Sex and CYP3A5 genotype influence total CYP3A activity: high CYP3A activity and a unique distribution of CYP3A5 variant alleles in Ethiopians.

The objectives of the this study were to assess the influence of CYP3A5 genotype and sex on the variability in total CYP3A activity and to compare 4ß-hydroxycholesterol and omeprazole sulfoxidation as phenotypic markers for CYP3A activity in Ethiopians. Healthy subjects (n=150) were genotyped for CYP3A5*3, *6 and *7 using allele-specific PCR and Taqman genotyping assays. Plasma levels of 4ß-hydroxycholesterol, 3 h post-dose omeprazole and omeprazole sulfone, were determined by gas chromatography-mass spectrometry and high performance liquid chromatography, respectively. The frequency of CYP3A5*1, *3, *6 and *7 was 20.5, 67.3, 12.2 and 0%, respectively. The mean plasma 4ß-hydroxycholesterol level was 35.4 ng ml⁻¹. The mean 4ß-hydroxycholesterol level (P=0.0001) and the 4ß-hydroxycholesterol/cholesterol ratio (P=0.004) were higher in women than in men. CYP3A5 genotype significantly correlated with the plasma 4ß-hydroxycholesterol concentration (P=0.003) and 4ß-hydroxycholesterol/cholesterol ratio (P=0.0002). The omeprazole/omeprazole sulfone ratio was significantly correlated with 4ß-hydroxycholesterol and 4ß-hydroxycholesterol/cholesterol ratio in CYP3A5*0/*0 genotypes but not in individuals carrying the CYP3A5*1 allele. No correlation of omeprazole/omeprazole sulfone ratio with sex or CYP3A5 genotype was observed. A clear gene-dose effect implies plasma 4ß-hydroxycholesterol level as a useful endogenous biomarker for total CYP3A activity (CYP3A5 plus CYP3A4) whereas the omeprazole/omeprazole sulfone ratio reflects mainly CYP3A4 activity. Sex and CYP3A5 genotype influence total CYP3A activity. Ethiopians display high total CYP3A activity and a unique distribution of CYP3A5 variant alleles not described hitherto.

Potential genotoxicity of plant extracts used in Ethiopian traditional medicine.

AIM OF THE STUDY: Although traditional herbal medicines are widely used in Ethiopia, no information is available on their potential genotoxicity. In the present study, hydroalcoholic extracts of Glinus lotoides, Plumbago zeylanica, Rumex steudelii and Thymus schimperi were evaluated for their DNA damaging effects using the comet assay. MATERIAL AND METHODS: Mouse lymphoma L5178Y cells were exposed to different concentrations of the extracts for 3h with and without metabolic activation (S9-mix) using 4-nitroquinoline-N-oxide and benzo(a)pyrene as positive controls, and vehicles as negative controls. RESULTS: In the absence of S9, all extracts were found to induce significant DNA damage without affecting the cell viability. T. schimperi and R. steudelii were the most potent DNA-damaging extracts, and G. lotoides and P. zeylanica the least potent. The addition of S9 had different effects on the DNA damage induced by the extracts: it lowered the DNA damaging effect of P. zeylanica, did not affect the DNA damaging effect of T. schimperi, and increased the DNA damaging effects of R. steudelii and G. lotoides. CONCLUSION: The findings of the present study suggest that all extracts evaluated have a genotoxic potential in vitro which needs to be substantiated by further studies.

Manifold decrease of sialic acid synthase in fetal Down syndrome brain.

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.

Long-term effect of moderate and profound hypothermia on morphology, neurological, cognitive and behavioural functions in a rat model of perinatal asphyxia.

