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At least 793 records · Page 44Linked to original sources

PubMed2006

Insights into social insects from the genome of the honeybee Apis mellifera.

Here we report the genome sequence of the honeybee Apis mellifera, a key model for social behaviour and essential to global ecology through pollination. Compared with other sequenced insect genomes, the A. mellifera genome has high A+T and CpG contents, lacks major transposon families, evolves more slowly, and is more similar to vertebrates for circadian rhythm, RNA interference and DNA methylation genes, among others. Furthermore, A. mellifera has fewer genes for innate immunity, detoxification enzymes, cuticle-forming proteins and gustatory receptors, more genes for odorant receptors, and novel genes for nectar and pollen utilization, consistent with its ecology and social organization. Compared to Drosophila, genes in early developmental pathways differ in Apis, whereas similarities exist for functions that differ markedly, such as sex determination, brain function and behaviour. Population genetics suggests a novel African origin for the species A. mellifera and insights into whether Africanized bees spread throughout the New World via hybridization or displacement.

Animals↗

PubMed2002

The intracellular antibody capture technology (IACT): towards a consensus sequence for intracellular antibodies.

We describe the application of an intracellular antibody capture technology (IACT) as a generic in vivo selection procedure for isolating intracellular antibodies or ICAbs. IACT was applied to the de novo selection of functional ICAbs against the microtubule-associated protein TAU, found in neurofibrillary lesions of Alzheimer's disease brains. A panel of 17 different ICAbs was created which bind TAU inside cells and the epitopes recognized by the selected ICAbs have been determined by an in vivo epitope mapping procedure. Finally, sequence analysis showed that the IACT-derived ICAbs are characterized by a common signature of conserved amino acid residues, suggesting that the IACT naturally selects a sort of "captured consensus sequence" for intracellular antibodies. The development of IACT, together with the possibility of scaling up in a high throughput and automated format, makes IACT a new enabling tool for target validation in functional genomics and global proteomics.

Alzheimer Disease↗

PubMed2006

Generation of mouse FANCL antibody and analysis of FANCL protein expression profile in mouse tissues.

Fanconi anemia complementation group L (FANCL) is a novel Fanconi anemia protein, which mono-ubiquitinates FANCD2 as a ubiquitin E3 ligase, and plays a crucial role in DNA damage repair and chromosome stability maintenance. FANCL is involved in the proliferation of primordial germ cells (PGC) in early embryonic stages, and may play a role in the development of germ cells by forming a novel testis-specific network with testis-specific proteins in the adult testis. FancL cDNA sequence was cloned by RT-PCR from mouse testis total RNA, and expressed in E. coli BL21(DE3). Rabbit FANCL polyclonal antiserum was generated using the recombinant protein as the antigen. To prepare an antigen column for affinity purification of FANCL-specific antibody, recombinant His-tagged FANCL was purified by Ni(2+)-charged HiTrap Chelating HP column and coupled to an NHS-activated HiTrap column. To confirm the activity and specificity of the FANCL antibody, we constructed plasmid pCMV-HA/FANCL to transfect HEK 293T cells. Transiently expressed HA-FANCL fusion protein was analyzed by immunoblotting with both the FANCL antibody and HA monoclonal antibody. The antibody was used in Western blotting to check the expression of FANCL protein in mouse tissues. We found wide expression of FANCL in brain, muscle, heart, lung, liver, spleen, kidney, testis, ovary and uterus, indicating the functional importance of this novel protein.

Animals↗

PubMed2005

Imaging mass spectrometry: principles and potentials.

Direct tissue profiling and imaging mass spectrometry (MS) allow for detailed mapping of the complex protein pattern across a tissue sample. Utilization of these tools provides spatial information across a tissue section for target protein expression and can be used to correlate changes in expression levels with specific disease states or drug response. Protein patterns can be directly correlated to known histological regions within the tissue, allowing for the direct monitoring of proteins specific for morphological regions within a tissue sample. Profiling and imaging MS have been used to characterize multiple tissues, including human gliomas and lung cancers, as well as tumor response to specific therapeutics, suggesting the use of proteomic information in assessing disease progression as well as predicting patient response to specific treatments. This article discusses both the technology and methods involved in analyzing proteins directly from tissue samples as well as several MS applications, including profiling human tumors, characterizing protein differences between tumor grades, and monitoring protein changes due to drug therapy.

