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At least 1,189 records · Page 66Linked to original sources
PubMed2006

Small heat shock proteins protect against alpha-synuclein-induced toxicity and aggregation.

Protein misfolding and inclusion formation are common events in neurodegenerative diseases, such as Parkinson's disease (PD), Alzheimer's disease (AD) or Huntington's disease (HD). Alpha-synuclein (aSyn) is the main protein component of inclusions called Lewy bodies (LB) which are pathognomic of PD, Dementia with Lewy bodies (DLB), and other diseases collectively known as LB diseases. Heat shock proteins (HSPs) are one class of the cellular quality control system that mediate protein folding, remodeling, and even disaggregation. Here, we investigated the role of the small heat shock proteins Hsp27 and alphaB-crystallin, in LB diseases. We demonstrate, via quantitative PCR, that Hsp27 messenger RNA levels are approximately 2-3-fold higher in DLB cases compared to control. We also show a corresponding increase in Hsp27 protein levels. Furthermore, we found that Hsp27 reduces aSyn-induced toxicity by approximately 80% in a culture model while alphaB-crystallin reduces toxicity by approximately 20%. In addition, intracellular inclusions were immunopositive for endogenous Hsp27, and overexpression of this protein reduced aSyn aggregation in a cell culture model.

Animals↗
PubMed2000

Nicotine enhances the biosynthesis and secretion of transthyretin from the choroid plexus in rats: implications for beta-amyloid formation.

Epidemiological studies indicated that cigarette smoking protects against the development of several neurodegenerative disorders, including Alzheimer's disease (AD). However, the molecular mechanism(s) underlying this is poorly understood. To gain insight into these protective effects, we used differential display PCR (DD-PCR) to amplify RNA from various brain regions of rats self-administering (SA) nicotine compared with yoked-saline controls. We found that the transthyretin (TTR) gene, whose product has been shown to bind to amyloid beta (Abeta) protein and prevent Abeta aggregation, was more abundantly expressed ( approximately 1.5- to 2.0-fold) in the brainstem and hippocampus (areas containing choroid plexus) of nicotine SA rats. Subsequently, quantitative reverse transcription-PCR analysis confirmed these DD-PCR findings and demonstrated that nicotine increased TTR mRNA levels in these regions in a time- and dose-dependent manner. Significantly higher TTR protein concentrations were also detected in the ventricular CSF of nicotine-treated rats. In contrast, no differences either in plasma TTR or in CSF and plasma retinol-binding protein were detected. Immunohistochemical analysis showed that immunoreactive TTR was 41.5% lower in the choroid plexus of nicotine-treated rats compared with the saline controls. On the basis of these data, we speculate that the protective effects of nicotine on the development of AD may be attributable, in part, to the increased biosynthesis and secretion of TTR from the choroid plexus. These findings also point toward new approaches that may take advantage of the potentially novel therapeutic effects of nicotinic agonists in patients with AD.

Amyloid beta-Peptides↗
PubMed1978

The ribosomes of Plasmodium berghei: isolation and ribosomal ribonucleic acid analysis.

Ribosomes and high molecular weight ribosomal ribonucleic acid (rRNA) from the blood stages of Plasmodium berghei parasites were studied in preparations free from host ribosome contamination. Purified malarial ribosomes were isolated in high yield from a population of ultrastructurally intact, viable parasites by hypertonic lysis with Triton X-100 and differential centrifugation. These ribosomes were shown to be derived from active polysomes and could be dissociated into subunits by puromycin-0.5 M KCl treatment. Malarial rRNA extracted from purified 40S and 60S ribosomal subunits was characterized by electrophoretic, sedimentation and base ratio analyses. Like certain other protozoa, the P. berghei 40S ribosomal subunit possessed an exceptionally large RNA species (mol. wt 0.9 X 10(6), while RNA isolated from the parasite's 60S subunit (mol. wt 1.5 X 10(6)) was specifically 'nicked' to produce one large component (mol.wt 1.2 X 10(6)) and one small component (mol.wt 0.3 X 10(6)) in equimolar quantities. These rRNA's migrate identically on polyacrylamide gels after heating to 63 degrees C for 5 min or under denaturing conditions in the presence of formamide, indicating an absence of aggregation and non-specific degradation of the rRNA species. Base composition studies showed P. berghei rRNA to be low in guanosine and cytosine content, as is the case for protozoa generally.