BACKGROUND: Perinatal asphyxia is a frequent cause of neurological handicap with no known therapy. However, hypothermic therapy has recently attracted attention owing to its neuroprotective property in brain of immature organisms. OBJECTIVES: Hypothermia appears to be promising in reversing the immediate effect of perinatal asphyxia, but data on long-term neuroprotection is still lacking. We therefore intended to test the long-term effect of moderate and profound hypothermia on brain morphology and functions using a well established rat model of perinatal asphyxia. METHODS: Rat pups delivered by caesarean section were placed into a water bath, still in patent membranes, at 37 degrees C and variable hypothermic conditions to induce asphyxia and thereafter given to surrogate mothers. Examinations were performed at the age of three months, consisting of a battery of motor, behavioural, cognition and reflex tests including rota-rod, Morris water maze, multiple T-maze, elevated plus maze and open field studies. Morphological alterations were evaluated by Nissl staining of brain areas known to be hypoxia sensitive. Neurotransmission system markers, including tyrosine hydroxylase, vesicular monoamine transporter, vesicular acetylcholine transporter and excitatory amino acid carrier1 were analyzed by immunohistochemistry. RESULTS: Survival increased with hypothermia. The Nissl stain revealed neuronal loss in hippocampus and hypothalamus of normothermic asphyxiated group (20/37) compared to controls (0/37), but no neuroprotective patterns emerged from hypothermia. An overall inconsistent protection of the neural systems was noted by variable periods of hypothermia. Motor function was significantly impaired in 20/37 as compared to 0/37. In the Morris water maze and multiple T-maze, results were comparable between the groups. In the elevated plus maze, time spent in the closed arm was reduced and in the open field, vertical behaviour was altered in the 20/37 group with horizontal motor behaviour being unaffected. Hypothermia reversed all abnormalities seen in 20/37, with short-term moderate and profound hypothermia being superior to long-term hypothermia. CONCLUSION: Hypothermia not only significantly increased survival, but also resulted in unimpaired motor as well as improved cognitive functions. Those findings are in contrast to altered brain morphology. As neuronal loss was present in various brain regions, we conclude that deficits may be compensated in the maturing animal. Intrahypoxic hypothermia was able to protect the rat from the devastating effect of perinatal asphyxia not in morphological, but in functional terms.

Mass spectrometrical analysis of galectin proteins in primary rat cerebellar astrocytes.

Galectins are a family of animal lectins with specificity for beta-galactosides and are involved in a host of cellular activities, ranging from development to cancer. The molecules are expressed by neural and non-neural cells intracellularly as well as extracellularly. Using two-dimensional gel electrophoresis coupled to tandem mass spectrometry, the present work aimed to identify and characterize galectins in primary rat cerebellar astrocytes. The protein-chemical method identified nine spots representing two members of the galectin family, namely galectin-1 and galectin-3. These findings suggest that high abundant expression of galectin in astrocytes is limited to the two abundant galectin family members. As these family members are linked to human astrocytic tumors, their reliable detection in astrocytes by proteomic techniques would enable us to further understand their role in neural development, injury, and regeneration in general and astrocytoma in particular.

Identification and characterisation of soluble epoxide hydrolase in mouse brain by a robust protein biochemical method.

The central nervous system is an important potential target for certain environmental prototoxins, but relatively little is known regarding brain-specific expression of biotransformation enzyme systems. On the other hand, developments in the field of molecular biology and advances in high-throughput screening methods continue to increase the number and amounts of available proteins. We used thus a robust and reliable technique, two-dimensional gel electrophoresis coupled to matrix assisted laser desorption/ionisation mass spectroscopy followed by tandem mass spectrometry and identified for the first time soluble epoxide hydrolase and added other biotransformation enzymes in the hippocampal region of mouse brain. Soluble epoxide hydrolase has an Mr of 61.5 kDa, pI of 5.9, twenty-six matching peptides and sequence coverage of 56% and was unambiguously identified by MS/MS. Since localised biotransformation events in regions of the central nervous system may account for pathologies and/or toxicities initiated by exposure to certain endogenous and/or environmental chemicals, identification of these enzymes would present an opportunity for developing novel therapeutic targets or would have critical toxicologic significance.

Aberrant protein expression in cerebral cortex of fetus with Down syndrome.

Down syndrome is the most common birth defect associated with mental retardation. Identifying proteins that are aberrantly expressed therefore helps to understand how chromosomal imbalance leads to subnormal intelligence in Down syndrome. In the present study, we generated a fetal brain map with the use of an analytical method based on two-dimensional electrophoresis coupled with mass spectrometry and searched the proteome for differential protein expression. Among 49 proteins analyzed in seven control and nine Down syndrome fetuses, we found 11 proteins that have been deregulated in cerebral cortex of fetal Down syndrome. While double-strand break repair protein rad 21 homologue, eukaryotic translation initiation factor 3 subunit 5, mixed lineage leukemia septin-like fusion protein-B and heat shock protein 75 were increased; beta-amyloid precursor-like protein 1, tropomyosin 4-anaplastic lymphoma kinase fusion oncoprotein type 2, Nck adaptor protein 2, Src homology domain growth factor receptor bound 2-like endophilin B2, beta tubulin, septin 7 and hematopoietic stem/progenitor cells 140 were decreased. The current data suggest that misexpression of proteins that have functions ranging from signaling to cellular structural organization could contribute to or reflect brain dysgenesis in Down syndrome.