Adenocarcinoma↗

PubMed2025

An integrative network approach for longitudinal stratification in Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by motor symptoms resulting from the loss of dopamine-producing neurons in the brain. Currently, there is no cure for the disease which is in part due to the heterogeneity in patient symptoms, trajectories and manifestations. There is a known genetic component of PD and genomic datasets have helped to uncover some aspects of the disease. Understanding the longitudinal variability of PD is essential as it has been theorised that there are different triggers and underlying disease mechanisms at different points during disease progression. In this paper, we perform longitudinal and cross-sectional experiments to identify which data modalities or combinations of modalities are informative at different time points. We use clinical, genomic, and proteomic data from the Parkinson's Progression Markers Initiative. We validate the importance of flexible data integration by highlighting the varying combinations of data modalities for optimal stratification at different disease stages in idiopathic PD. We show there is a shared signal in the DNAm signatures of participants with a mutation in a causal gene of PD and participants with idiopathic PD. We also show that integration of SNPs and DNAm data modalities has potential for use as an early diagnostic tool for individuals with a genetic cause of PD.

Parkinson Disease↗

PubMed2004

Analysis of differential protein expression in Actinobacillus pleuropneumoniae by Surface Enhanced Laser Desorption Ionisation--ProteinChip (SELDI) technology.

Actinobacillus pleuropneumoniae (APP) is the aetiological agent of porcine pleuropneumonia. An increased understanding of its molecular basis of pathogenicity and vaccine development will be facilitated by the availability of sequence data from a complete genome which, by analogy to other bacteria, is predicted to encode many proteins in the molecular mass range 3-20kDa. However, conventional techniques to study bacterial protein expression, such as SDS-PAGE and 2-dimensional electrophoresis, typically focus on the 15-200kDa range. In this study we have evaluated Surface Enhanced Laser Desorption Ionisation-ProteinChip (SELDI) technology for the analysis of protein expression, in particular those of <20kDa, of APP grown under different environmental conditions. Cytoplasmic/periplasmic and outer membrane protein fractions were obtained from the APP wildtype serotype 1 strain 4074 grown in Brain Heart Infusion (BHI) broth (+different concentrations of NAD), BHI containing pig serum or defined medium. Optimum conditions for SELDI profiles included a sample size of 1 microg and the use of sinapinic acid as the energy absorbing matrix. In the <20kDa range, the SELDI profiles obtained from wild-type bacteria grown in rich medium plus 33-66% pig serum were most similar to those grown in defined medium. The SELDI profiles of extracts of the wild-type and of an rpoE mutant were similar although there were clear differences. The results suggest that SELDI is a useful complementary approach to conventional proteomic analytical methods with APP, and presumably other bacterial pathogens, being particularly suited for analysis of proteins in the <20kDa mass range.

Actinobacillus pleuropneumoniae↗

PubMed2025

Impact of the ECM on the Mechanical Memory of Cancer Cells.

Besides genomic and proteomic analyses of bulk and individual cancer cells, cancer research focuses on the mechanical analysis of cancers, such as cancer cells. Throughout the oncogenic evolution of cancer, mechanical inputs are stored as epigenetic memory, which ensures versatile coding of malignant characteristics and a quicker response to external environmental influences in comparison to solely mutation-based clonal evolutionary mechanisms. Cancer's mechanical memory is a proposed mechanism for how complex details such as metastatic phenotypes, treatment resistance, and the interaction of cancers with their environment could be stored at multiple levels. The mechanism appears to be similar to the formation of memories in the brain and immune system like epigenetic alterations in individual cells and scattered state changes in groups of cells. Carcinogenesis could therefore be the outcome of physiological multistage feedback mechanisms triggered by specific heritable oncogenic alterations, resulting in a tumor-specific disruption of the integration of the target site/tissue into the overall organism. This review highlights and discusses the impact of the ECM on cancer cells' mechanical memory during their metastatic spread. Additionally, it demonstrates how the emergence of a mechanical memory of cancer can give rise to new degrees of individuality within the host organism, and a connection to the cancer entity is established by discussing a connection to the metastasis cascade. The aim is to identify common mechanical memory mechanisms of different types of cancer. Finally, it is emphasized that efforts to identify the malignant potency of tumors should go way beyond sequencing approaches and include a functional diagnosis of cancer physiology and a dynamic mechanical assessment of cancer cells.

