PCR cloning and sequencing of the coding portion of the thioredoxin-encoding gene from Streptomyces aureofaciens BMK.

The nucleotide sequence of the PCR-cloned coding portion of the thioredoxin-encoding gene from Streptomyces aureofaciens BMK was determined. The deduced 106-amino-acid sequence was compared with three other thioredoxins.

Brain vasopressin levels in Down syndrome and Alzheimer's disease.

Performing gene hunting in Down Syndrome fetal brain we detected an overexpressed sequence highly homologous to the human vasopressin gene. As this neuropeptide may be involved in the pathogenetic mechanism and, moreover, was described to play a role in memory and learning, we decided to study the brain gene product level in Down Syndrome (DS), controls and patients with Alzheimer's disease (AD). Subtractive hybridization was used to study the differential expression between steady state mRNA levels in fetal brain of DS and controls at the 23rd week of gestation. A radioimmunological method was used to determine vasopressin (AVP) in five brain regions of each 9 aged DS brains, 9 brains with AD and 9 control individuals, obtained from brain bank. An overexpressed nucleic acid sequence with 91% homology to the vasopressin gene was detected in both fetal brains with DS. AVP levels in controls were of the order cerebellum>occipital>frontal>parietal>temporal lobe and were significantly higher in temporal lobe and lower in cerebellum of patients with DS. AVP levels in brain of AD patients were also significantly increased in temporal lobe but were not reduced in cerebellum. The biological meaning of increased AVP remain unclear but may be linked to the neurodegenerative processes, proposed to be similar in both disorders. Data from gene hunting in fetal DS brain along with our data on aged DS and AD patients suggest the early involvement of AVP in the pathomechanism accompanying cholinergic, monoaminergic and neuropeptidergic deficits described in DS and AD.

Increased glyceraldehyde 3-phosphate dehydrogenase levels in the brain of patients with Down's syndrome.

Impaired glucose metabolism in Down's syndrome (DS) has been well-documented in vivo, although information on the underlying biochemical defect is limited and no biochemical studies on glucose handling enzymes have been carried out in the brain. In a previous study, we found by gene hunting in DS brain an overexpressed sequence homologous to the glyceraldehyde 3-phosphate dehydrogenase (G3PD) gene. Here we studied G3PD activity and expression levels, using two-dimensional gel analysis, in five brain regions of patients with DS and Alzheimer's disease (AD). The protein expression levels in four brain areas were approximately 1.5-fold higher in patients with DS in comparison with the controls. G3PD activity was significantly elevated in the frontal, parietal, occipital and temporal lobe of DS as well, but not in the corresponding AD brain regions. We conclude that our biochemical findings complement previously published data of impaired brain glucose metabolism in DS evaluated by positron emission tomography in clinical studies.

Expression of DNA excision-repair-cross-complementing proteins p80 and p89 in brain of patients with Down Syndrome and Alzheimer's disease.

Although deficient DNA repair was proposed for neurodegenerative disorders including Down syndrome (DS), repair proteins for nucleotide excision repair have not been studied in brain yet. As one of the hypotheses for the pathogenesis of brain damage in DS and Alzheimer's disease (AD), is oxidative stress, and cells of patients with DS were shown to be more susceptible to ionizing irradiation. We decided to study expression of excision repair-cross-complementing (ERCC) gene products, proteins 80 and 89, representatives of repair genes known to be involved in the repair of different types of DNA damage. ERCC2-protein 80 kDa and ERCC3-protein p89 were determined in five individual brain regions of controls, aged DS and AD patients. Although different in the individual regions, DNA repair proteins were consistently higher in temporal and frontal lobes of patients with DS and higher in all brain regions of patients with AD. Our results are the first to describe DNA repair gene protein patterns in human brain regions providing the basis for further studies in this area. We showed that DNA repair genes ERCC2 and ERCC3 (excision-repair-cross-complementing) for nucleotide excision repair were increased at the protein level with the possible biological meaning that this increase may be compatible with and indicate ongoing (oxidative?) DNA damage.

