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PubMedDate not supplied

The platelet microparticle proteome.

Platelet-derived microparticles are the most abundant type of microparticle in human blood and contribute to many biologically significant processes. Here, we report the first proteomic analysis of microparticles generated from activated platelets. Using 1D SDS-PAGE and liquid chromatography coupled to a linear ion trap mass spectrometer, the identification of 578 proteins was accomplished using a minimum of 5 MS/MS detections of at least two different peptides for each protein. These microparticles displayed many proteins intrinsic to and well-characterized on platelets. For example, microparticles in these experiments were found to contain membrane surface proteins including GPIIIa, GPIIb, and P-selectin, as well other platelet proteins such as the chemokines CXCL4, CXCL7, and CCL5. In addition, approximately 380 of the proteins identified were not found in two previous studies of the platelet proteome. Since several of the proteins detected here have been previously implicated in microparticle formation and/or pathological function, it is hoped that this study will help fuel future work concerning the possible role of microparticles in various disease states.

Animals↗

PubMed2005

Dominant role of copper in the kinetic stability of Cu/Zn superoxide dismutase.

Mutations in Cu/Zn superoxide dismutase (SOD) are involved in some cases of familial amyotrophic lateral sclerosis, and it appears that misfolding and aggregation, perhaps mediated by abnormal binding or loss of copper (Cu) and/or zinc (Zn), may play a pathological role. It is known that the absence of both metals kinetically destabilizes wild type and mutant SOD leading to a 60-fold increase in their rate of unfolding. Here, the individual contributions of Cu and Zn to the kinetic stability of SOD were investigated, and the results show that Cu plays a greater role. Thus, the deficiency of Cu or Zn, especially the former, will compromise the kinetic stability of SOD, thereby increasing the probability that pathogenic mutants and even the WT protein may misfold and self-assemble into toxic species.

Amyotrophic Lateral Sclerosis↗

PubMed2001

Interaction between neuronal intranuclear inclusions and promyelocytic leukemia protein nuclear and coiled bodies in CAG repeat diseases.

Neuronal intranuclear inclusions (NIIs) are a pathological hallmark of CAG repeat diseases. To elucidate the influence of NII formation on intranuclear substructures, we investigated the relationship of NIIs with nuclear bodies in brains of dentatorubral-pallidoluysian atrophy and Machado-Joseph disease. In both diseases, promyelocytic leukemia protein, a major component of the promyelocytic leukemia protein nuclear bodies, altered the normal distribution and was rearranged around NII, forming a single capsular structure. We further demonstrated that NIIs were present in close contact with coiled bodies, a highly dynamic domain that may be involved in the biogenesis of small nuclear ribonucleoproteins. The preferential association of intranuclear polyglutamine aggregates with coiled bodies was also confirmed in the dentatorubral-pallidoluysian atrophy transgenic mouse brain and culture cells expressing mutant atrophin-1. The results suggest that the interaction between NIIs and nuclear bodies may play a role in the pathogenesis of CAG repeat diseases.

Adult↗

PubMed2004

Biomarkers of neurodegenerative disorders: how good are they?

Biomarkers are very important indicators of normal and abnormal biological processes. Specific changes in pathologies, biochemistries and genetics can give us comprehensive information regarding the nature of any particular disease. A good biomarker should be precise and reliable, distinguishable between normal and interested disease, and differential between different diseases. It is believed that biomarkers have great potential in predicting chances for diseases, aiding in early diagnosis, and setting standards for the development of new remedies to treat diseases. New technologies have enabled scientists to identify biomarkers of several different neurodegenerative diseases. The followings, for instance, are only a few of the many new biomarkers that have been recently identified: the phosphorylated tau protein and aggregated Beta-amyloid peptide for Alzheimer's disease (AD), Alpha-synuclein contained Lewy bodies and altered dopamine transporter (DAT) imaging for Parkinson's disease (PD), SOD mutations for familial amyotrophic lateral sclerosis (ALS), and CAG repeats resulted from Huntington's gene mutations in Huntington's disease (HD). This article will focus on the most-recent findings of biomarkers belonging to the four mentioned neurodegenerative diseases.