Decreased brain levels of Lupus La protein and increased U5 small ribonucleoprotein-specific 40 kDa protein in fetal Down syndrome.

RNA-binding proteins have important role in the post-transcriptional regulation of gene expression. They are involved in events such as mRNA processing, transport, stability and translation. Studies in different species indicate that mutants with defect in RNA-binding proteins are defective in cell growth and differentiation. Expression of various RNA-binding proteins in prenatal life was analyzed by the highly sensitive two-dimensional electrophoresis coupled to matrix-assisted laser desorption ionization mass spectroscopy. No apparent change was obtained in levels of heterogeneous nuclear ribonucleoproteins (A3, C1-C2, L and M), nucleolin, polyadenylate binding protein-1, nuclear factor associated with double stranded RNA-2 and RNA-binding motif protein-4 between control and Down syndrome fetuses. By contrast, U5 small nuclear ribonucleoprotein-specific 40 kDa protein (p < 0.05) and Lupus La protein (p < 0.01) were significantly elevated and reduced, respectively in fetal DS. As a conclusion we can say U5 small nuclear ribonucleoprotein-specific 40 kDa protein appears to play important role in spliceosome assembly and disassembly, whereas La protein is involved in small nuclear riboncleoprotein complex biogenesis and transfer RNA maturation. Aberrant expression of these proteins points to the fact that dysregulation of the splicing and translation processes is apparent early in prenatal life, and may contribute to the defective growth and differentiation in Down syndrome.

Long-term sequelae of perinatal asphyxia in the aging rat.

Information on the consequences of perinatal asphyxia (PA) on brain morphology and function in the aging rat is missing although several groups have hypothesized that PA may be responsible for neurological and psychiatric deficits in the adult. We therefore decided to study the effects of PA on the central nervous system (CNS) in terms of morphology, immunohistochemistry, neurology and behavior in the aging animal. Hippocampus and cerebellum were evaluated morphologically by histological, immunohistochemical and magnetic resonance imaging and cerebellum also by stereological tests. Neurological function was tested by an observational test battery and rota rod test. Cognitive functions were examined by multiple-T-maze and the Morris water maze (MWM). Increased serotonin transporter (SERT) immunoreactivity in the CA2 region of the hippocampus and a significant difference in the escape latency, when the platform of the MWM was moved to a new location, were observed in asphyxiated rats. We showed that deteriorated cognitive functions accompanied by aberrant expression of hippocampal SERT and impaired relearning are long-term sequelae of perinatal asphyxia, a finding that may form the basis for understanding CNS pathology in the aging subject, animal or human.

Comparison between hypothermia and glutamate antagonism treatments on the immediate outcome of perinatal asphyxia.

This study investigated the influence of temperature or glutamate antagonism on the immediate outcome of perinatal asphyxia. Perinatal asphyxia was produced by water immersion of fetus-containing uterus horns removed by cesarean section from ready to deliver rats. The uterus horns were kept in a water bath for different time periods, before the pups were delivered and stimulated to breathe. After delivery, the pups were assessed for behavior and for systemic glutamate, aspartate, lactate and pyruvate levels measured with in vivo microdialysis, or ex vivo for energy-rich phosphates, including adenosine triphosphate (ATP), in brain, heart and kidney. In a series of experiments, asphyxia was initiated in a water bath at 37 degrees C, before the pup-containing uterus horns were moved for different time intervals to a 15 degrees C bath. In another series of experiments, the mothers were treated with N-methyl-D-aspartate (NMDA) antagonist, dizocilpine (MK-801), or alpha-amino-3-hydroxy-methylisoxazole-4-propionic acid (AMPA) antagonist,2,3-dihydroxy-6-nitro-7-sulfamoyl benzo(f) quinoxalin NBQX) 1 h before hysterectomy and asphyxia at 37 degrees C. The rate of survival rapidly decreased following exposure to more than 16 min of asphyxia, and no survival could be observed after 22 min of asphyxia. An LD50 was estimated to occur at approximately 19 min of asphyxia. The outcome was paralleled by a decrease in ATP in kidney, followed by a decrease in heart and brain. A maximal decrease in ATP was observed after 20 min of asphyxia in all tissues. Systemic microdialysis revealed that glutamate, aspartate and pyruvate levels were increased with a peak after 5 min of asphyxia. In contrast, lactate levels increased along with the length of the insult. Survival was increased when the pup-containing uterus horns were moved from a 37 degrees C to a 15 degrees C bath, at 15 min of asphyxia (the LD50 was thus increased to 30 min). If the shift occurred at 10 or 5 min of asphyxia, the LD50 increased to 80 or 110 min, respectively. The effect of glutamate antagonism was minor compared to hypothermia; the best effect (an increase in the LD50 to approximately 22 min) was observed after combining AMPA and NMDA antagonists.