Decreased transcription factor junD in brains of patients with Down syndrome.

JunD is a member of the Jun family of transcription factors (TF), recently shown to negatively regulate cell growth and antagonizes transformation by the protooncogene ras: c-jun decreases while junD is accumulating when fibroblasts become quiescent. Furthermore, overexpression of junD resulted in slower growth and an increase in cells in G0/G1. Performing gene hunting on fetal Down syndrome (DS) brain we found a sequence downregulated and homologous to junD. This observation made us examine junD protein levels in adult brain specimens. Western blot experiments were carried out in five brain regions of aged patients with DS (n = 9), controls (n = 9) and patients with Alzheimer's disease (AD, n = 9). We found that junD in AD brains were comparable to controls, whereas junD levels were significantly and remarkably reduced in frontal, temporal lobe and cerebellum of patients with DS. These findings may indicate a specific finding in DS and were not linked to the AD-like-neuropathological changes of plaques and tangles, observed in DS from the fourth decade, which is also suggested by the findings of downregulated junD at the mRNA level revealed by the gene hunting technique (subtractive hybridization) in fetal DS brain. We propose that junD plays a role for the impaired development and wiring of DS brain, maybe already early in life.

The transcription of liver thioredoxin following the ionizing irradiation of radioresistant and radiosensitive mice.

The radiation protective effect of thioredoxin (TRX) in a bacterial system has been reported and based upon this observation we were interested to examine TRX transcription in the mammalian system following ionizing irradiation. In order to answer the question whether radiation sensitive mice (BALB/c) showed TRX transcription different from radiation resistant mice (C3H), we exposed these strains to X-ray doses of 2 Gy, 4 Gy and 6 Gy. Groups consisting of 6 mice were sacrificed 5, 15 and 30 minutes after irradiation and livers were immediately taken into liquid nitrogen. Total RNA was isolated from the organs by the use of a commercially available kit and used for Northern blots and slot blots with a chemiluminescence technique. Northern blots revealed a single band at 538 bp for TRX and at 1.8 kb for beta-actin. Quantification of mRNA TRX by densitometry of slot blots revealed that C3H transcribed TRX significantly higher at an earlier time point (5 min) than BALB/c. This delayed transcription of TRX in the radiosensitive mouse strain showed a comparable pattern at three different radiation doses and may well be responsible for radioresistance although no quantitative differences of TRX transcription between BALB/c and C3H mice were detectable.

Impaired brain glucose metabolism in patients with Down syndrome.

A series of impaired metabolic functions in Down Syndrome (DS) including glucose handling has been described. Recent information from positron emission tomography studies in DS patients and our finding of downregulated phosphoglucose isomerase (PGI) in fetal brain with DS by gene hunting using subtractive hybridization, made us investigate PGI, a key enzyme of glucose metabolism, in brain of patients with DS, Alzheimer's disease (AD) and controls. PGI and phosphofructokinase (PFK) activities were determined in frontal, parietal, temporal, occipital lobe and cerebellum of 9 controls, 9 patients with DS and 9 patients with AD. PGI activity in DS brain was significantly decreased in frontal, temporal lobe and cerebellum, comparable to controls in parietal lobe and elevated in occipital lobe. Brain PGI activity of patients with AD was comparable to controls in all regions tested, PFK, a rate limiting enzyme of glucose metabolism, was comparable between all brain regions of all three groups. Data of this study confirm impaired glucose metabolism in DS proposed in literature and found by positron emission tomography (PET) studies. We show that changes in glucose handling in patients with AD as evaluated by PET studies are not supported by our data, although not contradictory, as determinants other than glucose metabolizing enzymes as e.g. vascular factors and glucose transport may account for these findings. Changes of downregulated PGI found by subtractive hybridization at the transcriptional level in fetal DS brain along with our findings in DS brain regions suggest a strong specific link between glucose metabolism and DS rather than AD.