Animals↗
PubMed1980

Purification of mouse immunoglobulin heavy-chain messenger RNAs from total myeloma tumor RNA.

A procedure is described for the large-scale purification of light (L) and heavy (H) chain mRNAs from plasmacytomas produced in mice. Intact RNA is selectively precipitated in high yield from frozen tumors homogenized in 3 M LiCl and 6 M urea. L and H-chain mRNAs were purified by oligo(dT)-cellulose chromatography and either sucrose gradient centrifugation in conditions preventing aggregation or by means of high-resolution preparative gel electrophoresis under non-denaturing conditions. gamma 2a and alpha H-chain mRNAs sedimented as major components at 15.5 S and 16.5 S respectively, when L-chain mRNAs sedimented as 12-S species. H-chain mRNAs isolated by continuous elution during preparative gel electrophoresis were completely separated from both L-chain mRNA and residual 18-S rRNA, and migrated as single components of 1900 +/- 50 nucleotides on analytical denaturing gels. The partially purified H-chain mRNAs were translated into major components of molecular weights of 56,000 (gamma 2a) and 60,000 (alpha) in an mRNA-dependent rabbit reticulocyte lysate, whereas L-chain mRNAs yielded polypeptides of molecular weights of 25,000 (gamma) and 27,000 (chi). Up to 95% of the translation products directed by the purified mRNAs were immunoprecipitated using specific antisera. The purity of L and H-chain mRNAs was assessed by hybridization of corresponding cDNAs with excess recombinant plasmid DNA. The results indicated a minimum purity of 47% (gamma 2a), 62% (alpha), for H-chain mRNAs and 60% (chi), for L-chain mRNAs.

Animals↗
PubMed1990

Albumin and collagen mRNA expression in normal and analbuminemic rodent liver: analysis by in situ hybridization using biotinylated probes.

We used in situ nucleic acid hybridization cytochemistry to examine cell types and subcellular sites expressing albumin (alb) or pro alpha 2 collagen (col) mRNA in livers from normal and analbuminemic rodents. Biotinylated cDNA or RNA probes were applied to aldehyde-fixed, non-frozen sections and the resulting DNA-RNA or RNA-RNA hybrids were subsequently visualized by enzymatic detection of either peroxidase or alkaline phosphatase conjugated to anti-biotin IgG or streptavidin. In normal rat liver, alb mRNA was expressed in all hepatocytes and was localized to discrete subcellular structures distributed as aggregates in the cytoplasm and in specific structures encircling the nucleus; these subcellular structures most likely represent the endoplasmic reticulum and nuclear envelope. In mouse liver, pro alpha 2 col mRNA was identified in a subpopulation of sinusoidal lining cells which have the morphological appearance of lipocytes. In liver from analbuminemic rats, a small number of hepatocytes, distributed throughout the hepatic lobule, expressed alb mRNA at high levels; the subcellular distribution of this alb mRNA was essentially identical to that observed in normal rat hepatocytes. Since non-radioactive in situ hybridization detected mRNA within the boundaries of individual cells and showed its precise subcellular location under conditions in which there was excellent preservation of tissue morphology, this procedure should be useful for a wide variety of histopathologic studies.

Albumins↗
PubMed1989

A developmentally regulated gene product from Dictyostelium discoideum shows high homology to human alpha-L-fucosidase.