Humans↗

PubMed2005

Phosphorylation of Numb family proteins. Possible involvement of Ca2+/calmodulin-dependent protein kinases.

To search for the substrates of Ca2+/calmodulin-dependent protein kinase I (CaM-KI), we performed affinity chromatography purification using either the unphosphorylated or phosphorylated (at Thr177) GST-fused CaM-KI catalytic domain (residues 1-293, K49E) as the affinity ligand. Proteomic analysis was then carried out to identify the interacting proteins. In addition to the detection of two known CaM-KI substrates (CREB and synapsin I), we identified two Numb family proteins (Numb and Numbl) from rat tissues. These proteins were unphosphorylated and were bound only to the Thr177-phosphorylated CaM-KI catalytic domain. This finding is consistent with the results demonstrating that Numb and Numbl were efficiently and stoichiometrically phosphorylated in vitro at equivalent Ser residues (Ser264 in Numb and Ser304 in Numbl) by activated CaM-KI and also by two other CaM-Ks (CaM-KII and CaM-KIV). Using anti-phospho-Numb/Numbl antibody, we observed the phosphorylation of Numb family proteins in various rat tissue extracts, and we also detected the ionomycin-induced phosphorylation of endogenous Numb at Ser264 in COS-7 cells. The present results revealed that the Numb family proteins are phosphorylated in vivo as well as in vitro. Furthermore, we found that the recruitment of 14-3-3 proteins was the functional consequence of the phosphorylation of the Numb family proteins. Interaction of 14-3-3 protein with phosphorylated Numbl-blocked dephosphorylation of Ser304. Taken together, these results indicate that the Numb family proteins may be intracellular targets for CaM-Ks, and they may also be regulated by phosphorylation-dependent interaction with 14-3-3 protein.

14-3-3 Proteins↗

PubMed2005

Cerebellar protein expression in three different mouse strains and their relevance for motor performance.

The present study uses a proteomic approach to link motor function to cerebellar protein expression in 129X1/SvJ, C57BL/6J and nNOS WT mice. Poor performance on the Rota rod, the standard test for motor coordination, was detected in 129X1/SvJ mice. No gross impairments of neurological, cognitive and behavioural functions were observed. Identification and quantification of 48 proteins revealed reduced expression of calbindin, septin 5 and syntaxin binding protein 1 in 129X1/SvJ. In nNos WT glucose-6-phosphate 1 dehydrogenase X was decreased whereas dihydropyrimidinase-related protein-4 was increased. In C57BL/6J stress-70 protein, alpha enolase, NAD-dependent deacetylase sirtuin 2, septin 2, dihydropyrimidinase-related protein-2 and brain derived neurotrophic factor showed elevated levels. Neurological examination, Rota rod test, Morris Water Maze, Multiple-T-Maze, Open field and Elevated plus-maze were employed to study motor, cognitive and behavioural function. Mice were sacrificed and cerebellar tissue was homogenized. Proteins were extracted and separated on two-dimensional gel electrophoresis with subsequent in-gel digestion followed by mass spectrometrical analysis of peptides (MALDI-TOF/TOF-TOF). Quantification of spots was carried out by specific software. A strong association of impaired motor function with altered cerebellar protein expression of calbindin, septin 5 and syntaxin binding protein 1in 129X1/SvJ was observed and is in agreement with previous observations of motor deficiencies in a calbindin knock-out mouse. These results have to be taken into account when using 129X1/SvJ for biochemical, toxicological or gene targeting experiments as well as when studying the above-mentioned proteins or corresponding pathways and cascades in this mouse strain.

Animals↗

PubMed2003

Hematopoietic growth factor inducible neurokinin-1 type: a transmembrane protein that is similar to neurokinin 1 interacts with substance P.