Differential expression of molecular chaperones in brain of patients with Down syndrome.

Heat shock proteins (HSPs) in their molecular capacity as chaperones have been reported to regulate the apoptotic pathway and also play a critical role in protein conformational diseases such as Alzheimer's disease (AD). As all Down syndrome (DS) brains display AD-like neuropathology, neuronal loss in DS was shown to be mediated by apoptosis. We decided to investigate the expression patterns of HSPs in seven brain regions of adults with DS using two-dimensional polyacrylamide gel electrophoresis (2-DE). Following 2-DE, approximately 120 protein spots were successfully identified by matrix-assisted laser desorption/ionization--mass spectrometry (MALDI-MS) followed by quantification of the identified proteins. We unambiguously identified and quantified nine different chaperone proteins. Accordingly, all but three chaperone proteins did exhibit a significant change in expression. HSP 70 RY, heat shock cognate (HSC) 71 and glucose-regulated protein (GRP) 75 showed a significant decrease (P < 0.05) in DS temporal cortex whereas HSP 70.1 and GRP 78 were significantly increased (P<0.05) in cerebellum. Whilst T-complex 1 (TCP-1) epsilon subunit showed a significant decrease (P< 0.05) in parietal cortex, a similar extent of increase (P<0.05) as that observed in cerebellum was obtained in parietal levels of GRP 78. Alpha-crystallin B, HSP 60 and GRP 94 did not show any detectable changes in expression patterns. This report presents the first approach to quantify nine different chaperones simultaneously at the protein level in different brain regions and provides evidence for aberrant chaperone expression patterns in DS. The relevance of this aberrant expression patterns are discussed in relation to the biochemical and neuropathological abnormalities in DS brain.

Expression of apoptosis related proteins in brains of patients with Alzheimer's disease.

An increasing number of proteins are implicated in apoptosis and several of them have been shown to be altered in Alzheimer's disease (AD) brain. Because of this apoptosis is thought to be the underlying mechanism of neuronal cell loss in AD. To further substantiate this hypothesis we investigated the expression of a recently identified apoptosis related proteins and other apoptosis regulators in frontal cortex and cerebellum of AD by Western blot and enzyme-linked immunsorbent assay technique. Quantitative analysis revealed unaltered levels of Bax and RAIDD (Receptor interacting protein associated ICH-1 (caspase-2)/CED-3 (Caenorhabditis elegans death protease-3)-homologous protein with death domain) in both regions. ZIP (Zipper interacting protein) kinase, Bim/BOD (Bcl-2 interacting mediator of cell death/Bcl-2 related ovarian death gene) and p21 were significantly increased only in AD frontal cortex (P < 0.05, in all cases). Cerebellar Bcl-2 levels were significantly increased in AD (P < 0.01) while in AD frontal cortex, although the levels tended to increase did not reach significance level. The results indicate that apoptosis indeed account for the neuronal loss in AD. However, it does not seem to involve Bax and RAIDD.

Alteration of caspases and apoptosis-related proteins in brains of patients with Alzheimer's disease.