Altered gene expression in fetal Down syndrome brain as revealed by the gene hunting technique of subtractive hybridization.

Information on gene expression in brain of patients with Down Syndrome (DS, trisomy 21) is limited and molecular biological research is focussing on mapping and sequencing chromosome 21. The information on gene expression in DS available follows the current concept of a gene dosage effect due to a third copy of chromosome 21 claiming overexpression of genes encoded on this chromosome. Based upon the availability of fetal brain and recent technology of gene hunting, we decided to use subtractive hybridization to evaluate differences in gene expression between DS and control brains. Subtractive hybridization was applied on two fetal brains with DS and two age and sex matched controls, 23rd week of gestation, and mRNA steady state levels were evaluated generating a subtractive library. Subtracted sequences were identified by gene bank and assigned by alignments to individual genes. We found a series of up- and downregulated sequences consisting of chromosomal transcripts, enzymes of intermediary metabolism, hormones, transporters/channels and transcription factors (TFs). We show that trisomy 21 or aneuploidy leads to the deterioration of gene expression and the derangement of transcripts describes the impairment of transport, carriers, channels, signaling, known metabolic and hormone imbalances. The dys-coordinated expression of transcription factors including homeobox genes, POU-domain TFs, helix-loop-helix-motifs, LIM domain containing TFs, leucine zippers, forkhead genes, maybe of pathophysiological significance for abnormal brain development and wiring found in patients with DS. This is the first description of the concomitant expression of a large series of sequences indicating disruption of the concerted action of genes in this disorder.

Gene expression in fetal Down syndrome brain as revealed by subtractive hybridization.

Information on gene expression in brain of patients with Down Syndrome (DS, trisomy 21) is limited and molecular biological research is focussing on mapping and sequencing chromosome 21. The information on gene expression in DS available follows the current concept of a gene dosage effect due to a third copy of chromosome 21 claiming overexpression of genes encoded on this chromosome. Based upon the availability of fetal brain and recent technology of gene hunting, we decided to use subtractive hybridization to evaluate differences in gene expression between DS and control brains. Subtractive hybridization was applied on two fetal brains with DS and two age and sex matched controls, 23rd week of gestation, and mRNA steady state levels were evaluated generating a subtractive library. Subtracted sequences were identified by gene bank and assigned by alignments to individual genes. We found a series of up- and downregulated sequences consisting of chromosomal transcripts, enzymes of intermediary metabolism, hormones, transporters/channels and transcription factors (TFs). We show that trisomy 21 or aneuploidy leads to the deterioration of gene expression and the derangement of transcripts described describes the involvement of chromosomes other than chromosome 21, explains impairment of transport, carriers, channels, signaling, known metabolic and hormones imbalances. The dys-coordinated expression of transcription factors including homeobox genes, POU-domain TFs, helix-loop-helix-motifs, LIM domain containing TFs, leucine zippers, forkhead genes, maybe of pathophysiological significance for abnormal brain development and wiring found in patients with DS. This is the first description of the concomitant expression of a large series of sequences indicating disruption of the concerted action of genes in that disorder.

Reduced aldehyde dehydrogenase levels in the brain of patients with Down syndrome.

Aldehyde dehydrogenase (ALDH) is a key enzyme in fructose, acetaldehyde and oxalate metabolism and represents a major detoxification system for reactive carbonyls and aldehydes. In the brain, ALDH exerts a major function in the metabolism of biogenic aldehydes, norepinephrine, dopamine and diamines and gamma-aminobutyric acid. Subtractive hybridization studies in Down Syndrome (DS) fetal brain showed that mRNA for ALDH are downregulated. Here we studied the protein levels in the brain of adult patients. The proteins from five brain regions of 9 aged patients with DS and 9 controls were analyzed by two-dimensional (2-D) gel electrophoresis and identified by matrix-assisted laser desorption ionization mass spectrometry. ALDH levels were reduced in the brain regions of at least half of the patients with Down Syndrome, as compared to controls. The decreased ALDH levels in the DS brain may result in accumulation of aldehydes which can lead to the formation of plaques and tangles reflecting abnormally cross-linked, insoluble and modified proteins, found in aged DS brain. Furthermore, we constructed a 2-Dmap including approximately 120 identified human brain proteins.

