q22 is sufficient to cause the DS phenotype including immunodeficiency, premature aging, neurodegenerations, mental retardation and an increased risk of leukemia. Chromosomal aberrations caused by X-ray irradiation were observed in DS lymphocytes and DS fibroblasts, but the correlation to cell death or repair deficiency was not clear. We approached this problem and report here on a profound X-ray repair deficiency of DS cells. With a colorimetric viability assay we observed an UV sensitivity of DS fibroblasts at doses beyond 14 Jm-2 but no significant X-ray sensitivity. By the nucleoid sedimentation technique, a deficient restoration of nucleoids in DS cells after X-ray irradiation was demonstrated. The same features apply for cells, which contain an overexpressed Cu/Zn-superoxide dismutase (SOD-1) gene. Radiation sensitivity of DS cells and SOD-1 overexpressing cells resemble those of ataxia telangiectasia (AT) fibroblasts. Additionally, DS and AT cells exert lack of inhibition of DNA synthesis after X-ray irradiation.

The RUNX3 gene--sequence, structure and regulated expression.

The RUNX3 gene belongs to the runt domain family of transcription factors that act as master regulators of gene expression in major developmental pathways. In mammals the family includes three genes, RUNX1, RUNX2 and RUNX3. Here, we describe a comparative analysis of the human chromosome 1p36.1 encoded RUNX3 and mouse chromosome 4 encoded Runx3 genomic regions. The analysis revealed high similarities between the two genes in the overall size and organization and showed that RUNX3/Runx3 is the smallest in the family, but nevertheless exhibits all the structural elements characterizing the RUNX family. It also revealed that RUNX3/Runx3 bears a high content of the ancient mammalian repeat MIR. Together, these data delineate RUNX3/Runx3 as the evolutionary founder of the mammalian RUNX family. Detailed sequence analysis placed the two genes at a GC-rich H3 isochore with a sharp transition of GC content between the gene sequence and the downstream intergenic region. Two large conserved CpG islands were found within both genes, one around exon 2 and the other at the beginning of exon 6. RUNX1, RUNX2 and RUNX3 gene products bind to the same DNA motif, hence their temporal and spatial expression during development should be tightly regulated. Structure/function analysis showed that two promoter regions, designated P1 and P2, regulate RUNX3 expression in a cell type-specific manner. Transfection experiments demonstrated that both promoters were highly active in the GM1500 B-cell line, which endogenously expresses RUNX3, but were inactive in the K562 myeloid cell line, which does not express RUNX3.

Architecture and anatomy of the genomic locus encoding the human leukemia-associated transcription factor RUNX1/AML1.

The RUNX1 gene on human chromosome 21q22.12 belongs to the 'runt domain' gene family of transcription factors (also known as AML/CBFA/PEBP2alpha). RUNX1 is a key regulator of hematopoiesis and a frequent target of leukemia associated chromosomal translocations. Here we present a detailed analysis of the RUNX1 locus based on its complete genomic sequence. RUNX1 spans 260 kb and its expression is regulated through two distinct promoter regions, that are 160 kb apart. A very large CpG island complex marks the proximal promoter (promoter-2), and an additional CpG island is located at the 3' end of the gene. Hitherto, 12 different alternatively spliced RUNX1 cDNAs have been identified. Genomic sequence analysis of intron/exon boundaries of these cDNAs has shown that all consist of properly spliced authentic coding regions. This indicates that the large repertoire of RUNX1 proteins, ranging in size between 20-52 kDa, are generated through usage of alternatively spliced exons some of which contain in frame stop codons. The gene's introns are largely depleted of repetitive sequences, especially of the LINE1 family. The RUNX1 locus marks the transition from a ~1 Mb of gene-poor region containing only pseudogenes, to a gene-rich region containing several functional genes. A search for RUNX1 sequences that may be involved in the high frequency of chromosomal translocations revealed that a 555 bp long segment originating in chromosome 11 FLI1 gene was transposed into RUNX1 intron 4.1. This intron harbors the t(8;21) and t(3;21) chromosomal breakpoints involved in acute myeloid leukemia. Interestingly, the FLI1 homologous sequence contains a breakpoint of the t(11;22) translocation associated with Ewing's tumors, and may have a similar function in RUNX1.