Dysregulated programmed cell death or apoptosis is suggested to be involved in the pathogenesis of Alzheimer's disease (AD). Caspases, the major effectors of apoptosis, are cysteine proteases that cleave crucial substrate proteins exclusively after aspartate residues. The activity of caspases are delicately regulated by a variety of proteins that possess distinct domains for protein-protein interaction. To further substantiate the role of apoptosis in AD, we investigated the levels of nine different proteins involved in apoptosis by Western blot technique in frontal cortex and cerebellum of control and AD subjects. The protein levels of caspase-3, -8, and -9, DFF45 (DNA fragmentation factor 45), and FLIP (Fas associated death domain (FADD)-like interleukin-1beta-converting enzyme inhibitory proteins) were decreased, whereas those of ARC (apoptosis repressor with caspase recruitment domain) and RICK (Receptor interacting protein (RIP)-like interacting CLARP kinase) increased in AD. In contrast, cytochrome c and Apaf-1 (apoptosis protease activating factor-1) were unchanged. Regression analysis revealed no correlation between levels of protein and postmortem interval. However, inconsistent correlation was found between age and levels of proteins as well as among the levels of individual proteins. The current findings showed that dysregulation of apoptotic proteins indeed exists in AD brain and support the notion that it may contribute to neuropathology of AD. The study further hints that apoptosis in AD may occur via the death receptor pathway independent of cytochrome c. Hence, therapeutic strategies that ablate caspase activation may be of some benefit for AD sufferers.

Selective upregulation of the ubiquitin-proteasome proteolytic pathway proteins, proteasome zeta chain and isopeptidase T in fetal Down syndrome.

The ubiquitin-proteasome proteolytic pathway is involved in an important non-lysosomal proteolytic pathway that is responsible for the highly selective turnover of cellular proteins both under basal metabolic conditions as well as stress. Protein degradation by this pathway is attributed to the 20S proteasome that forms the catalytic core of the complex. Recently there has been increasing interest in the proteasome because of its possible role in neuron degeneration and death. Fetal Down syndrome (DS) neurons were demonstrated to degenerate and undergo apoptosis in vitro. We therefore investigated the expression of different proteins involved in this degradative pathway, including subunits of the 20S proteasome, ubiquitinating and deubiquitinating enzymes, and regulatory subunits of the 26S proteasome in control and DS fetal brains by two-dimensional electrophoresis (2-DE). After 2-DE, approximately 389 protein spots were successfully identified by matrix-associated laser desorption ionization mass spectroscopy (MALDI-MS) and this was followed by quantification of twenty three proteins of the pathway. The results indicate that all but two proteins exhibited no apparent alterations in their pattern of expression. Proteasome zeta chain, an alpha subunit of the 20S proteasome (P < 0.05) and ubiquitin carboxy-terminal hydrolase T (Isopeptidase T), a deubiquitinating enzyme (P < 0.001) were significantly increased in fetal DS compared to controls. Whilst the expression of proteasome iota (n = 9, r = -0.9489, P = 0.0004) and proteasome epsilon (n = 9, r = -0.7227, P = 0.0311) chains was decreased with age in fetal DS brain, no significant correlation was obtained in the other proteins with age. The data suggest that such selective upregulation may have relevance to the developmental abnormalities that characterize this disorder.

Unaltered expression of Fas (CD95/APO-1), caspase-3, Bcl-2 and annexins in brains of fetal Down syndrome: evidence against increased apoptosis.

Apoptosis is the mechanism by which cells are programmed to die under a wide range of physiological and developmental stimuli. Accumulating evidence indicates that enhanced apoptosis (programmed cell death) in Down syndrome (DS) may play a role in mental retardation and precocious neurodegeneration of the Alzheimer-type. In this regard, alteration of several apoptosis related proteins have been reported in adult DS brain. Fetal DS neurons exhibited increased reactive oxygen species leading to early apoptosis, however, expression of apoptosis related proteins in fetal DS, has never been considered. To address this issue, we investigated the expression of proteins involved in apoptosis including Fas (CD95, APO-1), caspase-3, Bcl-2 and annexins in the cerebral cortex of control and DS fetal brain by western blot and two dimensional electrophoresis. Here, we report that no detectable changes were obtained in fetal DS brain in the expression of Fas, caspase-3, Bcl-2 and Annexins (I, II, V, and VI) compared to controls. In parallel experiment, we also examined the expression of neuron specific enolase (NSE), a neuronal marker found to be decreased in adult DS brain, to see if there is any neuronal loss and no difference was observed between the two groups. Protein expression did not correlate with age. The unchanged levels of Fas, Bcl-2 and annexins together with unaltered caspase-3 expression, a predominant caspase that executes apoptosis in the developing nervous system, suggest that enhanced apoptosis may not be apparent in fetal DS brain as demonstrated for adult DS brain.