Downregulation of the transcription factor scleraxis in brain of patients with Down syndrome.

Performing gene hunting in fetal Down Syndrome (DS) brain, we found a downregulated sequence with 100% homology to the basic-helix-loop-helix transcription factor (TF) scleraxis (Scl). It was the aim of the study to evaluate Scl-mRNA steady state levels in adult DS brain with Alzheimer's disease (AD) neuropathological changes, brain of patients with AD, and controls in order to find out whether Scl-downregulation is linked to DS per se or simply to neurodegeneration, common to both disorders. Determination of Scl-mRNA steady state levels was carried out by a blotting method in frontal, parietal, temporal, occipital lobe and cerebellum. We found significantly decreased Scl-transcripts in brain of DS and AD, both, when normalized versus the house-keeping gene beta actin or total RNA. We demonstrate the significant decrease of Scl-mRNA steady state levels in the pathogenesis of DS and AD suggesting a tentative role for this transcription factor in the development of the neurodegenerative processes known to occur in both disorders. More specifically, the biological meaning of the downregulation of Scl may be the involvement in the pathogenesis of impaired neuronal plasticity and wiring observed in DS and AD, phenomena regulated by the concerted action of the many transcription factors expressed in human brain.

Overexpression of DNAse I in brain of patients with Down syndrome.

Human DNAse I (EC 3.1.21.1) is an enzyme most probably involved in apoptotic processes. Splicing of the DNAse I primary transcript in normal and apoptotic cells into up to 20 splicing forms and the recent description of a different family of caspase-activated DNAses, hampered studies on the role of DNAse I in apoptosis research. Performing gene hunting in fetal brain of patients with DS we found a sequence with 100% homology to DNAse I and this formed the Rationale for studies in adult DS brain. It was therefore the aim of the study to evaluate DNAse I-mRNA steady state levels in DS brain using adult brain without brain pathologies and Alzheimer's Disease (AD) brain as control, in order to rule out that DNAse I--overexpression may not be specific for DS but rather reflecting apoptosis per se, a hallmark of both disorders. Determination of DNAse I-mRNA steady state levels was carried out by a blotting method in frontal, parietal, temporal occipital lobe and cerebellum. We found significantly increased DNAse I transcripts in brain of DS and AD both, when normalized versus the house-keeping gene beta actin or total RNA. We demonstrate the significant increase of DNAse I--transcript in the pathogenesis of DS and AD suggesting a role for this enzyme in the apoptotic process known to occur in both disorders. We are now going to carry out protein and enzyme activity levels in our laboratory to confirm our findings at the transcriptional level.

cAMP upregulates the transposable element mys-1: a possible link between signaling and mobile DNA.

Mys represents one of the many families of transposable elements abundant in the mammalian genome. Transposable elements (transposons, retrotransposons, Tr) are best described as "mobile DNA". Mechanisms for the transposition process have been well-described and recently two human Tr have been identified as the progenitors of disease producing insertions. A functional role, however, has never been proposed. Studying overexpression of genes induced by cAMP using the technique of subtractive hybridization, a clone Sch. p15 was isolated and sequenced. Computer assisted analysis of the sequence revealed strong homology to mys-1. In a parallel clone cAMP related and cAMP inducible genes were found by this technique. The fact that a mammalian Tr is modulated by the cell's signalling / second messenger system made us hypothesize that transposition may well be under physiological control and that Tr may play physiological roles as e.g. rearranging, reshuffling or programmed erasing of genes. Although methodologically sound, the interpretation of our data remains hypothetical due to the absence of any previous studies on transposition function in eukaryotes.

