Expression of cystathionine beta-synthase, pyridoxal kinase, and ES1 protein homolog (mitochondrial precursor) in fetal Down syndrome brain

Down syndrome (DS) is the most common human chromosomal abnormality caused by an extra copy of chromosome 21 and characterized by somatic anomalies and mental retardation. The phenotype of DS is thought to result from overexpression of genes encoded on chromosome 21. Although several studies reported mRNA levels of genes localized on chromosome 21, mRNA data cannot be simply extrapolated to protein levels. Furthermore, most protein data have been generated using immunochemical methods. In this study we investigated expression of three proteins (cystathionine beta-synthase (CBS), pyridoxal kinase (PDXK), ES1 protein homolog, mitochondrial precursor (ES1)) whose genes are encoded on chromosome 21 in fetal DS (n = 8; mean gestational age of 19.8 +/- 2.0 weeks) and controls (n = 7; mean gestational age of 18.8 +/- 2.2 weeks) brains (cortex) using proteomic technologies. Two-dimensional electrophoresis (2-DE) with subsequent in-gel digestion of spots and matrix-assisted laser desorption ionization (MALDI) spectroscopic identification followed by quantification of spots with specific software was applied. Subsequent quantitative analysis of CBS and PDXK revealed levels comparable between DS and controls. By contrast, ES1 was two-fold elevated (P < 0.01) in fetal DS brain. This protein shows significant homology with the E. coli SCRP-27A/ELBB and zebrafish ES1 protein and contains a potential targeting sequence to mitochondria in its N-terminal region. Based on the assumption that structural similarities reflect functional relationship, it may be speculated that ES1 is serving a basic function in mitochondria. Although no function of the human ES1 protein is known yet, ES1 may be a candidate protein involved in the pathogenesis of the brain deficit in DS.     

Molecular cloning of mouse p47, a second group mammalian RuvB DNA helicase-like protein: homology with those from human and Saccharomyces cerevisiae

A 47k protein (p47) in a high-salt buffer extract of a rat liver nuclear matrix fraction was purified by means of a wheat germ agglutinin affinity column, reversed phase HPLC, and SDS-PAGE, and partial amino acid sequences were analyzed. Based on these sequences, the mouse cDNA of the protein was cloned and sequenced, and its amino acid sequence was deduced. Mouse p47 consists of 463 amino acid residues with a molecular weight of 51,112. The amino acid sequences of human and Saccharomyces cerevisiae p47s were also deduced from the nucleotide sequences of "expressed sequence tag" fragments and genomic DNA, respectively. These sequences contain helicase motifs and show homology to bacterial RuvB DNA helicases acting in homologous recombination. They also show homology with the putative mammalian helicases p50/TIP49 and RUVBL1. Comparison of the amino acid sequences of p47 group proteins and those of p50/TIP49 group proteins revealed the p47 group proteins to comprise a group distinct from the p50/TIP49 proteins. Ultracentrifugation and gel filtration analyses showed that p47 in the rat liver cytosol fraction exists as large complexes of 697k.     

Varying brain insulin concentrations differentially regulate the fetal brain insulin receptor

We investigated the downregulating effect of varying states (physiologic and pharmacologic) of systemic and intracranial hyperinsulinism on the 28 to 30 day fetal rabbit brain insulin receptor. Alloxan-induced maternal diabetes (n = 5) produced mild fetal hyperinsulinemia (D) (plasma insulin concentrations = 59.80 +/- 8.10 microU/ml, control = 26.25 +/- 3.70; p less than 0.01), whereas systemic administration (IMI) of 1.0 U (n = 4) and 2.0 U (n = 4) of insulin to the fetus resulted in moderate (103.13 +/- 34.63 microU/ml) and severe (288.3 +/- 51 microU/ml) fetal hyperinsulinemia respectively. All three states of systemic hyperinsulinemia neither altered the fetal brain insulin content nor the brain insulin receptor number and affinity. 0.01 U (n = 4) of intracranial insulin administration (ICI) increased the brain insulin content four-fold (p less than 0.01) but did not alter the brain insulin receptor number or affinity. 0.1 (n = 5) and 2.0 U (n = 7) of intracranial insulin increased the brain insulin content to supraphysiologic concentrations (p less than 0.01) and decreased the fetal brain insulin receptor number (p less than 0.01), the affinity remaining constant. We conclude that 1) regardless of the ability of insulin to cross the blood brain barrier, the downregulation of the brain insulin receptor is insulin dose-dependent and 2) the downregulation of the fetal brain insulin receptor is not a physiologic but a pharmacologic effect of insulin.     

