Loss of correlations among proteins in brains of the Ts65Dn mouse model of down syndrome

The Ts65Dn mouse model of Down syndrome (DS) is trisomic for orthologs of 88 of 161 classical protein coding genes present on human chromosome 21 (HSA21). Ts65Dn mice display learning and memory impairments and neuroanatomical, electrophysiological, and cellular abnormalities that are relevant to phenotypic features seen in DS; however, little is known about the molecular perturbations underlying the abnormalities. Here we have used reverse phase protein arrays to profile 64 proteins in the cortex, hippocampus, and cerebellum of Ts65Dn mice and littermate controls. Proteins were chosen to sample a variety of pathways and processes and include orthologs of HSA21 proteins and phosphorylation-dependent and -independent forms of non-HSA21 proteins. Protein profiles overall show remarkable stability to the effects of trisomy, with fewer than 30% of proteins altered in any brain region. However, phospho-proteins are less resistant to trisomy than their phospho-independent forms, and Ts65Dn display abnormalities in some key proteins. Importantly, we demonstrate that Ts65Dn mice have lost correlations seen in control mice among levels of functionally related proteins, including components of the MAP kinase pathway and subunits of the NMDA receptor. Loss of normal patterns of correlations may compromise molecular responses to stimulation and underlie deficits in learning and memory.     

Adoptive transfer of the hematopoietic system of trisomic mice with limited life span: Stem cells from six different trisomies are capable of survival

The life span of murine trisomies is limited to the fetal or early postnatal period. However, rescue of the hematopoietic system of fetal mice with trisomies (Ts) 12, 13, 14, 16, 18, and 19 is possible by transplanting hematopoietic stem cells from the liver into lethally irradiated adult hosts. Thus, radiation chimeras with permanent and almost complete trisomic hematopoietic and lymphocytopoietic systems were constructed. The longest documented survival of a trisomic graft was 12 months in Ts 19 chimeras. Blood counts in trisomic chimeras reveal a marked anemia in Ts 16 chimeras; lymphocytopenia in Ts 12, Ts 16, and Ts 19 chimeras; and granulocytopenia in Ts 18 chimeras. Survival rates of Ts 12, Ts 18, and Ts 19 chimeras were not different from those of the respective controls, whereas survival rates of chimeras with Ts 13 and Ts 16 hematopoiesis were markedly reduced and that of Ts 14 chimeras only slightly reduced. These results indicate that transplanted hematopoietic stem cells from Ts 13, Ts 14, and Ts 16 fetuses exhibit relevant genetically determined defects, resulting in a reduced restoration capacity of hematopoietic organs and/or deficiencies of differentiated blood cells. © 1992 Wiley-Liss, Inc.     

Adoptive transfer of the hematopoietic system of trisomic mice with limited life span: stem cells from six different trisomies are capable of survival.

The life span of murine trisomies is limited to the fetal or early postnatal period. However, rescue of the hematopoietic system of fetal mice with trisomies (Ts) 12, 13, 14, 16, 18, and 19 is possible by transplanting hematopoietic stem cells from the liver into lethally irradiated adult hosts. Thus, radiation chimeras with permanent and almost complete trisomic hematopoietic and lymphocytopoietic systems were constructed. The longest documented survival of a trisomic graft was 12 months in Ts 19 chimeras. Blood counts in trisomic chimeras reveal a marked anemia in Ts 16 chimeras; lymphocytopenia in Ts 12, Ts 16, and Ts 19 chimeras; and granulocytopenia in Ts 18 chimeras. Survival rates of Ts 12, Ts 18, and Ts 19 chimeras were not different from those of the respective controls, whereas survival rates of chimeras with Ts 13 and Ts 16 hematopoiesis were markedly reduced and that of Ts 14 chimeras only slightly reduced. These results indicate that transplanted hematopoietic stem cells from Ts 13, Ts 14, and Ts 16 fetuses exhibit relevant genetically determined defects, resulting in a reduced restoration capacity of hematopoietic organs and/or deficiencies of differentiated blood cells.     