Alzheimer Disease↗

PubMed1999

Embryonic Stem Cells in vitro - Prospects for Cell and Developmental Biology, Embryotoxicology and Cell Therapy.

Embryonic stem (ES) cells are able to differentiate in vitro via embryo-like aggregates, so-called "embryoid bodies", into derivatives of the endodermal, ectodermal and mesodermal lineage. We established standardised protocols for cardiogenesis, myogenesis, neurogenesis and vascular smooth muscle cell differentiation in vitro. The developmentally controlled expression of tissue-specific genes, proteins, ion channels and receptors during ES cell differentiation is the basis of several in vitro approaches: (1) "Loss of function" assays with ES cells containing homozygous mutations of specific genes, (2) "Gain of function" assays with ES cells overexpressing exogenous genes, (3) Developmental analysis of teratogenic/embryotoxic compounds in vitro, (4) Pharmacological assays and the establishment of model systems for pathological cell functions, and (5) The application of differentiation and growth factors for induction of selectively differentiated cells which, in the future, may be used as a source for tissue grafts. We propose the ES cell technology as valuable in vitro system to substitute and reduce the use of animals in basic and applied research.

Journal Article↗

PubMed1997

Binding of Abeta to alpha- and beta-synucleins: identification of segments in alpha-synuclein/NAC precursor that bind Abeta and NAC.

NAC, a 35-residue peptide derived from the neuronal protein alpha-synuclein/NAC precursor, is tightly associated with Abeta fibrils in Alzheimer's disease amyloid, and alpha-synuclein has recently been shown to bind Abeta in vitro. We have studied the interaction between Abeta and synucleins, aiming at determining segments in alpha-synuclein that can account for the binding, as well as identifying a possible interaction between Abeta and the beta-type synuclein. We report that Abeta binds to native and recombinant alpha-synuclein, and to beta-synuclein in an SDS-sensitive interaction (IC50 approx. 20 microM), as determined by chemical cross-linking and solid-phase binding assays. alpha-Synuclein and beta-synuclein were found to stimulate Abeta-aggregation in vitro to the same extent. The synucleins also displayed Abeta-inhibitable binding of NAC and they were capable of forming dimers. Using proteolytic fragmentation of alpha-synuclein and cross-linking to 125I-Abeta, we identified two consecutive binding domains (residues 1-56 and 57-97) by Edman degradation and mass spectrometric analysis, and a synthetic peptide comprising residues 32-57 possessed Abeta-binding activity. To test further the possible significance in pathology, alpha-synuclein was biotinylated and shown to bind specifically to amyloid plaques in a brain with Alzheimer's disease. It is proposed that the multiple Abeta-binding sites in alpha-synuclein are involved in the development of amyloid plaques.

Amyloid↗

PubMed1994

Pediatric malignant glioma with tubuloreticular inclusions and MYCN amplification. Report of a case with immunohistochemical, ultrastructural, flow cytometric, karyotypic, and Southern blot analysis.

BACKGROUND: The authors described unusual pathologic features in a left frontal lobe malignant glioma in a 31/2-year-old boy. The pathology was similar in the initial excision and two subsequent recurrences at 9 and 11 months and at autopsy, when extensive subarachnoid spread was noted. METHODS: The tumor was studied by conventional histology, immunohistochemistry, flow cytometry, transmission electron microscopy (TEM), immune electron microscopy (IEM), and cytogenetic and Southern blot analysis. RESULTS: The tumor revealed two different histologic patterns. One component showed large cells with eosinophilic cytoplasm, vesicular nuclei with prominent nucleoli, eosinophilic perinuclear inclusions, and immunoreactivity for glial fibrillary acidic protein (GFAP) and vimentin. The other component consisted of undifferentiated cells with hyperchromatic nuclei and scanty cytoplasm. By TEM, the perinuclear aggregates were composed of tubuloreticular inclusions, which were also observed in endothelial cells within the tumor vasculature. By IEM, the intermediate filaments in the tumor cell cytoplasm were decorated with GFAP. Flow cytometric results revealed a marked increase in the S-phase (48%), whereas cytogenetic analysis of short-term cultures showed an abnormal karyotype containing marker chromosomes and double minutes. In the second resection, additional karyotypic abnormalities were noted, including 1p- and several additional markers. The first and second resections showed MYCN amplification by Southern Blot analysis in the 60- to 80-fold range. CONCLUSIONS: This tumor presents unique histologic, ultrastructural, and cytogenetic findings as well as MYCN amplification that is notable for a pediatric malignant glioma. Tubuloreticular inclusions were a prominent feature in this tumor, which again is unique for a glioma.