Nutrition and fetal brain maturation : II. Impact of maternal starvation on changing levels of acetylcholinesterase and enolase in vitro

The impact of maternal starvation during Days 17-20 of gestation was examined in 20-day fetal rat brain tissue cultured for 6 days in MEM and 10% adult rat serum. Acetylcholinesterase (AChE) activities were consistently greater in fetal brain cell cultures from starved mothers. When fetal tissues from starved mothers were continuously exposed to 72-h fasted serum, AChE activities increased from 1.03 +/- 0.14 to 1.59 +/- 0.21 mumol/h/mg protein (P less than 0.001). In fetal tissues from fed mothers, lower AChE activities were increased from 0.78 +/- 0.09 to 1.04 +/- 0.07 mumol/h/mg protein (P less than 0.05) when 72-h fasted serum was used to replace the fed serum during incubation. When fetal brain cell cultures from fed mothers were exposed for 6 days to graded concentrations of fed serum (2.5-15%), the activities of AChE fell reciprocally from 1.34 +/- 0.10 to 0.82 +/- 0.12 mumol/h/mg protein (P less than 0.05). The levels of AChE activity in tissues exposed to fasted serum were consistently greater, but fell similarly from 1.62 +/- 0.10 to 0.97 +/- 14 mumol/h/mg protein (P less than 0.01), when serum concentrations were increased from 2.5 to 15%. AChE activities were 30% higher in tissues incubated with cycloheximide 10(-3) M (P less than 0.02). Unlike AChE, fetal brain enolase activities were unaffected by maternal starvation. In fetal brain cell cultures from fed mothers, enolase fell from 1.85 +/- 0.10 to 1.37 +/- 0.12 mumol/min/mg protein following exposure to fasted instead of fed serum (P less than 0.02). In fetal cultures from starved mothers, enolase activities were depressed similarly from 1.76 +/- 0.08 to 1.41 +/- 0.09 mumol/min/mg protein when fasted replaced fed serum (P less than 0.02). Thus, the fetal brain cell cultures appear to maintain enzymatic realignments imposed by maternal starvation for at least 6 days. In addition, serum from fasted animals has significant growth inhibiting properties manifested by heightened activities of AChE and lower activities of enolase.     

AMP Sensing by DEAD-Box RNA Helicases

In eukaryotes, cellular levels of adenosine monophosphate (AMP) signal the metabolic state of the cell. AMP concentrations increase significantly upon metabolic stress, such as glucose deprivation in yeast. Here, we show that several DEAD-box RNA helicases are sensitive to AMP, which is not produced during ATP hydrolysis by these enzymes. We find that AMP potently inhibits RNA binding and unwinding by the yeast DEAD-box helicases Ded1p, Mss116p, and eIF4A. However, the yeast DEAD-box helicases Sub2p and Dbp5p are not inhibited by AMP. Our observations identify a subset of DEAD-box helicases as enzymes with the capacity to directly link changes in AMP concentrations to RNA metabolism.     

Rearrangement of structured RNA via branch migration structures catalysed by the highly related DEAD-box proteins p68 and p72

RNA helicases, like their DNA-specific counterparts, can function as processive enzymes, unwinding RNA with a defined step size in a unidirectional fashion. Recombinant nuclear DEAD-box protein p68 and its close relative p72 are reported here to function in a similar fashion, though the processivity of both RNA helicases appears to be limited to only a few consecutive catalytic steps. The two proteins resemble each other also with regard to other biochemical properties. We have found that both proteins exhibit an RNA annealing in addition to their helicase activity. By using both these activities the enzymes are able in vitro to catalyse rearrangements of RNA secondary structures that otherwise are too stable to be resolved by their low processive helicase activities. RNA rearrangement proceeds via protein induced formation and subsequent resolution of RNA branch migration structures, whereby the latter step is dependent on ATP hydrolysis. The analysed DEAD-box proteins are reminiscent of certain DNA helicases, for example those found in bacteriophages T4 and T7, that catalyse homologous DNA strand exchange in cooperation with the annealing activity of specific single strand binding proteins.     