A cDNA library of poly(A+)-RNA has been prepared from membrane-bound polysomes of Dictyostelium discoideum and screened for clones hybridizing to mRNA species that encode developmentally regulated proteins. The clone investigated in this paper recognizes a 1.8 kb transcript that accumulates strongly between the growth phase and aggregation stage. Stimulation of cells with pulses of cAMP enhances the accumulation. The amino acid sequence derived from a complete cDNA and from a genomic clone displays extensive sequence identity to human liver alpha-L-fucosidase. The D. discoideum DNA sequence encodes a 50.5 kDa polypeptide with a hydrophobic signal peptide at the N-terminus. Antibodies against a synthetic peptide corresponding to amino acids 262-275 of the deduced protein sequence recognize a developmentally regulated 50 kDa protein in D. discoideum that is recovered in the particulate fraction.

Amino Acid Sequence↗
PubMed1991

Differential expression of VLA-4 integrin by resident and peripheral blood B lymphocytes. Acquisition of functionally active alpha 4 beta 1-fibronectin receptors upon B cell activation.

Very-late antigen (VLA)-4(CD49d/CD29) constitutes the only member of the beta 1 integrin family that plays a role in the interaction of lymphoid cells with both extracellular matrix and endothelial cells through two identified ligands: fibronectin (FN) and VCAM-1, respectively. The expression and functional activity of VLA-4 has been studied in different maturation and activation stages of B cells from several cellular compartments. Resident B lymphocytes of different lymphoid organs were almost negative for VLA-4 as detected by both immunoperoxidase staining and flow cytometry analysis. However, a high expression of both chains of this heterodimer was observed when tonsillar B cells were activated in vitro with different stimuli, such as phorbol esters or Staphylococcus aureus Cowan I (SAC). Both nonactivated and in vitro activated B cells from peripheral blood constitutively expressed high levels of this surface antigen. The induced expression of VLA-4 after activation of tonsillar B lymphocytes was accompanied by the acquisition of the capacity to bind to a 38-kDa proteolytic fragment, containing the connecting segment I domain, of FN. Interestingly, nonactivated peripheral blood B cells were unable to attach to this FN fragment, in spite of their constitutive expression of VLA-4, and only acquired this functional capacity after cell activation with phorbol esters and SAC. This FN-binding acquisition was not affected by preincubation with inhibitors of protein and RNA synthesis. These results underline that the FN-binding activity of VLA-4 is dependent on processes affecting cellular activation as described for other members of the integrin family. By contrast, VLA-4-mediated homotypic aggregation of peripheral blood B cells could be triggered by anti-alpha 4 monoclonal antibodies independently of the cell activation state.

B-Lymphocytes↗
PubMed1997

Multiple cadherins are expressed in human fibroblasts.

Although the cell-cell adhesiveness of fibroblasts is thought to be related to wound healing, the molecular basis of this adhesiveness is still unknown. We isolated five kinds of cadherin fragments from the cDNA of human fibroblasts by polymerase chain reaction (PCR). Two of the five were known cadherins: PC43, a protocadherin containing six cadherin repeats in the extracelluar domain, and human Fat, which is the human homologue of the Drosophila tumor suppressor Fat. The other three were novel cadherin fragments, and we named them cadherins FIB1, FIB2, and FIB3. The expressions of cadherins including E-, P-, and N-cadherin, PC43, human Fat, and cadherins FIB1, FIB2, and FIB3 were compared in human fibroblasts, human melanocytes, and human epidermal keratinocytes. The latter six cadherins were expressed in human fibroblasts, and cadherins FIB1 and FIB2 were fibroblast-specific. These results suggest that diverse cadherin molecules may contribute to the cell-cell adhesion in human fibroblasts.

Amino Acid Sequence↗
PubMedDate not supplied

Cell wall proteins of the ectomycorrhizal basidiomycete Pisolithus tinctorius: identification, function, and expression in symbiosis.

Specific cell-cell and cell-substrate interactions direct the growth of ectomycorrhizal fungi to their host root targets. These elaborate mechanisms lead to the differentiation of distinct multihyphal structures, the mantle, and the Hartig net. In the ectomycorrhizal basidiomycete Pisolithus tinctorius, the use of two-dimensional gel electrophoresis, immunocytochemical microscopy, and RNA blot analysis has demonstrated the differential expression of cell wall proteins (CWPs), such as hydrophobins, adhesins, and mannoproteins, during symbiotic interaction. In other fungi, these CWPs have been suggested to play a role in hyphae aggregation, intracellular signaling cascades, and cytoskeletal changes. The recent cloning of the genes for several of these CWPs in P. tinctorius allows us to address their function in symbiosis. This review summarizes our knowledge of CWPs in P. tinctorius and considers parallels with other biotrophic fungi as a possible framework for future work.