Alteration of caspases and other apoptosis regulatory proteins in Down syndrome.

Apoptosis has been implicated in the selective neuronal loss of Down syndrome (DS). Apoptosis activates a family of cysteine proteases with specificity for aspartic acid residues referred to as caspases that play a key role in dismantling a cell committed to die. Caspase activity is regulated by a variety of proteins that possess a domain resembling the prodomains of caspases. Little is known, however, about the changes of caspases and their regulatory proteins in DS. Here, we investigated levels of nine such different proteins by western blot technique in frontal cortex and cerebellum of control and DS subjects. The protein levels of DFF45 (DNA fragmentation factor 45), and FLIP (FADD like interleukin-1beta-converting enzyme inhibitory proteins) were significantly decreased whereas that of RICK (RIP-like interacting CLARP kinase) increased in both regions of DS. In contrast, cytochrome c, Apaf-1 (apoptosis protease activating factor-1), procaspase-9 and ARC (apoptosis repressor with caspase recruitment domain) were unchanged. Procaspase-3 and -8 were significantly decreased in frontal cortex but no significant change was observed in cerebellum. Regression analysis revealed no correlation between postmortem interval and levels of the investigated proteins. However, inconsistent correlation was found between age and levels of proteins as well as amongst the density of individual proteins. These findings demonstrate that dysregulation of apoptotic proteins does exist in DS brain and may underlie the neuropathology of DS. The study further suggests that apoptosis in DS may occur via the death receptor pathway independent of cytochrome c. Hence, therapeutic strategies that target caspase activation may prove useful in combating neuronal loss in this disorder.

Expression of apoptosis related proteins: RAIDD, ZIP kinase, Bim/BOD, p21, Bax, Bcl-2 and NF-kappaB in brains of patients with Down syndrome.

Down syndrome (DS) is a genetic disease that exhibits significant neuropathological parallels with Alzheimer's disease (AD). One of the features of DS, neuronal loss, has been hypothesized to occur as a result of apoptosis. An increasing number of proteins are implicated in apoptosis and several of them were shown to be altered in AD, however, the knowledge in DS is far from complete. To further substantiate the hypothesis that apoptosis is the underlying mechanism for neuronal loss and contribute towards the current knowledge of apoptosis in DS, we analyzed the expression of apoptosis related proteins in frontal cortex and cerebellum of DS by western blot and ELISA techniques. Quantitative analysis revealed a significant increase in DS frontal (P < 0.0001) and cerebellar (P < 0.05) Bim/BOD (Bcl-2 interacting mediator of cell death/Bcl-2 related ovarian death gene), cerebellar Bcl-2 (P < 0.01) as well as p21 (P < 0.05) levels compared to controls. No significant change was detected in Bax, RAIDD (receptor interacting protein (RIP)-associated ICH-1/CED-3-homologus protein with death domain), ZIP (Zipper interacting protein) kinase and NF-kappaB p65 levels in both regions, although frontal cortex levels of RAIDD, Bcl-2 and p21 levels tended to increase. In addition, a 45 kDa truncated form of NF-kappaB p65 displayed a significant elevation (P < 0.05) in DS cerebellum. No significant correlation had been obtained between postmortem interval and level of the proteins analyzed. With regard to age, it was only NF-kappaB p65 that showed significant correlation (r = -0.8964, P = 0.0155, n = 9) in frontal cortex of controls. These findings provide further evidence that apoptosis indeed accounts for the neuronal loss in DS but Bax and RAIDD do not appear to take part in this process.

Changes in nicotinic acetylcholine receptor subunits expression in brain of patients with Down syndrome and Alzheimer's disease.