Brain TRH and Cyclo (His-Pro) and brain protein in the newborn rat are altered by maternal liquid protein feeding

Liquid protein diet (LPD) has been shown previously to produce maternal and fetal weight loss and fetal congenital anomalies, including cataracts and craniofacial malformations. Therefore, to examine the effects of LPD in pregnancy upon the central nervous system of pups, pregnant dams were fed either a 20% casein diet ad libitum, a 20% LPD, or pair-fed with a 20% casein diet. LPD was associated with significant maternal weight loss, and pups had significantly lower birth weights (5.14 +/- 0.64) than pups from the pair-fed controls (5.70 +/- 0.46, p less than 0.05). Total brain protein content was reduced significantly in pups of both sexes from pregnant fed LPD. Moreover, the concentrations of two brain peptides neurotransmitters, thyrotropin-releasing hormone (TRH), and its biologically active metabolite, histidyl-proline diketopiperazine Cyclo (His-Pro), were elevated in the pups from LPD-fed mothers. In contrast, there was no significant difference in brain protein or brain peptides in pups from pair-fed mothers vs. pups from mothers fed ad libitum. These data suggest that qualitative alterations of the protein component in maternal dietary composition have deleterious effects upon the ontogeny of the rat fetal CNS, as reflected by reduced total protein and elevated concentrations of TRH and Cyclo (His-Pro).     

Thyroid stimulating hormone-receptor overexpression in brain of patients with Down syndrome and Alzheimer's disease

Thyroid hormone abnormalities are strongly associated with Down Syndrome (DS) with elevated thyroid stimulating hormone (TSH) levels as the most consistent finding. Using subtractive hybridization for gene hunting we found significant overexpression of mRNA levels for the TSH-receptor (TSH-R) in brain of a fetus with DS. Based upon this observation we determined TSH-R protein levels in five brain regions of patients with DS (n=8), Alzheimer disease (AD, n=8) and controls (C, n=8). Western blots revealed significantly elevated immunoreactive TSH-R protein(s) 40 kD and 61 kD in temporal and frontal cortex of patients with DS and, unexpectedly, in AD. Levels for the 40 kD protein in temporal cortex were 1.00+/-0.036 (arbitrary units+/-SD) in C, 1.35+/-0.143 in DS, 1.52+/-0.128 in AD; in frontal cortex: 1.00+/-0.046 in C, 1.10+/-0.03 in DS, 1.10+/-0.038 in AD. Levels for the 61 kD protein in temporal cortex were 1.01+/-0.015 in C, 1.47+/-0.013 in DS, 1.623+/-0.026 in AD; in frontal cortex: 1.02+/-0.020 in C, 1.18 +/-0.123 in DS, 1.48+/-0.020 in AD. These results show that elevated brain immunoreactive TSH-R is not specific for DS and maybe reflecting apoptosis, a hallmark of both neurodegenerative disorders, as it is well-documented that the thyroid hormone system is involved in the control of programmed cell death.     

Regulation of alternative splicing by the ATP-dependent DEAD-box RNA helicase p72

Although a number of ATP-dependent RNA helicases are important for constitutive RNA splicing, no helicases have been implicated in alternative RNA splicing. Here, we show that the abundant DEAD-box RNA helicase p72, but not its close relative p68, affects the splicing of alternative exons containing AC-rich exon enhancer elements. The effect of p72 was tested by using mini-genes that undergo different types of alternative splicing. When the concentration of p72 was increased in transient transfections, the inclusion of enhancer-containing CD44 alternative exons v4 and v5 increased using a mini-gene that contained these exons and their flanking introns inserted into a beta-globin gene. Other types of alternative splicing were not impacted by altering p72 concentrations. Mutation of the p72 helicase ATP-binding site or deletion of the carboxy-terminal region of the protein reduced the ability of the transfected protein to affect CD44 variable exon splicing. Use of in vitro extracts overexpressing p72 indicated that p72 becomes associated with complexes containing precursor RNA. Helicases have been implicated both in altering RNA-RNA interactions and in remodeling RNA-protein complexes. CD44 exon v4 contains a potential internal secondary structure element that base pairs the 5' splice site with a region inside the exon located between enhancer elements. Mutations that destroyed this complementarity modestly increased inclusion in the absence of p72 but still responded to increasing p72 concentration like the wild-type exon, suggesting that p72 might have effects on protein-RNA interactions. In agreement with this hypothesis, p72 was not able to restore the inclusion of an exon mutated for its major enhancer element. Our results suggest that RNA helicases may be important alternative splicing regulatory factors.     