Neurokinin 1 (NK-1) is a member of seven transmembrane G protein-coupled receptors. NK-1 interacts with peptides belonging to the tachykinin family and showed preference for substance P (SP). NK-1 is induced in bone marrow (BM) stroma. NK-1-SP interactions could lead to changes in the functions of lymphohematopoietic stem cell (LHSC). This report describes the cloning and characterization of a cDNA clone isolated after screening of three cDNA libraries with an NK-1-specific probe. Based on its expression, the cDNA clone was designated hematopoietic growth factor inducible neurokinin-1 type (HGFIN). Computational analyses predicted that HGFIN is transmembrane with the carboxyl terminal extracellular. Proteomic studies with purified HGFIN and SP showed noncovalent interactions. HGFIN-SP interactions were supported by transient expression of HGFIN in CHO cells. Transient expression of HGFIN in unstimulated BM fibroblasts led to the induction of endogenous NK-1. Since NK-1 expression in BM fibroblasts requires cell stimulation, these studies suggest that there might be intracellular crosstalk between NK-1 and HGFIN. Northern analyses with total RNA from different BM cell subsets showed that HGFIN was preferentially expressed in differentiated cells. This suggests that HGFIN might be involved in the maturation of LHSC. HGFIN was detected in several other tissues, but not in brain where NK-1 is constitutively expressed.

Animals↗

PubMed2006

Glial fibrillary acidic protein is a major target of glycoxidative and lipoxidative damage in Pick's disease.

Pick's disease is a subset of fronto-temporal dementia characterised by severe atrophy of the temporal and frontal lobes due to marked neuronal loss accompanied by astrocytic gliosis enriched in glial acidic protein. The remaining neurones have intracytoplasmic inclusions composed of hyperphosphorylated tau, called Pick bodies, in addition to hyperphosphorylated tau in astrocytes and oligodendrocytes. Gel electrophoresis and western blotting using markers of glycoxidation (advanced glycation end products, N-carboxyethyl-lysine and N-carboxymethyl-lysine: AGE, CEL, CML, respectively) and lipoxidation (4-hydroxy-2-nonenal: HNE, and malondialdehyde-lysine: MDAL) were used in the frontal and occipital cortex in three Pick's disease cases and three age-matched controls. In Pick's disease, increased AGE, CML, CEL, HNE and MDAL bands of about 50 kDa were observed in the frontal cortex (but not in the occipital cortex) in association with increased density of glial acidic protein bands. Bi-dimensional gel electrophoresis and western blotting also disclosed increased amounts and numbers of glial acidic protein isoforms in the frontal cortex in Pick's disease. Moreover, redox proteomics showed glycoxidation, as revealed with anti-CEL antibodies and lipoxidation using anti-HNE antibodies, of at least three glial acidic protein isoforms. The present results demonstrate that glial acidic protein is a target of oxidative damage in the frontal cortex in Pick's disease.

Aldehydes↗

PubMed2005

Genes and homology in nervous system evolution: comparing gene functions, expression patterns, and cell type molecular fingerprints.

The evolution of the nervous system is one of the most fascinating, but also most nebulous fields of homology research. We do not know for example whether the last common ancestors of human, squid, and fly already possessed an elaborate brain and eyes, or rather had a simple, diffuse nervous system. Nevertheless, in the past decade molecular data has greatly advanced our understanding of bilaterian nervous system evolution. In this methodological review, I explain the four levels on which molecular genetic studies advance the quest for homologies between animal nervous systems. (I) Bioinformatic homology research elucidates the evolutionary history of gene families relevant for nervous system evolution such as the opsin superfamily. It tells us when and in what order genes and their functions have emerged. Based on this, we can (II) infer the organismal complexity of some remote ancestor from the functional diversity of its reconstructed proteome. (III) Most common in molecular homology research has been the comparison of expression patterns of developmental control genes. This approach matches and aligns embryonic regions along the body axes, between remote bilaterians. It does not tell us much, however, about the complexity of structures that developed from these regions in Urbilateria. (IV) This is overcome by a novel variant of molecular homology research, the comparison of cell types. Here, a similar "molecular fingerprint" of cells is taken as indication of cross-bilaterian homology. This approach makes it possible to reconstruct the cell-type repertoire of the urbilaterian nervous system.

Animals↗

PubMed2006

Decreased striatal levels of PEP-19 following MPTP lesion in the mouse.