Cholinergic deficit associated with loss of nicotinic acetylcholine receptors (nAChRs) has been described in Alzheimer's disease (AD) by receptor binding assays, positron emission tomography and immunoblotting. However, little is known about the alteration of these receptors in a related disease, Down syndrome (DS) which might be of importance for therapeutic strategies. The protein levels of neuronal nAChR alpha and beta subunits in human postmortem brain samples (frontal cortex and cerebellum) of control, adult DS, and AD were investigated by making use of western blot analysis. Two major bands at 26 and 45 kDa for alpha3, one at 50 kDa for alpha4 and beta2, and one at 45 kDa for alpha7 were detected by the respective antibodies. Specific alteration in individual subunits was also apparent in DS and AD. In frontal cortex, the 45kDa alpha3 subunit was significantly increased in DS (121%) (P < 0.05) and AD (93%) (P < 0.05), whereas the 26kDa, an isoform/truncated form of alpha3, displayed a reversed pattern. It was significantly decreased in DS (75%) (P < 0.001) and AD (52.6%) (P < 0.05). Alpha4 was comparable in all groups by contrast, alpha7 was significantly decreased in AD (64%) (P < 0.05). In DS, however, although the levels tended to be lower (17.3%) the reduction was not significant. Beta2 was unchanged in AD but showed a significant increase in DS frontal cortex (98.1%) (P < 0.01). In cerebellum, no significant alteration was observed in any of the subunits except beta2. It exhibited a significant increase (161%) (P < 0.01) in DS. Derangement in expression of nAChRs is apparent in DS, as in AD that may have some relevance to DS neuropathology. Furthermore, the increase in beta2 expression indicate that these subunits may have more than a structural role. Hence, therapeutic strategies tailored towards these end might be of some benefit for cognitive enhancement in these disorders.

Beta-amyloid precursor protein, ETS-2 and collagen alpha 1 (VI) chain precursor, encoded on chromosome 21, are not overexpressed in fetal Down syndrome: further evidence against gene dosage effect.

Down syndrome (DS) is the most common human chromosomal abnormality caused by an extra copy of chromosome 21 and characterized clinically by somatic anomalies, mental retardation and precocious dementia. The phenotype of DS is thought to result from overexpression of a gene or genes located on the triplicated chromosome or chromosome region. Reports that challenge this notion, however, have been published. To add to this body of evidence, the expression of beta-amyloid precursor protein (APP), ETS-2 and collagen alpha1 (VI) chain precursor, encoded on chromosome 21, was investigated in fetal brain by western blot and two-dimensional electrophoresis (2-DE). Western blot detected APP and ETS-2 that migrated at approximately 75 and 50kDa, respectively. Subsequent densitometric analysis of APP and ETS-2 immunoreactivity did not produce any significant change between controls and DS. Since the metabolic fate of APP determines the propensity of amyloid beta production, the expression of the secreted forms of APP (sAPP) had been examined. Neither the expression of sAPPalpha nor sAPPbeta showed any detectable changes among the two groups. Collagen alpha1 (VI) chain precursor, a protein resolved as a single spot on 2D gel was identified by matrix associated laser desorption ionization mass spectroscopy. Quantitative analysis of this spot using the 2D Image Master software revealed a significant decrease in fetal DS (P < 0.01) compared to controls. Linear regression analysis did not show any correlation between protein levels and age. The current data suggest that overexpression per se can not fully explain the DS phenotype.

Expression of the multidrug resistance P glycoprotein (Pgp) and multidrug resistance associated protein (MRP1) in Down syndrome brains.

Transport by ATP-dependent efflux pumps such as P glycoprotein (Pgp) and multidrug resistance associated protein (MRP), encoded by multidrug resistant (MDR) associated genes, is an increasingly recognized mechanism by which cells maintain substrate homeostasis and evade drug therapy. Pgp and MRP are members of the so-called ATP binding cassette (ABC) transporters superfamily, which are associated with many biological processes in both prokaryotes and eukaryotes, as well as clinical problems. The observation of upregulated sequences that are homologous to the Mycobacterium smegmatis phage resistance (mpr) gene and putative ABC transporters subunits in fetal Down syndrome (DS) using the gene hunting technique, subtractive hybridization formed the Rationale for this study. The expression of Pgp and MRP1 is therefore investigated in different brain regions of controls and adult DS patients with western blot technique. No apparent changes were observed between controls and DS in levels of Pgp in all brain regions examined. By contrast, MRP1 detection using the rat monoclonal antibody (MRPr1) produced a significant elevation in DS temporal cortex (P < 0.01) and parietal cortex (P < 0.05). Although MRP1 detected with the mouse monoclonal antibody (MRPm6) tended to increase in most of the regions of DS brain, it failed to reach significance level. Age or postmortem interval did not correlate with protein levels in both controls as well as DS. Taken together, the current data provide evidence for the presence of MDR related pumps in different regions of the human brain. In addition, overexpression of MRP1 in DS brain may have some relevance to the disorder either by deranging substrate homeostasis or limiting drug access.