Blotting, Southern↗

PubMed1994

Transglutaminase facilitates the formation of polymers of the beta-amyloid peptide.

One of the major pathological characteristics of Alzheimer's disease is the increased number of amyloid-containing senile plaques within the brain. The dense cores of these plaques are composed primarily of highly insoluble aggregates of a 39-43-residue peptide referred to as the beta-amyloid peptide (beta A). The mechanisms by which these insoluble extracellular deposits of beta A are formed remain unknown. In this study, the cross-linking of beta A by the calcium-dependent enzyme, transglutaminase was examined. Transglutaminases are a family of enzymes which are found in brain, and catalyse the cross-linking of specific proteins into insoluble polymers. Synthetic beta A (1-40) was readily cross-linked by transglutaminase, forming multimers in a time-dependent fashion. Furthermore, a second peptide with a substitution similar to that in the Dutch-type hereditary amyloidosis mutation (Glu22 to Gln) was also found to be a substrate for transglutaminase. Since transglutaminase covalently cross-links proteins through glutamine residues, it is suggested that transglutaminase contributes to amyloid deposition in Dutch-type hereditary amyloidosis, and possibly Alzheimer's disease.

Alzheimer Disease↗

PubMed1983

Selective binding of heat- and antigen-aggregated IgG to Blue-Sepharose. Radioimmunoassay of circulating immune complexes.

In an attempt to find a direct method for the determination of circulating immune complexes in pathological sera we studied the ability of Cibacron Blue 3GA covalently bound to Sepharose 4B to bind heat- or antigen-aggregated human IgG. Blue-Sepharose was found to bind significant amounts of aggregated IgG (about 1.5 mg/ml packed gel) but very low amounts of monomeric IgG (about 0.1 mg/ml gel). The ability of Blue-Sepharose to bind immune complexes was tested on human pathological sera containing variable amounts of circulating immune complexes. Using both a turbidimetric measurement and Barkas' method we found that Blue-Sepharose is able to remove a very high percent of circulating immune complexes (greater than 80%) from sera. Considering the above observations a new method is described for the direct determination of circulating immune complexes using Blue-Sepharose as serum adsorbent and either 125I-labeled protein A or 125I-labeled rabbit IgG anti-human IgG to demonstrate the binding of immune complexes.

Animals↗

PubMed1980

[Structural and functional bases of hemostasis and its pathology].

The multiphase process of blood coagulation occurs owing to the interaction of three links of hemostasis--thrombocyte apparatus, plasma factors, and vascular wall components--and is regulated by the neuro-reflectory mechanisms of the coagulating and anticoagulating systems. At the early stage of hemostasis, a set of reactions begins in the thrombocyte apparatus leading to cell aggregation, secretion of the content of granules, synthesis of prostaglandines and eventually to the formation of thrombocyte thrombus. Disorders in ultrastructures and membranes of thrombocytes, particularly in receptor glycoproteins lead to the development of molecular disease of thrombocyte and to the disturbance of hemostasis. Plasma factors are activated by some proteolysis reactions in the realization of which a great role is played by phospholipids; Ca ions and regulatory proteins. The key reaction of hemostasis is activation of prothrombin into thrombin. Hemostasis disorders at this stage are of congenital (factor deficiency) or acquired (vitamin K deficiency) nature and occur at the molecular level. Hemostasis is terminated by a step-wise formation of fibrin. Disorders of one of the stages of this process due to congenital abnormality of fibrinogen results in coagulation defect due to the molecular disease of fibrinogen. The problem of hemostasis is inseparable from that of the mechanism causing the living host resistance to thrombus formation. The regulation of the liquid state of the blood and its coagulated is performed by the combined functioning of the coagulating and anticoagulating systems.