Neurochemical characterization of embryonic brain development in trisomy 19 (Ts19) mice: implications of selective deficits observed for abnormal neural development in aneuploidy

In this study, we examined the neurochemical profiles of selected brain regions (cerebral hemispheres, diencephalon/brainstem) in fetal (day 14 to 18 gestation) trisomy 19 (Ts19) mice. The neurochemical characteristics we observed in Ts19 mice were quite different from those we observed previously in Ts16 mice. Choline acetyltransferase (ChAT) activity was reduced significantly in the cerebral hemispheres, but not in the brainstem/diencephalon, of the fetal Ts19 mouse brain, suggesting a selective vulnerability of telencephalic cholinergic neurons. Additionally, the activity of glutamic acid decarboxylase (GAD) was reduced significantly in both hemispheres and diencephalon/brainstem of late gestation Ts19 fetuses, suggesting a selective vulnerability of GABAergic neurons as well. While the levels of catecholaminergic and dopaminergic markers were reduced significantly at late gestational ages, the relative rate of turnover of dopamine (DA), measured by the ratio of DOPAC/DA, was elevated significantly in Ts19 mice. Neither reduction in the thickness of various cellular zones of the cerebral cortex nor reduced cell density of the cerebral cortex accounts for the alterations in neurochemical parameters observed in Ts19 mice. These results suggest that the effects of the triplication of specific genes on the respective chromosomes, rather than a generalized disruption of developmental homeostasis resulting from extra chromosomal material, may produce selective alterations in neurochemical and neuroanatomical markers observed in these two mouse trisomies.     

Identification of oncofetal antigen/immature laminin receptor protein epitopes that activate BALB/c mouse OFA/iLRP-specific effector and regulatory T cell clones

During tumor development in mice and humans, oncofetal Ag/immature laminin receptor (OFA/iLRP)-specific Th1, CTL, and IL-10-secreting T (Ts) cells are induced. The presence of too many Ts or too few effector T cells appears to predict a poor prognosis. We established clones of OFA/iLRP-specific splenic Th1, CTL, and Ts cells from the OFA/iLRP+ MCA1315 fibrosarcoma-bearing BALB/c mice or from BALB/c mice vaccinated with 1 or 10 microg of rOFA/iLRP. The MCA1315 tumor cell-reactive T cell clones were characterized as to surface Ag phenotype, cytokine secretion profile, and specificity for OFA/iLRP presented by syngeneic splenic APC. OFA/iLRP-specific Th1 and Ts clones were established from all mice. OFA/iLRP-specific CTL could be established from all mice except for mice immunized with 10 microg of rOFA/iLRP. Analysis of the proliferation profile of the OFA/iLRP-specific clones to overlapping OFA/iLRP 12-mer peptides that spanned the OFA/iLRP protein sequence defined the epitopes to which the T cell clones responded. There was a similar spatial distribution of the epitopes to which the two types of CD8 T cell clones responded. The nonapeptide epitopes of the Ts clones were located between aa 36 and 147 of OFA/iLRP, while the epitopes of the CTL clones were located between aa 52 and 163. Even though the CTL and Ts epitopes shared part of the protein, all of the CD8 CTL epitopes were distinct and separable from those of CD8 Ts cells.     

A simple pattern classification method for alcohol-responsive proteins that are differentially expressed in mouse brain

Proteomic analysis of brain tissues obtained from two inbred mice, C57BL/6J (B6, an alcohol-preferring strain) and DBA/2J (D2, an alcohol-avoiding strain), that were orally administered 1.5 g/kg ethanol, was performed to investigate alcohol-responsive proteins. To analyze relationships of alcohol-responsive protein spots between B6 and D2 mice, we have developed a simple spot classification method (SCM) for the fully matched spot data sets produced by the Melanie 4 analysis software using the paired two-dimensional (2-D) gels of two strains over time. By applying SCM, 55 protein spots that were differentially expressed in brain tissue were classified into 16 patterns as mirror images (2x8 patterns), and additionally in an ordered fashion such as 'fast turn over' and 'slow turn over' forms, depending on the frequency of repetition and rate of changed expression profile in 2-D gels over time. Searching for any interaction proteins through databases of interacting proteins using the classified data set has led to the construction of a linkage map, which reveals the interrelationship of the alcohol-responsive proteins between different species. Thus, it is suggested that the different responses for alcohol between B6 and D2 may come from differences of the response rates and interactions of different variants of the alcohol-responsive protein family.     