Amino Acid Sequence↗
PubMed1987

Oncogene expression in differentiating F9 mouse embryonal carcinoma cells.

Differentiation of F9 mouse embryonal carcinoma cells in culture is accompanied by a decrease in growth rate and loss of tumorigenicity. Cells differentiating in monolayer culture (to parietal endoderm-type cells) or in aggregates (to visceral endoderm-type cells) show qualitatively similar changes in transcript levels from several c-oncogenes. In contrast with other studies with F9 cells, we find an early decrease in c-myb RNA but not in c-myc RNA. This and a later increase in c-src RNA may be associated with decreasing cell growth rate. Before differentiation, induction and maintenance of elevated c-abl RNA levels depend on the presence of retinoic acid in the medium. After differentiation c-abl RNA levels decline only partially when retinoic acid is removed. Increased RNA from c-fos is seen late in differentiation in monolayer cultures only, a change also seen with appearance of similar endoderm cell types in the developing mouse embryo.

Animals↗
PubMed1992

Isolation and characterization of hamster brain polyribosome-cytomatrix complexes.

We have developed a method for the isolation of a brain subcellular fraction enriched in both highly aggregated polyribosomes and cytoskeletal proteins. This method is based on gentle dispersion of brain tissue and low speed centrifugation. This fraction is enriched in typical cytoskeletal proteins as glial fibrillary protein, neurofilament proteins and actin. Messenger RNA did not seem to be involved in the polyribosome association to the cytomatrix as shown by the effect of exposure to micrococcal nuclease. On the other hand, in vivo disruption of protein synthesis by acute experimental phenylketonuria, hypothermia or heat-shock did not cause the release of ribosomes from the cytomatrix.

Actins↗
PubMed2001

Suppression of a sialyltransferase by antisense DNA reduces invasiveness of human colon cancer cells in vitro.

Transfer of terminal alpha 2,6-linked sialic acids to N-glycans is catalyzed by beta-galactoside alpha 2,6-sialyltransferase (ST6Gal I). Expression of ST6Gal I and its products is reportedly increased in colon cancers. To investigate directly the functional effects of ST6Gal I expression, human colon cancer (HT29) cells were transfected with specific antisense DNA. ST6Gal I mRNA and protein were virtually undetectable in six strains of transfected HT29 cells. ST6Gal activity was reduced to 14% of control (P<0.005) in transfected cells. Expression of terminal alpha 2,6- and alpha 2,3-linked sialic acids, and unmasked N-acetyllactosamine oligosaccharides, respectively, was assessed using flow cytometry and fluoresceinated Sambucus nigra, Maackia amurensis and Erythrina cristagalli lectins. Results indicated a major reduction in expression of alpha 2,6-linked sialic acids and counterbalancing increase in unmasked N-acetyllactosamines in antisense DNA-transfected cells, without altered expression of alpha 2,3-linked sialic acids or ganglioside profiles. The ability of transfected cells to form colonies in soft agar and to invade extracellular matrix material (Matrigel), respectively, in vitro was reduced by approx. 98% (P<0.0001) and more than 3-fold (P<0.005) compared to parental HT29 cells. These results indicate that N-glycans bearing terminal alpha 2,6-linked sialic acids may enhance the invasive potential of colon cancer cells.

Cell Aggregation↗
PubMed1976

Fine structural observations on oocyte development in monogeneans.