Blood Coagulation↗

PubMed2006

Identification of candidate substrates for ectodomain shedding by the metalloprotease-disintegrin ADAM8.

ADAM proteases are type I transmembrane proteins with extracellular metalloprotease domains. As for most ADAM family members, ADAM8 (CD156a, MS2) is involved in ectodomain shedding of membrane proteins and is linked to inflammation and neurodegeneration. To identify potential substrates released under these pathologic conditions, we screened 10-mer peptides representing amino acid sequences from extracellular domains of various membrane proteins using the ProteaseSpot system. A soluble ADAM8 protease containing a pro- and metalloprotease domain was expressed in E. coli and purified as active protease owing to autocatalytic prodomain removal. From 34 peptides tested in the peptide cleavage assay, significant cleavage by soluble ADAM8 was observed for 14 peptides representing membrane proteins with functions in inflammation and neurodegeneration, among them the beta-amyloid precursor protein (APP). The in vivo relevance of the ProteaseSpot method was confirmed by cleavage of full-length APP with ADAM8 in human embryonic kidney 293 cells expressing tagged APP. ADAM8 cleaved APP with similar efficiency as ADAM10, whereas the inactive ADAM8 mutant did not. Exchanging amino acids at defined positions in the cleavage sequence of myelin basic protein (MBP) revealed sequence criteria for ADAM8 cleavage. Taken together, the results allowed us to identify novel candidate substrates that could be cleaved by ADAM8 in vivo under pathologic conditions.

ADAM Proteins↗

PubMed1988

Hemorheologic variables in breast cancer patients at the time of diagnosis and during treatment.

The increased risk of thrombosis seen in patients with malignancy also was recently confirmed in breast cancer patients undergoing hormonal or chemotherapeutic treatment. Besides changes within the coagulation system, alterations of hemorheologic variables have been implicated in the genesis of thrombosis. We evaluated plasma viscosity, erythrocyte aggregation, fibrinogen level, sedimentation rate, hematocrit concentration, and protein concentration in patients with breast cancer at the time of primary diagnosis and during follow-up with or without treatment. We then compared the results to a control group without malignant or infectious disease. Plasma viscosity and erythrocyte aggregation were significantly higher in patients with malignant disease, with a further increase at the time of dissemination. Plasma fibrinogen level was significantly higher only at the time of dissemination. The influence of therapy on hemorheologic variables was minor. Tumor volume was the most important factor. As individual values vary considerably and form a continuous spectrum, no cutoff line between normal and pathologic values can be defined. However, high values should induce further measures to diagnose metastatic disease. Second, these factors could explain the relative inefficiency of thrombosis prophylaxis in this patient group and suggest the addition of rheologically active drugs to the treatment regimen.

Blood Sedimentation↗

PubMed1998

Mantle cell lymphoma.

Mantle cell lymphomas comprise 2 to 8% of non-Hodgkin's lymphoma in the United States. They occur in older adults with a distinct male predominance, who present with generalized lymphadenopathy, and often have disseminated disease at the time of diagnosis. Pathologically, mantle cell lymphomas are characterized by a proliferation of small lymphocytes, with irregular nuclei, clumped chromatin, and sparse cytoplasm that can grow in nodular or diffuse patterns in lymph nodes, that localize to the splenic white pulp and that produce interstitial, paratrabecular, and intertrabecular lymphoid aggregates in the bone marrow. Phenotypically, mantle cell lymphomas are B cell neoplasms that express pan B cell lineage antigens, CD5 and CD43, and that are negative for CD10 and CD23. On a genetic level, many cases of mantle cell lymphomas have the t(11;14)(q13;q32) that causes overexpression of cyclin-D1, a protein that can be demonstrated by immunohistochemistry in many examples of mantle cell lymphoma and that can be exploited diagnostically to distinguish mantle cell lymphomas from other low-grade B cell lymphoproliferative disorders. The differential diagnosis of mantle cell lymphoma includes small B cell lymphoma, lymphoplasmacytic lymphoma, nodal, extranodal, and splenic marginal zone lymphomas, and follicular small cleaved cell lymphoma. In most instances, these disorders can be separated from one another by morphology, distinctive immunophenotypic profiles, and genetic features.