Genetic variability of proteins from mitochondria and mitochondrial fractions of mouse organs

Proteins of whole mitochondria from mouse liver and brain and proteins of liver mitochondrial fractions (plasma and rough membrane fraction) were separated by two-dimensional electrophoresis. Protein patterns of two inbred strains of mouse, C57BL/6J and DBA/2J, and of F₁ mice of these two strains were studied. The protein patterns obtained from the different mitochondrial materials were analyzed with regard to their protein composition and the genetic variability of proteins (qualitative and quantitative protein variants). Included in this analysis are data previously obtained from the cytosols and plasma membranes of the same organs and mouse strains. The results showed the following. (1) Mitochondria and organelle-free cell components (cytosol and plasma membranes) have only a few percent of their proteins in common, while two organs, liver and brain, reveal up to approximately 50% organ-nonspecific proteins. The frequency of proteins common to solubilized and structure-bound proteins ranges below 20%. (2) Genetic variability in protein amount occurs much more frequently than genetic variability in protein structure. Liver proteins reveal more genetic variants than brain proteins. Proteins solubilized in the cell show more genetic variation than structure-bound proteins. Furthermore, the results show that with regard to the composition and the genetic variability of proteins, liver and brain differ more in their mitochondria than in their cytosol and plasma membranes.This work was supported by grants from the Deutsche Forschungsgemeinschaft awarded to Sonderforschungsbereich 29.     

Comparative proteomic analysis of longan (Dimocarpus longan Lour.) seed abortion

Two-dimensional gel electrophoresis (2-DE), coupled with mass spectroscopy, was used to study seed abortion in Dimocarpus longan Lour. (cv. Minjiao 64-1) by comparing normal and aborted seeds at three developmental stages. More than 1,000 protein spots were reproducibly detected in 2-DE gels, with 43 protein spots being significantly altered in their intensity between normal and aborted seeds at least at one stage. Thirty-five proteins were identified by matrix-assisted laser desorption ionization-time of flight-tandem mass spectrometry (MALDI-TOF-MS/MS) analysis and protein database searching. Most of the identified proteins were associated with a variety of functions, including energy and metabolism (30%), programed cell death (9%), antioxidative processes (14%), chaperonin (23%), cell division, amino acid metabolism, secondary metabolism, and other functional classes. Furthermore, the expression patterns of HSP70 and cytosolic ascorbate peroxidase (cAPX) were validated by immunoblotting analysis. This study provides a novel, global insight into proteomic differences between normal and aborted seeds in longan. We anticipate that identification of the differentially expressed proteins may lead to a better understanding of the molecular basis for seed abortion in longan.     

Protein Dynamics Associated with Failed and Rescued Learning in the Ts65Dn Mouse Model of Down Syndrome

Down syndrome (DS) is caused by an extra copy of human chromosome 21 (Hsa21). Although it is the most common genetic cause of intellectual disability (ID), there are, as yet, no effective pharmacotherapies. The Ts65Dn mouse model of DS is trisomic for orthologs of ∼55% of Hsa21 classical protein coding genes. These mice display many features relevant to those seen in DS, including deficits in learning and memory (L/M) tasks requiring a functional hippocampus. Recently, the N-methyl-D-aspartate (NMDA) receptor antagonist, memantine, was shown to rescue performance of the Ts65Dn in several L/M tasks. These studies, however, have not been accompanied by molecular analyses. In previous work, we described changes in protein expression induced in hippocampus and cortex in control mice after exposure to context fear conditioning (CFC), with and without memantine treatment. Here, we extend this analysis to Ts65Dn mice, measuring levels of 85 proteins/protein modifications, including components of MAP kinase and MTOR pathways, and subunits of NMDA receptors, in cortex and hippocampus of Ts65Dn mice after failed learning in CFC and after learning was rescued by memantine. We show that, compared with wild type littermate controls, (i) of the dynamic responses seen in control mice in normal learning, >40% also occur in Ts65Dn in failed learning or are compensated by baseline abnormalities, and thus are considered necessary but not sufficient for successful learning, and (ii) treatment with memantine does not in general normalize the initial protein levels but instead induces direct and indirect responses in approximately half the proteins measured and results in normalization of the endpoint protein levels. Together, these datasets provide a first view of the complexities associated with pharmacological rescue of learning in the Ts65Dn. Extending such studies to additional drugs and mouse models of DS will aid in identifying pharmacotherapies for effective clinical trials.     