Transgenic mice with elevated level of CuZnSOD are highly susceptible to malaria infection.

Copper/zinc superoxide dismutase (CuZnSOD) catalyses the conversion of O2.- into H2O2. Constitutive overexpression of CuZnSOD in cells and animals creates an indigenous oxidative stress that predisposes them to added insults. In this study, we used transgenic CuZnSOD (Tg-CuZnSOD) mice with elevated levels of CuZnSOD to determine whether overexpression of CuZnSOD affected the susceptibility of these mice to plasmodium infection. Acute malaria is associated with oxidative stress, mediated by redox-active iron released from the infected RBC. Two independently derived Tg-CuZnSOD lines showed higher sensitivity than control mice to infection by Plasmodium berghei (P. berghei), reflected by an earlier onset and increased rate of mortality. Nevertheless, while Tg-CuZnSOD mice were more vulnerable than control mice, the levels of parasitemia were comparable in both strains. Moreover, treatment of infected red blood cells (RBC) with oxidative stress inducers, such as ascorbate or paraquat, reduced the viability of parasites equally in both transgenic and control RBC. This further confirms that increased CuZnSOD does not support plasmodia development. The data are consistent with the possibility that the combination of increased redox-active iron and elevated H2O2 in the plasmodium-infected Tg-CuZnSOD mice, led to an enhanced Fenton's reaction-mediated HO. production, and the resulting oxidative injury renders the transgenic mice more vulnerable to parasite infection.

Expression of manganese superoxide dismutase is not altered in transgenic mice with elevated level of copper-zinc superoxide dismutase.

In evaluating the relative expression of copper-zinc and manganese superoxide dismutase (CuZnSOD and MnSOD) in vivo in states like Down syndrome in which one dismutase is present at increased levels, we measured activities of both enzymes, in tissues of control and transgenic mice constitutively expressing increased levels of CuZnSOD, during exposure to normal and elevated oxygen tensions. Using SOD gel electrophoresis assay, CuZnSOD and MnSOD activities of brain, lung, heart, kidney, and liver from mice exposed to either normal (21%) or elevated (> 99% oxygen, 630 torr) oxygen tensions for 120 h were compared. Whereas CuZnSOD activity was elevated in tissues of transgenic relative to control mice under both normoxic or hyperoxic conditions, MnSOD activities in organs of transgenic mice were remarkably similar to those of controls under both conditions. To confirm the accuracy of this method in quantitating MnSOD relative to CuZnSOD expression, two other methods were utilized. In lung, which is the organ exposed to the highest oxygen tension during ambient hyperoxia, a sensitive, specific ELISA for MnSOD was used. Again, MnSOD protein was not different in transgenic relative to control mice during exposure to air or hyperoxia. In addition, lung MnSOD protein was not changed significantly by exposure to hyperoxia in either group. In kidney, a mitochondrion-rich organ, SOD assay, before and after inactivation of CuZnSOD with diethyldithiocarbamate, was used. MnSOD activity was not different in organs from air-exposed transgenic relative to control mice. The data indicated that expression of MnSOD in vivo was not affected by overexpression of the CuZnSOD and, therefore, the two enzymes are probably regulated independently.

Late degeneration of nigro-striatal neurons in ATM-/- mice.