The transcription of the XRCC1 gene in spleen following ionizing irradiation in radiosensitive and radioresistant mice.

The XRCC1 gene was described to play a role for the sensitivity of mammalian cell lines towards ionizing irradiation. Cells with a mutation of this gene present with decreased single strand break repair, reduced recombination repair, show increased double strand breaks and sister chromatid exchange is increased up to tenfold. The goal of our study was to investigate the transcription of this gene in the spleen following ionizing irradiation in the mouse. Furthermore, we intended to examine whether radiation sensitive (RS) mice would show a transcriptional pattern different from radiation resistant (RR) mice. Radiation sensitive BALB/c/J Him mice and radiation resistant C3H He/Him mice were untreated or whole body irradiated with X-ray at 4 and 6 Gy and sacrificed 5, 15 and 30 min after irradiation. mRNA was isolated from the spleen and hybridized with probes for XRCC1 and beta-actin as a house keeping gene control. Transcription of XRCC1 was not different in unirradiated or 4 Gy-irradiated mouse RR or RS mouse strains. When irradiated at 6 Gy, RR mice showed an approximately threefold increase of mRNA XRCC1/mRNA beta actin as early as 15 min after irradiation. We conclude that radiation resistant mice show a higher transcription level for the XRCC1 gene in the spleen early after high dose X-ray whole body irradiation. This finding is the first in vivo study on XRCC1 of this kind and may in part explain the differences in the radiation sensitivity between the two strains studied.

Alpha-methyl-proline restores normal levels of bone collagen Type I synthesis in ovariectomized rats.

Collagen type I is the major protein of bone matrix and significantly reduced in osteoporosis. We tested the effect of alpha - methyl - proline on collagen synthesis in the model of the ovariectomized rat. Collagen synthesis was studied at the transcriptional level using Northern and dot blotting and at the protein level using hydroxyproline determination and a specific dye binding collagen assay. Alpha - methyl- proline treatment significantly increased collagen synthesis as compared to untreated ovariectomized and estradiol treated ovariectomized rats and restored collagen synthesis to levels of sham operated rats. Proline analogues were described to stimulate procollagen synthesis at the transcriptional level, however, if incorporated, lead to negative collagen production due to rapid intracellular degradation of the deficient collagen. Our synthesized analogue is not being incorporated, thus not interfering with collagen conformation and can therefore induce collagen production.

Deficient transcription of subunit RPA 40 of RNA polymerase I and III in heart of rats with neonatal asphyxia.

RNA polymerases transcribe nuclear genes for ribosomal RNA thus representing ribosomal biogenesis. RNA polymerase I transcribes class I genes, coding for large ribosomal RNA and is located in the nucleolus. RNA polymerase III transcribes class III genes, those that encode a number of small ribosomal RNA molecules. Both RNA polymerases form ribosomal biogenesis in a concerted action and have a common subunit, RPA40, essential for function and integrity. The aim of our study was to study the influence of hypoxia/asphyxia on transcription of this subunit as deterioration of ribosomal biogenesis may not be compatible with life. To test this hypothesis we used a nonsophisticated model of neonatal asphyxia. Rat pups were exposed to various asphyctic periods up to twenty minutes and heart tissue was taken for the evaluation of mRNA RPA40 levels, pH measurements and histological evaluation of the nucleolus by silver staining. mRNA RPA40 levels gradually decreased with the length of the asphyctic period paralleling the decrease of pH. Silver staining was remarkably decreased at the asphyctic period of 20 minutes. Our findings of decreased transcription of this essential RNA polymerase subunit indicate impairment of the ribosomal RNA synthetizing machinery and the histological findings suggest its structural relevance. This is the first in vivo observation of deteriorated RNA polymerase in asphyxia/hypoxia.

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.

Increased steady state mRNA levels of DNA-repair genes XRCC1, ERCC2 and ERCC3 in brain of patients with Down syndrome.