PEP-19 is a neuronal calmodulin-binding protein, and as such, a putative modulator of calcium regulated processes. In the present study, we used proteomics technology approaches such as peptidomics and imaging MALDI mass spectrometry, as well as traditional techniques (immunoblotting and in situ hybridization) to identify PEP-19 and, specifically, to measure PEP-19 mRNA and protein levels in an animal model of Parkinson's disease. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration in mice resulted in a significant decrease in striatal PEP-19 mRNA. Capillary nano-flow liquid chromatography electrospray mass spectrometry analysis of striatal tissue revealed a significant decrease of the PEP-19 protein level. Moreover, imaging MALDI mass spectrometry also showed that PEP-19 protein was predominantly localized to the striatum of the brain tissue cross sections. After MPTP administration, PEP-19 levels were significantly reduced by 30%. We conclude that PEP-19 mRNA and protein expression are decreased in the striatum of a common animal model of Parkinson's disease. Further studies are needed to show the specific involvement of PEP-19 in the neurodegeneration seen in MPTP lesioned animals. Finally, this study has shown that the combination of traditional molecular biology techniques with novel, highly specific and sensitive mass spectrometry methods is advantageous in characterizing molecular events of many diseases, including Parkinson's disease.

Animals↗

PubMed2006

Protein expression changes in the nucleus accumbens and amygdala of inbred alcohol-preferring rats given either continuous or scheduled access to ethanol.

Chronic ethanol (EtOH) drinking produces neuronal alterations within the limbic system. To investigate changes in protein expression levels associated with EtOH drinking, inbred alcohol-preferring (iP) rats were given one of three EtOH access conditions in their home-cages: continuous ethanol (CE: 24h/day, 7days/week access to EtOH), multiple scheduled access (MSA: four 1-h sessions during the dark cycle/day, 5 days/week) to EtOH, or remained EtOH-naïve. Both MSA and CE groups consumed between 6 and 6.5g of EtOH/kg/day after the 3rd week of access. On the first day of EtOH access for the seventh week, access was terminated at the end of the fourth MSA session for MSA rats and the corresponding time point (2300h) for CE rats. Ten h later, the rats were decapitated, brains extracted, the nucleus accumbens (NAcc) and amygdala (AMYG) microdissected, and protein isolated for 2-dimensional gel electrophoretic analyses. In the NAcc, MSA altered expression levels for 12 of the 14 identified proteins, compared with controls, with six of these proteins altered by CE access, as well. In the AMYG, CE access changed expression levels for 22 of the 27 identified proteins, compared with controls, with 8 of these proteins altered by MSA, as well. The proteins could be grouped into functional categories of chaperones, cytoskeleton, intracellular communication, membrane transport, metabolism, energy production, or neurotransmission. Overall, it appears that EtOH drinking and the conditions under which EtOH is consumed, differentially affect protein expression levels between the NAcc and AMYG. This may reflect differences in neuroanatomical and/or functional characteristics associated with EtOH self-administration and possibly withdrawal, between these two brain structures.

Alcohol Drinking↗

PubMed2004

Changes in magnetization transfer MRI correlate with spreading depression-induced astroglial reactivity and increased protein expression in mice.

OBJECTIVE: Gliosis refers to a range of glial cell transformations that vary according to specific brain pathologic states. Disease, however, is not a prerequisite for gliosis because glial reactivity may also be seen in regions of increased physiologic activity. Our study tests the hypothesis that high-field-strength magnetization transfer MRI is a sensitive technique for detecting transient glial reactivity after experimental spreading depression, a relatively benign perturbation unaccompanied by cell injury. MATERIALS AND METHODS: Unilateral neocortical spreading depression was elicited in mouse cerebral hemispheres and confirmed by transcranial blood flow and extracellular potential measurements. After 3 days, mice were imaged at 4 T using magnetization transfer techniques. Astroglial reactivity was determined immunohistochemically, and protein expression in control and experimental hemispheres was compared using proteomic techniques. RESULTS: Sixteen ([mean +/- SD] +/- 3) spreading depressions (n = 10) were recorded in experimental hemispheres. Spreading depression was never observed in control hemispheres. At 3 days, an 8% decrease (p < 0.05, n = 4) in magnetization transfer signal intensity was measured in experimental hemispheres, which was associated with a 37% increase (p < 0.001, n = 4) in the intensity of glial fibrillary acidic protein staining. Proteomic analysis performed 3 days after the induction of spreading depression showed upregulation of at least 56 proteins, including extracellular and intracellular elements. CONCLUSION: Magnetization transfer at 4.0-T MRI is a sensitive method for detecting glial reactivity and changes in protein expression not associated with cell injury. These results suggest magnetization transfer MRI techniques may have potential for detecting glial reactivity in physiologic processes such as learning and in early disease states.