CD72几种新的剪切形式的发现及它们在红斑狼疮模型鼠中的差异表达

CD72是一个重要的B细胞特异性受体,它以多种选择性剪切形式存在.在小鼠脾细胞中发现并鉴定了8种新的CD72选择性剪切形式,这些剪切形式中包含有2种独特的插入片段,一种选择性剪切保留了一个内含子(intron1),而这个内含子被翻译成氨基酸序列后并没有改变前后外显子的读码框,另一种使用了一个位于内含子之内的3′剪切位点,从而产生移码,提前终止了蛋白质的开放读码框,称为3′AS(3′alternative splicingsite).比较了CD72所有剪切形式在BALB/C小鼠和NZB/W小鼠中的差异表达,发现:a.含有3′AS的剪切形式的表达都很少;b.WT, In1, In1-Ex3和-Ex3的表达在BLAB/C小鼠中比在NZB/W小鼠中高;c.没有ITIM2的-Ex2-Ex3剪切形式在NZB/W小鼠中有特异性高表达.这些结果提示,CD72的多种选择性剪切形式在调控B细胞受体信号转导过程中可能发挥着不同的作用,并与系统性红斑狼疮的发病密切相关.     

Influenza-specific suppression: contribution of major viral proteins to the generation and function of T suppressor cells

Suppressor cells were generated in BALB/c mice by two sequential injections of PR8 influenza virus (A/Puerto Rico/8/34[H1N1]) and were tested for their ability to inhibit proliferative cellular responses towards multiple viral and nonviral antigens. In this way, suppression specific to PR8 as compared with purified protein derivative (PPD) and keyhole limpet hemocyanin (KLH) antigenic responses was illustrated. Experiments involving adoptive transfer of suppression to naive hosts with subfractionated lymphocyte populations demonstrated that the suppressors were Lyt-2+ T cells. Two major questions were addressed with this system. First, a determination was made of which anti-viral protein proliferative responses were affected by the PR8-induced T suppressor (Ts) cells. Ts cells were found to inhibit proliferating cells with specificities for isolated hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), and matrix (M) antigens. Second, experiments were conducted to analyze the viral proteins contributing to the induction of PR8-specific Ts cells. Inoculations with either isolated HA or a combination of M + NP proteins induced T suppression specific to proliferative responses towards PR8. These experiments illustrate the contribution of external (HA and NA) as well as internal (M + NP) viral proteins to Ts cell generation and function.     

Two-dimensional electrophoretic protein patterns of reciprocal hybrids of the mouse strains DBA and C57BL

Two-dimensional electrophoretic patterns of cytoplasmic liver proteins of the mouse were investigated in reciprocal hybrids of the two inbred mouse strains DBA and C57BL in order to establish whether strain-specific protein variants reveal a mode of inheritance compatible with the concept of genomic imprinting. Variants of this type were found to account for about 11% of approximately 200 variant protein spots scrutinized. Transmission of the maternal form of a variant protein was more frequent than transmission of the paternal form. Maternal/paternal transmission was observed only for proteins showing strain variations in their amount. The results are discussed in terms of the frequency of imprinted genes.     

Genetic variability of proteins from plasma membranes and cytosols of mouse organs

The genetic variability of membrane proteins (structure-bound proteins) and cytosol proteins (water-soluble proteins) was investigated in two inbred strains of the mouse, C57BL/6J and DBA/2J. Membrane proteins and cytosol were isolated from the brain and liver of the mouse. The proteins were separated by two-dimensional electrophoresis. A high number of genetic variant proteins (brain, 30; liver, 72) was found in the cytosol. Most of these variants represented changes in the amount of proteins. Electrophoretic mobility changes occurred only in about 1% (brain, 6; liver, 9) of all protein spots of a two-dimensional pattern. In contrast to the cytosol proteins, no genetic variation was detected among the membrane proteins, not even for the quantitative characteristics of the protein spots. The results obtained for the two classes of proteins suggest that the degree of variability in the amount of proteins is related to the degree of variability in the structure of proteins.     

Composition and genetic variability of heparin-sepharose CL-6B protein fractions obtained from the solubilized proteins of mouse organs

The solubilized proteins of liver and brain from mice of two inbred strains (C57BL/6J and DBA/2J) and their hybrids were subfractionated by heparin Sepharose (H-S) CL-6B affinity chromatography. The H-S binding and nonbinding proteins were separated by two-dimensional electrophoresis. The protein patterns obtained were analyzed with regard to their protein composition and their genetic variability (qualitative and quantitative variants). Eighty to ninety percent of the H-S binding proteins were unique to this class of proteins. This class was rich in organ-specific proteins. Compared to the nonbinding proteins the portion of basic proteins was only slightly increased, suggesting that most of the H-S binding proteins interact specifically with heparin. The frequency of qualitative protein variants revealed that H-S binding proteins are more conservative than H-S nonbinding proteins. The quantitative genetic variability was higher in liver than in brain. Quantitative protein variants occurred more frequently than qualitative variants.     