The generation of an Atm -/- mouse model of the human ataxia-telangiectasia (AT) opened new avenues toward a better understanding of the molecular and cellular basis of AT. We have recently reported that 5-month-old Atm-/- mice exhibit severe loss of tyrosine hydroxylase-positive, dopaminergic nigro-striatal neurons, down to 26% of age-matched controls. In the present study we analyzed development of the dopaminergic cell loss in the context of the nigro-striatal system. We found that dopaminergic neurons are formed normally in the Atm-/- mouse, and degenerate during the first few months of life; there was no difference between 1-month-old Atm-/- and control mice in the number of dopaminergic cells that were retrogradely labeled by an injection of fluorescent tracer into the striatum. On the other hand, a dramatic reduction in the number of labeled cells was found in 5-month-old Atm-/- mice. This cell loss was significant in areas A9 and A10 but not in area A9-I. These findings indicate that midbrain dopaminergic neurons in Atm-/- mice initially send normal axons to the striatum, only to degenerate later in life. In addition, an age-dependent as well as topographic, medial-to-lateral loss of GAD, met-enkephaline and substance-P immunopositive cells was found in the striatum of the Atm-/- mice. This phenomenon was significant only in the 5-month-old Atm-/- mice (3 months after the beginning of detectable dopaminergic cell loss). In both the striatum and the substantia nigra, the apparent cell loss was accompanied by gliosis. In addition, alpha-synuclein immunopositive bodies were observed in the cortex, striatum and substantia nigra of these mice. The present data indicate that Atm-/- mice exhibit a progressive, age-dependent, reduction in dopaminergic cells of the substantia nigra, followed by a reduction in projection neurons of the striatum. Thus, the Atm-/- mouse may model the extrapyramidal motor deficits seen in AT patients.

RGC death in mice after optic nerve crush injury: oxidative stress and neuroprotection.

PURPOSE: To establish a method for morphometric analysis of retrogradely labeled retinal ganglion cells (RGCs) of the mouse retina, to be used for the study of molecular aspects of RGC survival and neuroprotection in this model; to evaluate the effect of overexpression of Cu-Zn-superoxide dismutase (CuZnSOD) on RGC survival after severe crush injury to the optic nerve, and to assess the effect of the alpha2-adrenoreceptor agonist brimonidine, recently shown to be neuroprotective, on RGC survival. METHODS: A severe crush injury was inflicted unilaterally in the orbital portion of the optic nerves of wild-type and transgenic (Tg-SOD) mice expressing three to four times more human CuZnSOD than the wild type. In each mouse all RGCs were labeled 72 hours before crush injury by stereotactic injection of the neurotracer dye FluoroGold (Fluorochrome, Denver, CO) into the superior colliculus. Survival of RGCs was then assessed morphometrically, with and without systemic injection of brimonidine. RESULTS: Two weeks after crush injury, the number of surviving RGCs was significantly lower in the Tg-SOD mice (596.6 +/- 71.9 cells/mm(2)) than in the wild-type control mice (863. 5 +/- 68 cells/mm(2)). There was no difference between the numbers of surviving RGCs in the uninjured retinas of the two strains (3708 +/- 231.3 cells/mm(2) and 3904 +/- 120 cells/mm(2), respectively). Systemic injections of brimonidine significantly reduced cell death in the Tg-SOD mice, but not in the wild type. CONCLUSIONS: Overexpression of CuZnSOD accelerates RGC death after optic nerve injury in mice. Activation of the alpha2-adrenoreceptor pathway by brimonidine enhances survival of RGCs in an in vivo transgenic model of excessive oxidative stress.

A large variety of alternatively spliced and differentially expressed mRNAs are encoded by the human acute myeloid leukemia gene AML1.