Adolescent↗

PubMed2003

Spatial and temporal distribution of intracellular free cholesterol in brains of a Niemann-Pick type C mouse model showing hyperphosphorylated tau protein. Implications for Alzheimer's disease.

Niemann-Pick type C (NPC) disease is a fatal hereditary neurovisceral disorder with diagnostically relevant intracellular accumulation of cholesterol in non-brain tissue, for example the spleen and fibroblasts. In the brain, many ballooned neurons are seen. Using filipin microfluorodensitometry, significant accumulations of free cholesterol in specified neurons have been described in NPC patients. The present study demonstrates spatial and temporal accumulation of free cholesterol in the brains of homozygous NPC (-(npc)/-(npc)) mice, a widely acknowledged mouse model, and in primarily cultured neurons therefrom. Intraneuronal storage of free cholesterol was already prominent at a pre-clinical stage in various grey matter areas of the murine cerebral cortex. Hippocampal areas showed differential development of the pathological distribution of free cholesterol. The pyramidal cells in the CA3 sector of Ammon's horn were affected much earlier than in CA1. Some of the deeper cerebral nuclei were affected only slightly, even at the final stage. Neurons (E15-E17) cultured in a cholesterol-free medium also showed massive accumulation of intracellular free cholesterol. In addition, brains from the murine NPC model for Alzheimer's disease (AD)-like changes in the microtubule-associated protein tau were tested using the Gallyas silver technique and AT8-immunolabelling, since both human diseases are accompanied by intraneuronal tangles made up of tau protein aggregations. Although the analysis failed to show classical silver-stainable tangles of the AD type in the NPC mice, tau protein phosphorylated at epitopes considered to represent early stages of AD was found. This further strengthens the concept that an alteration in cholesterol metabolism may play an important role in AD. The NPC mouse model may thus serve as a tool to analyse the role of cholesterol in initial changes of tau that eventually lead to the formation of tangles in both NPC and AD.

Alzheimer Disease↗

PubMedDate not supplied

Channel catfish, Ictalurus punctatus rafinesque, neutrophil adhesion to selected extracellular matrix proteins, lipopolysaccharide, and catfish serum.

Adhesion of leukocytes to endothelium and extracellular matrix proteins is an important step in the inflammatory process. Therefore, the adhesion of channel catfish neutrophils to a surface coated with extracellular matrix proteins, LPS, and non-immune catfish serum was evaluated. Stimulation of neutrophils with phorbol dibutyrate (PDBU) resulted in at least two-fold increases in cellular adhesion to all substrates tested except laminin. When EDTA was included during or after PDBU stimulation, neutrophil adhesion to fibrinogen and LPS coated surfaces was reduced to the level of unstimulated neutrophils or to 50-60% of that for stimulated neutrophils. Similarly, EDTA and Ca2+/Mg2+ deficient medium reduced homotypic aggregation of PDBU stimulated neutrophils to background levels. Adhesion of stimulated neutrophils to fibrinogen coated surfaces was inhibited 44, 33, and 50% when soluble fibrinogen, fibronectin, and serum, respectively, were used to block the adhesion assay. The tripeptide integrin adhesion recognition sequence, Arg-Gly-Asp (RGD), caused 83% reduction and the fibrinogen-binding inhibitor protein caused 10% reduction in binding of stimulated neutrophils to fibrinogen coated surfaces. Two hexapeptides tested did not reduce neutrophil adhesion to fibrinogen. The binding of channel catfish neutrophils to the matrices used in the present study is suggestive that integrin mediated adhesion occurs during biological and pathological processes of teleosts.

Animals↗

PubMed2004

Domain swapping in N-truncated human cystatin C.