Periodontal conditions in individuals with Down's syndrome

Periodontal disease in Down's syndrome (DS) population seems to be a more common and serious problem than caries. The aim of this study was to assess the state of periodontal structures in patients with DS. The Community Periodontal Index of Treatment Needs was used for periodontal status assessment in 71 DS subjects aged 9-34 years. A control group consisted of 71 age- and sex-matched healthy individuals. Both groups were divided into three age groups: 9-15 (n = 24); 16-25 (n = 32); and 26-34 (n = 15) years. The results showed a similar percentage of subjects with bleeding and calculus. The intact periodontium was significantly higher in control than in DS (16.9% vs. none; p < 0.01). Deep pockets were more frequent in DS group than in the control group (14.1% vs. 1.4%; p < 0.01). The mean number of sextants with healthy tissue was lower, and of those with bleeding, calculus and shallow pockets significantly higher in DS patients than in controls (p < 0.01), so all DS subjects required some periodontal treatment (p < 0.01). It can be concluded that the severity of periodontal disease and the treatment needs seem to be significantly greater in DS than in healthy subjects.     

Influence of age on activities of antioxidant enzymes and lipid peroxidation products in erythrocytes and neutrophils of Down syndrome patients

Thirty-seven individuals with Down syndrome (DS) were divided into four age categories: (i) 1 to < 6 years, (ii) 6 to < 13 years, (iii) 13 to < 20 years, and (iv) over 20 years. Activities of antioxidant enzymes found in individual age categories were different, but the differences between age groups were not statistically significant. We confirmed significantly higher activities of Cu/Zn superoxide dismutase (SOD) and glutathione peroxidase (GPx) in blood cells of people with DS as compared to 35 controls, which consisted, for the first time, of siblings of children with DS. No significant differences were found in activities of catalase and glutathione reductase in DS vs. controls. A significant difference was observed in serum concentration of malondialdehyde (MDA) in DS vs. controls (8.39 +/- 0.34 micromol/l vs. 7.34 +/- 0.27 micromol/l; p = .021) and concentration of MDA in erythrocytes of individuals with DS between the third and fourth age group (p = .05). In DS persons, an elevated ratio of SOD to catalase plus GPx with respect to the controls in all age categories was found, suggesting oxidative imbalance, potentially contributing to accelerated aging observed in these persons.     

Thymic regulation of primate fetal ovarian-adrenal differentiation

We report that fetal thymectomy inhibits oogenesis and induces abnormal ovarian differentiation in rhesus monkeys. In utero thymectomy (n = 5) elevated plasma follicle-stimulating hormone (7.8 +/- 1.1 microgram/ml vs. 4.2 +/- 0.5 microgram/ml; P less than 0.05) and decreased plasma prolactin (24.5 +/- 3.3 ng/ml vs. 76.3 +/- 11.2 ng/ml; P less than 0.05) concentrations compared with intact controls (n = 12), but did not change plasma luteinizing hormone, estradiol, cortisol, dehydroepiandrosterone sulfate, or thymosin-alpha 1 concentrations. In utero thymectomy reduced the weight of neonatal ovaries and adrenal glands, but not hepatic, renal, splenic, or total body weights. After fetal thymectomy, newborn ovaries (n = 8) contained a reduced total number of germ cells (123,926 +/- 11,651 vs. 432,034 +/- 40,311; P less than 0.001). The percentages of individual germ cell types were similar between thymectomized and intact groups (n = 11) except for an increased percentage of preantral-antral follicles in the thymectomy group (P less than 0.01). Our results indicate that the primate fetal thymus regulates antenatal ovarian follicular development, perhaps by interactions between the nascent immunologic and pituitary-ovarian systems.     

Belle is a Drosophila DEAD-box protein required for viability and in the germ line

DEAD-box proteins are ATP-dependent RNA helicases that function in various stages of RNA processing and in RNP remodeling. Here, we report identification and characterization of the Drosophila protein Belle (Bel), which belongs to a highly conserved subfamily of DEAD-box proteins including yeast Ded1p, Xenopus An3, mouse PL10, human DDX3/DBX, and human DBY. Mutations in DBY are a frequent cause of male infertility in humans. Bel can substitute in vivo for Ded1p, an essential yeast translation factor, suggesting a requirement for Bel in translation initiation. Consistent with an essential cellular function, strong loss of function mutations in bel are recessive lethal with a larval growth defect phenotype. Hypomorphic bel mutants are male-sterile. Bel is also closely related to the Drosophila DEAD-box protein Vasa (Vas), a germ line-specific translational regulator. We find that Bel and Vas colocalize in nuage and at the oocyte posterior during oogenesis, and that bel function is required for female fertility. However, unlike Vas, Bel is not specifically enriched in embryonic pole cells. We conclude that the DEAD-box protein Bel has evolutionarily conserved roles in fertility and development.     