Animals↗

PubMed2005

Neurogenetics II: complex disorders.

The genetic analysis of common neurological disorders will be a difficult and protracted endeavour. Genetics is only one of many disciplines that will be required but it has already thrown considerable light on the aetiology of several major neurological disorders through the analysis of rare inherited subgroups. The identification of individual susceptibility genes with variants of smaller effect will be more difficult but there is no sharp demarcation between large and small genetic effects, so that many new and important insights will emerge using existing and new technologies. The availability of improved neuroimaging, better animal models of disease and new genetic tools, such as high-throughput gene chips, expression microarrays and proteomics, are extending the range of traditional genetic mapping tools. Finally, an understanding of the genetic and epigenetic mechanisms that restrain the differentiation and integration of human neural stem cells into mature neuronal networks could have a major impact on clinical practice. These approaches will be illustrated in the context of Alzheimer disease, Parkinson disease and synucleinopathies, tauopathies, amyotrophic lateral sclerosis and stroke.

Alleles↗

PubMed2001

Genetic engineering of neural function in transgenic rodents: towards a comprehensive strategy?

As mammalian genome projects move towards completion, the attention of molecular neuroscientists is currently moving away from gene identification towards both cell-specific gene expression patterns (neuronal transcriptions) and protein expression/interactions (neuronal proteomics). In the long term, attention will increasingly be directed towards experimental interventions which are able to question neuronal function in a sophisticated manner that is cognisant of both transcriptomic and proteomic organization. Central to this effort will be the application of a new generation of transgenic approaches which are now evolving towards an appropriate level of molecular, temporal and spatial resolution. In this review, we summarize recent developments in transgenesis, and show how they have been applied in the principal model species for neuroscience, namely rats and mice. Current concepts of transgene design are also considered together with an overview of new genetically-encoded tools including both cellular indicators such as fluorescent activity reporters, and cellular regulators such as dominant negative signalling factors. Application of these tools in a whole animal context can be used to question both basic concepts of brain function, and also current concepts of underlying dysfuction in neurological diseases.

Animals↗

PubMed2002

Region-specific alternative splicing in the nervous system: implications for regulation by the RNA-binding protein NAPOR.

Alternative RNA splicing generates extensive proteomic diversity in the nervous system, yet few neural-specific RNA binding proteins have been implicated in splicing control. Here we show that the biochemical properties and spatial expression of mouse neuroblastoma apoptosis-related RNA-binding protein (NAPOR; also called NAPOR-1) are consistent with its roles in the regulation of the exon 5 and exon 21 splicing events of the N-methyl-D-aspartate (NMDA) receptor R1 transcript. NAPOR, which is closely related to CUG binding protein 2 (CUG-BP2), promotes exon 21 and represses exon 5 splicing in functional coexpression assays. These NMDA mRNA isoforms are distributed, in vivo, in a region-specific manner in rat brain, such that high levels of exon 21 selection and exon 5 skipping coincide with high NAPOR mRNA expression in the forebrain. Within the forebrain, this spatial correspondence is most striking in the visual cortex. In contrast, low NAPOR expression coincides with the reciprocal pattern of alternative splicing in the hindbrain. Complementary experiments demonstrate a tissue-specific distribution of NAPOR, CUG-BP, and other highly related proteins within the nervous system as assayed by probing forebrain and hindbrain nuclear extracts with monoclonal antibody, mAb 3B1. Thus, NAPOR may be one of a group of closely related proteins involved in splicing regulation within the brain. An intronic RNA element responsible for the silencing of exon 21 splicing is identified by mutational analysis and shown to bind directly to recombinant NAPOR protein, suggesting a model in which exon 21 selection is positively regulated by an antirepression mechanism of action.

Alternative Splicing↗