Although deficient DNA-repair was proposed for neurodegenerative disorders including Down Syndrome (DS), repair genes for nucleotide excision repair or X-ray repair have not been studied in brain yet. As one of the hypotheses for the pathogenesis of brain damage in DS is oxidative stress and cells of patients with DS are more susceptible to ionizing irradiation, we decided to study ERCC2, ERCC3 and XRCC1, representatives of repair genes known to be involved in the repair of oxidative DNA-damage. mRNA steady state levels of ERCC2, ERCC3, XRCC1, a transcription activator (TAF-DBP) and an elongation factor (EF1A) were determined and normalized versus the housekeeping gene beta-actin in five individual brain regions of nine controls and nine DS patients. Although different in the individual regions, DNA-repair genes were consistently higher in temporal, parietal and occipital lobes of patients with DS accompanied by comparable changes of TFA-DBP and EF1A. Our results are the first to describe DNA-repair gene patterns in human brain regions providing the basis for further studies in this area. We showed that DNA-repair genes ERCC2 and ERCC3 (excision-repair-cross-complementing-) for nucleotide excision repair and XRCC1 (X-ray-repair-cross-complementing-) for X-ray-repair, were increased at the transcriptional level with the possible biological meaning that this increase may be compatible with permanent (oxidative?) DNA damage.

Genes involved in the pathophysiology of perinatal asphyxia.

Mechanisms in the pathogenesis of perinatal asphyxia (PA) at the gene level are only beginning to be elucidated, although gene hunting using differential display has revealed differences in gene expression between hypoxic and normoxic cells in vitro. As no information on gene expression was available from in vivo studies, we decided to use a non-invasive and clinically relevant animal model of PA for mRNA hunting applying the subtractive hybridization method. mRNAs from normoxic rat brain and brain of rat pups with 20 min of asphyxia were isolated and compared by this technique. The resulting subtracted mRNAs were converted to cDNA, sequenced and identified by gene bank data. A series of transcripts representing transcription factors, transporters, metabolic factors, were found to be up- or downregulated providing insight into mechanisms of PA, and on the other hand, genes with unknown functions could be given a preliminary role i.e. in PA. Results obtained with this powerful tool are now challenging quantitative determination of these genes and gene products at the protein and activity level to confirm their role in PA.

mRNA levels of the hypoxia inducible factor (HIF-1) and DNA repair genes in perinatal asphyxia of the rat.

Hypoxia inducible factor 1 (HIF-1) is a transcription factor which is expressed, when mammalian cells are subjected to hypoxia, activating the transcription of genes encoding proteins thought important for maintaining oxygen hemostasis. The aim of the study was to evaluate HIF-1 mRNA levels in a non-invasive model of perinatal asphyxia (PA). Brain was taken for studies on HIF-1 alpha and beta 10 min following the asphyctic period. To rule out influences by the redox status we also determined antioxidant enzyme mRNA levels for superoxide dismutase, catalase, glutathion peroxidase and performed electron spin resonance studies. To study the link to protein phosphorylation as previously proposed, we evaluated mRNA levels for protein kinase C. As DNA breaks were reported to occur in PA, we determined mRNA levels of two genes representing DNA nucleotide excision repair, ERCC2 and ERCC3, and a DNA repair gene involved in the repair of oxidation mediated DNA damage, XRCC1. mRNAs for HIF-1 were not detectable following 5-20 minutes of asphyxia. The antioxidant enzymes did not show any changes during the asphyctic periods either and electron spin resonance failed to detect the presence of the hydroxyl radical. PKC significantly decreased with the length of the asphyctic period. ERCC2 and XRCC1 mRNAs were inducible during the acute phase of asphyxia indicating early repair phenomena. HIF-1 may not be relevant for periods of PA up to 20 minutes, the maximal survival time in our model. Neonatal factors may be responsible for that phenomenon although we cannot rule out that HIF-1 changes may occur at the protein level.