Human cystatin C (HCC) inhibits papain-like cysteine proteases by a binding epitope composed of two beta-hairpin loops and the N-terminal segment. HCC is found in all body fluids and is present at a particularly high level in the cerebrospinal fluid. Oligomerization of HCC leads to amyloid deposits in brain arteries at advanced age but this pathological process is greatly accelerated with a naturally occurring Leu68Gln variant, resulting in fatal amyloidosis in early adult life. When proteins are extracted from human cystatin C amyloid deposits, an N-terminally truncated cystatin C (THCC) is found, lacking the first ten amino acid residues of the native sequence. It has been shown that the cerebrospinal fluid may cause this N-terminal truncation, possibly because of disintegration of the leucocytes normally present in this fluid, and the release of leucocyte proteolytic enzymes. HCC is the first disease-causing amyloidogenic protein for which oligomerization via 3D domain swapping has been observed. The aggregates arise in the crystallization buffer and have the form of 2-fold symmetric dimers in which a long alpha-helix of one molecule, flanked by two adjacent beta-strands, has replaced an identical domain of the other molecule, and vice versa. Consistent with a conformational change at one of the beta-hairpin loops of the binding epitope, the dimers (and also any other oligomers, including amyloid aggregates) are inactive as papain inhibitors. Here, we report the structure of N-truncated HCC, the dominant form of cystatin C in amyloid deposits. Although the protein crystallized under conditions that are drastically different from those for the full-length protein, the structure reveals dimerization by the same act of domain swapping. However, the new crystal structure is composed of four independent HCC dimers, none of which has the exact 2-fold symmetry of the full-length dimer. While the four dimers have the same overall topology, the exact relation between the individual domains shows a variability that reflects the flexibility at the dimer-specific open interface, which in the case of 3D domain-swapped HCC consists of beta-interactions between the open hinge loops and results in an unusually long intermolecular beta-sheet. The dimers are engaged in further quaternary interactions resulting in spherical, closed octameric assemblies that are identical to that present in the crystal of the full-length protein. The octamers interact via hydrophobic patches formed on the surface of the domain-swapped dimers as well as by extending the dimer beta-sheet through intermolecular contacts.

Cystatin C↗

PubMed2004

Differential involvement and heterogeneous phosphorylation of tau isoforms in progressive supranuclear palsy.

We found previously that aggregated insoluble tau protein in progressive supranuclear palsy (PSP) brains exhibits a heterogeneous pattern that is not segregated by the type of clinical presentation. Here we have investigated tau isoform composition from 20 PSP cases and found marked variation between different brains. Cases were classified into three groups, each comprising essentially of (1) 1N4R; (2) 1N4R and 1N3R; or (3) 1N4R, 1N3R and 0N4R tau isoforms. There was also an absence of a simple relationship between isoform composition and the pattern of insoluble tau before dephosphorylation. We conclude that there is distinct molecular heterogeneity in the involvement of tau isoforms in the tau pathology in PSP.

Aged↗

PubMed2000

Characterization of the Schizosaccharomyces pombe orthologue of the human survival motor neuron (SMN) protein.

Childhood onset spinal muscular atrophy (SMA) is a common autosomal recessive disorder primarily characterized by the loss of lower alpha motor neurons. The underlying chromosomal defects causing SMA have been found in the survival motor neuron (SMN) gene. SMN has been shown previously to play a role in both snRNP biogenesis and mRNA processing, although direct evidence for the relationship between SMN and disease pathology has not been elucidated. SMN orthologues have been isolated in many species including Caenorhabditis elegans and Danio rerio. To study the function of SMN, we have identified and characterized the Schizosaccharomyces pombe orthologue of human SMN, smn1 (+). We have demonstrated that smn1 (+) is essential for viability in S.pombe and yeast expressing missense mutations in Smn1p, which mimic mutations in patients with Type I SMA, show significant mislocalization of the protein and a decrease in cell viability. Wild-type Smn1p is localized predominantly in the nucleus whereas yeast expressing Smn1p with missense mutations or deletions of specific domains of the protein accumulate cytoplasmic aggregates. Overexpression of Smn1p results in an increase in the growth rate of cells. Furthermore, mutations within two highly conserved protein interaction domains have a dominant-negative effect on growth, indicating that each domain is of functional significance in S.pombe. These dominant phenotypes can be suppressed by overexpression of murine Smn in the same cell. Given the structural and functional similarities between the protein in fission yeast and higher eukaryotes, S.pombe will be an ideal organism to study the role of SMN in RNA processing.

Amino Acid Sequence↗