The human chromosome 21 acute myeloid leukemia gene AML1 is frequently rearranged in the leukemia-associated translocations t(8;21) and t(3;21), generating fused proteins containing the amino-terminal part of AML1. In normal blood cells, five size classes (2-8 kb) of AML1 mRNAs have been previously observed. We isolated seven cDNAs corresponding to various AML1 mRNAs. Sequencing revealed that their size differences were mainly due to alternatively spliced 5' and 3' untranslated regions, some of which were vast, exceeding 1.5 kb (5') and 4.3 kb (3'). These untranslated regions contain sequences known to control mRNA translation and stability and seem to modulate AML1 mRNA stability. Further heterogeneity was found in the coding region due to the presence of alternatively spliced stop codon-containing exons. The latter led to production of polypeptides that were smaller than the full-length AML1 protein; they lacked the trans-activation domains but maintained DNA binding and heterodimerization ability. The size of these truncated products was similar to the AML1 segment in the fused t(8;21) and t(3;21) proteins. In thymus, only one mRNA species of 6 kb was detected. Using in situ hybridization, we showed that its expression was confined to the cortical region of the organ. The 6-kb mRNA was also prominent in cultured peripheral blood T cells, and its expression was markedly reduced upon mitogenic activation by phorbol myristate acetate (TPA) plus concanavalin A (ConA). These results and the presence of multiple coding regions flanked by long complex untranslated regions, suggest that AML1 expression is regulated at different levels by several control mechanisms generating the large variety of mRNAs and protein products.

Altered brain glucose metabolism in transgenic-PFKL mice with elevated L-phosphofructokinase: in vivo NMR studies.

The gene for the liver-type subunit of phosphofructokinase (PFKL) resides on chromosome 21 and is overexpressed in Down syndrome (DS) patients. Transgenic PFKL (Tg-PFKL) mice with elevated levels of PFKL were used to determine whether, as in DS, overexpression of PFKL was also associated with altered sugar metabolism. We found that Tg-PFKL mice had an abnormal glucose metabolism with reduced clearance rate from blood and enhanced metabolic rate in brain. Transgenic-PFKL mice exhibited elevated activity of phosphofructokinase in both blood and brain, as compared to control non-transgenic (ntg) mice. Following glucose infusion, the rate of glucose clearance from the blood of Tg-PFKL mice was significantly slower than that of control ntg mice, although the basal blood glucose levels were similar. However, unlike the slower rate of glucose metabolism in blood, the initial rate of glucose utilization in the brain of the transgenic mice, was 58% faster than in control ntg mice. This was determined by infusion of [1-13C]-glucose followed by in vivo nuclear magnetic resonance (NMR) measurements of brain glucose metabolism. The faster utilization of glucose in Tg-PFKL brain is similar to the increased rate of cerebral glucose metabolism found in the brain of young adult DS patients, which may play a role in the etiology of their cognitive disabilities.

Developmental pattern of muscarinic receptors in normal and Down's syndrome fetal brain--an autoradiographic study.

The ontogeny of muscarinic cholinergic receptors in developing human brain was analyzed by in vitro receptor autoradiography with [3H]Quinuclidinyl Benzilate. It was found that muscarinic receptors develop relatively early; the levels at 24 weeks of gestation were comparable or even higher then the values in the adult brain, and that the levels of both M1 and M2 receptors increase with age. M1 receptors were concentrated mainly in forebrain regions while M2 receptors dominated in the thalamus. Scatchard analysis revealed Kd and Bmax values which are comparable to the adult values. Three brains of aborted Down's syndrome fetuses were examined in parallel and exhibited comparable levels and similar distribution to normal non-Down fetuses except for a modest increase of receptor levels which was observed in the striatum.

Fetal human brain exhibits a prenatal peak in the density of serotonin 5-HT1A receptors.

High densities of serotonergic 5-HT1A receptors, in excess of adult levels, were found in the human fetal brain between the 16th and 22nd weeks of gestation, 5-HT1A receptors were measured by quantitative autoradiography using brain sections of fetuses aborted at gestational ages 16-22 weeks. The highest receptor concentrations were detected in the cortex and hippocampus. Two brains obtained from fetuses with Down's syndrome at 22 and 24 weeks gestation exhibited abnormal receptor levels compared to age matched controls. The presence of an early, prenatal peak of 5-HT1A receptors in fetal cortex and hippocampus suggests that these receptors play a role in human brain development and may also be involved in developmental disorders such as Down's syndrome.