The ultrastructural changes accompanying oocyte differentiation in the ovaries of the monogeneans, Diclidophora merlangi, Diplozoon paradoxum and Calicotyle kröyeri have been described. In each case, oogenesis in the ovary proceeds as far as meiotic prophase in the primary oocyte. A three-stage sequence of development based on oocyte morphology is proposed: (1) Oogonia and early, immature primary oocytes are typically undifferentiated, with chromatin-laden nuclei occupying most of the cell volume. The cytoplasm contains small clumps of mitochondria and unattached ribosomal aggregates. There is evidence of mitosis and, in later stages, meiotic prophase is indicated by the appearance of nuclear synaptonemal complexes. (2) Maturing primary oocytes are characterized by increased nucleolar volume associated with the production of RNA for export to the cytoplasm. An organized GER and Golgi apparatus are established and involved in the synthesis and packaging of membrane-limited cortical granules. Annulate lamellae and nucleolus-like bodies appear in the cytoplasm and, with development, the cells increase in size and, peripherally, become interdigitated. (3) Mature primary oocytes represent a resting phase when cellular activity is minimal. Golgi disappear and the ER fragments or becomes reduced in dimensions. Mitochondria and free ribosomes are numerous and cortical granules move to the cell periphery. The cells separate and, when mature, are released from the ovary. There are minor species differences in oocyte ultrastructure and development.

Animals↗
PubMed1999

Origin of the integrin-mediated signal transduction. Functional studies with cell cultures from the sponge Suberites domuncula.

Sponges (phylum Porifera) represent the phylogenetically oldest metazoan animals. Recently, from the marine sponge Geodia cydonium a first cDNA encoding a putative integrin receptor molecule was isolated. In the present study basic functional experiments have been conducted to test the hypothesis that in sponges integrin polypeptides also function as adhesion molecules and as outside-in signaling molecules. The sponge Suberites domuncula has been used for the experiments because from this sponge only has a cell culture been established. Here we report that aggregation factor (AF)-mediated cell-cell adhesion is blocked by the RGDS peptide which is known to interact with beta integrin. Both RGDS and AF were found to stimulate DNA synthesis within 24 h. The beta subunit of the integrin receptor was cloned from S. domuncula; the estimated 91-kDa molecule comprises the characteristic signatures. Evolutionary conservation of the beta integrin was assessed by comparison with corresponding beta integrin subunits from evolutionary higher metazoan taxa. Addition of RGDS or of AF to isolated cells of S. domuncula causes a rapid (within 1-2 min) increase in the intracellular Ca2+ concentration which is further augmented in the presence of Ca2+. Furthermore, incubation of the cells with RGDS or AF causes an activation of the GTP-binding protein Ras. In addition it is shown that after a prolonged incubation of the cells with RGDS and AF the expression of the genes coding for Ras and for calmodulin is upregulated. These results suggest that the integrin receptor functions in the sponge system not only as adhesion molecule but also as a molecule involved in outside-in signaling.

Amino Acid Sequence↗
PubMed2004

Proteasome inhibition and aggregation in Parkinson's disease: a comparative study in untransfected and transfected cells.

Dysfunction of the ubiquitin-proteasome system (UPS) has been implicated in Parkinson's disease (PD) and other neurodegenerative disorders. We have investigated the effect of UPS inhibition on the metabolism of alpha-synuclein (SYN) and parkin, two proteins genetically and histopathologically associated to PD. Pharmacological inhibition of proteasome induced accumulation of both parkin and SYN in transfected PC12 cells. We found that this effect was caused by increased protein synthesis rather than impairment of protein degradation, suggesting that inhibition of the UPS might lead to non-specific up-regulation of cytomegalovirus (CMV)-driven transcription. To investigate whether endogenous parkin and SYN can be substrate of the UPS, untransfected PC12 cells and primary mesencephalic neurones were exposed to proteasome inhibitors, and parkin and SYN expression was evaluated at both protein and mRNA level. Under these conditions, we found that proteasome inhibitors did not affect the level of endogenous parkin and SYN. However, we confirmed that dopaminergic neurones were selectively vulnerable to the toxicity of proteasome inhibitors. Our results indicate that studies involving the use of proteasome inhibitors, particularly those in which proteins are expressed from a heterologous promoter, are subjected to potential artefacts that need to be considered for the interpretation of the role of UPS in PD pathogenesis.

Animals↗