Proteomic analysis of human osteoarthritic chondrocytes reveals protein changes in stress and glycolysis

Osteoarthritis (OA) is characterized by cartilage degradation. The chondrocyte is the only cell type present in mature cartilage, and it is important in the control of cartilage integrity. The aim of this study was to analyze, by a proteomic approach, the changes that are characteristic of OA chondrocytes, and to identify new OA-related proteins. Chondrocytes were isolated from the cartilage of ten OA patients undergoing joint replacement and ten donors with no history of joint disease. Whole-cell proteins were resolved by 2-DE and stained with SYPRO Ruby. Protein expression patterns of 2-DE gels from OA and normal chondrocyte proteins were analyzed with PDQuest 7.3.1 software. OA-related proteins were identified by MALDI-TOF or MALDI-TOF/TOF MS. The results were validated for ANXA1, GSTO1, GRP78, and HSP90beta in cells by Western blotting and in tissue cartilage by immunohistochemistry. Results showed an average of 700 protein spots that were present in the 2-DE gels. Compared to normal chondrocytes, 19 protein spots were found to be significantly increased in OA cells (ratio OA:N> or =2.0, p<0.05), whereas nine were decreased in OA chondrocytes (ratio OA:N< or =0.5, p<0.05). Three stress response proteins were increased (HSP90beta, GRP78, and GRP94) and three proteins involved in glycolysis were decreased (enolase, glyceraldehyde 3-phosphate dehydrogenase, and fructose biphosphate aldolase). Functionally, almost all proteins could be classified as proteins involved in cellular metabolism (33%), structure (21%), or protein targeting (21%).     

Stability of liver nuclear proteins in inbred strains of mice

1. A remarkable similarity in the gel patterns of liver nuclear proteins between four inbred strains of mice (A.CA, B10.A, CBA and DBA/2) was observed. 2. Only a very few quantitative differences were detected in the protein spot patterns of nucleoplasmic (spot of about 41 kDa) and chromatin (spot of about 37 kDa) non-histone proteins between those strains of mice. 3. Comparison of two-dimensional gel patterns of non-histone proteins from males and females revealed a few sex-linked spots. Nucleoplasmic protein with molecular weight of about 59 kDa and chromatin proteins with molecular weights of approximately 47 and 57 kDa were more abundant in liver nuclei of male mouse.     

Proteomic analysis of diet-induced hypercholesterolemic mice

We report here a proteomic analysis of differentially expressed liver proteins of both C57BL/6J (B6, atherosclerosis-susceptible strain) and C3H/HeJ mice (C3H, atherosclerosis-resistant strain), which were fed either control or a high-fat enriched atherogenic diet for eight weeks. We observed differential patterns of plasma lipids between the two strains when both were fed atherogenic diets. That is, although low density lipoprotein cholesterol level was highly elevated in both, the levels of total cholesterol and triglyceride in B6 mice were much lower than those in C3H mice when they were fed atherogenic diets. However, the high density lipoprotein cholesterol level was increased in the latter but decreased in the former. Histopathological observation revealed that more prominent lipid droplets were present in B6 mice than in C3H mice, when they were maintained on the atherogenic diets. Proteomic analysis of liver tissues of these two strains showed that a total of 30 proteins were significantly changed in the livers obtained from both strains after being fed the atherogenic diet. Of these, 14 protein spots including carbonic anhydrase III, senescence marker protein 30 and selenium binding protein 2 were differentially changed only in B6 mice, which was also confirmed in part by Western blotting. An additional 16 protein spots including glutathione S-transferase subclass, apolipoprotein E and chaperonin proteins were changed in both strains. We also identified 28 proteins that were differentially expressed in the livers of both B6 and C3H mice, regardless of diet feeding condition. Of these, 4 protein spots in B6 mice and 11 protein spots in C3H mice were up-regulated. Thirteen strain specific protein spots including antioxidant protein 2, apolipoprotein E and apolipoprotein A-I were also detected in different positions in two-dimensional electrophoresis. These results suggest a clear distinction in differential expression of oxidative stress proteins and lipid metabolism related proteins between the two strains in response to atherogenic diet feeding, which might account for their difference in susceptibility to atherogenesis.