Nutrition and fetal brain maturation : I. Responses in vitro and in vivo

The glycolytic enzyme enolase increases during the perinatal period of brain development and was utilized as a marker for examining the effect of culture environment on differentiation of cells from 20-day fetal rat brain. Enolase activity in cell cultures increased from 0.91 +/- 0.03 (Day 0) to 2.11 +/- 0.10 mumol/min/mg protein (Day 6). Comparable levels were not reached in vivo until neonatal pups were 15 days old. The in vitro increase was inhibited by both cycloheximide and actinomycin D. Enolase activity in the cells responded to alterations in both incubation media and homologous serum. After 6 days in culture, cells incubated in rat serum (10%) added to MEM or RPMI produced twice as much enolase activity as cells incubated similarly in Ham's medium, i.e., 1.96 +/- 0.09 and 1.85 +/- 0.21 vs 1.02 +/- 0.09, P less than 0.001. Results of a comparable magnitude were obtained when fetal calf serum replaced adult rat serum, but enolase production was somewhat lower when newborn calf serum replaced adult rat or fetal calf serum. When cells were incubated for 6 days with graded concentrations of adult rat serum (2.5-15%), enolase activity increased progressively. The pattern of enolase response suggests that the fetal rat brain cell model described herein will provide a sensitive probe with which to gain insight into nutrition and fetal brain development.     

Protein expression of BACE1, BACE2 and APP in Down syndrome brains

Down syndrome (DS) is the most common human chromosomal abnormality caused by an extra copy of chromosome 21. The phenotype of DS is thought to result from overexpression of a gene or genes located on the triplicated chromosome or chromosome region. Several reports have shown that the neuropathology of DS comprises developmental abnormalities and Alzheimer-like lesions such as senile plaques. A key component of senile plaques is amyloid beta-peptide which is generated from the amyloid precursor protein (APP) by sequential action of beta-secretases (BACE1 and BACE2) and gamma-secretase. While BACE1 maps to chromosome 11, APP and BACE2 are located on chromosome 21. To challenge the gene dosage effect and gain insight into the expressional relation between beta-secretases and APP in DS brain, we evaluated protein expression levels of BACE1, BACE2 and APP in fetal and adult DS brain compared to controls. In fetal brain, protein expression levels of BACE2 and APP were comparable between DS and controls. BACE1 was increased, but did not reach statistical significance. In adult brain, BACE1 and BACE2 were comparable between DS and controls, but APP was significantly increased. We conclude that APP overexpression seems to be absent during the development of DS brain up to 18-19 weeks of gestational age. However, its overexpression in adult DS brain could lead to disturbance of normal function of APP contributing to neurodegeneration. Comparable expression of BACE1 and BACE2 speaks against the hypothesis that increased beta-secretase results in (or even underlies) increased production of amyloidogenic A beta fragments. Furthermore, current data indicate that the DS phenotype cannot be fully explained by simple gene dosage effect.     

Bent out of shape: RNA unwinding by the DEAD-box helicase Vasa

RNA helicases of the DEAD-box family are involved in essentially all RNA-dependent cellular processes. In this issue of Cell, Sengoku et al. (2006) solve the structure of the DEAD-box protein Vasa in the presence of RNA and a nonhydrolyzable ATP analog and provide important insights into how this family of helicases unwinds RNA.     

Manifold decrease of sialic acid synthase in fetal Down syndrome brain

Summary. Background: Down syndrome (DS, trisomy 21) is the most common genetic cause of mental retardation. A large series of biochemical defects have been observed in fetal and adult DS brain that help in unraveling the molecular mechanisms underlying mental retardation. Aims: As sialylation of glycoconjugates plays an important role in brain development, this study aimed to look at the sialic acid metabolism by measuring sialic acid synthase (SAS; N-acetylneuraminate synthase) in early second trimester fetal control and DS brain. Results: In this regard, protein profiling was performed by two-dimensional gel electrophoresis coupled to matrix-assisted laser desorption/ionization mass-spectrometry followed by database search and subsequent quantification of spot using specific software. SAS, the enzyme catalyzing synthesis of N-acetyl-neuraminic acid (syn: sialic acid) was represented as a single spot and found to be significantly and manifold reduced (P < 0.01) in cortex of fetuses with DS (control vs. DS, 0.052 ± 0.025 vs. 0.012 ± 0.006). Conclusion: The intriguing finding of the manifold decrease of SAS in DS fetal cerebral cortex as early as in the second trimester of pregnancy may help to explain the brain deficit observed in DS. Decreased SAS may well lead to altered sialic acid metabolism, required for brain development and, more specifically, for sialylation of key brain proteins, including neuronal cell adhesion molecule and myelin associated glycoprotein.