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At least 487 records · Page 27Linked to original sources
PubMed1995

Deletion detection in the dystrophin gene by multiplex gap ligase chain reaction and immunochromatographic strip technology.

The purpose of this study is to demonstrate the value of a multiplex amplification and readout system. The validation was done using as a model system the detection of deletions in nine possible dystrophin exons: 4, 8, 12, 17, 19, 44, 45, 48, and 51. The amplification system was gap ligase chain reaction, adapted to amplify selected regions of multiple exons simultaneously. The amplified products were read out with an immunochromatographic methodology, adapted from that used in the Abbott product line commercialized under the name Test Pack Plus. In each amplification, the beta-globin gene was incorporated and served as a procedural control. The complete process takes < 3 hr from DNA sample to result. The procedure is therefore rapid and simple, as well as being potentially very cost effective. The combination of these two technologies is shown to be a useful tool for the determination of deletions in the nine exons of the dystrophin gene. The results of a 100-patient sample study showed concordance with cDNA and PCR in current use. Equivalent performance at two sites was shown.

Base Sequence↗
PubMed1985

Characterization of a membrane-associated glycoprotein complex implicated in cell adhesion to fibronectin.

We have characterized a 140-kDa glycoprotein complex purified by a monoclonal antibody and implicated in cell adhesion to the extracellular molecule fibronectin. Three major polypeptide components were purified by monoclonal antibody JG22E, which had apparent molecular weights of 155,000 (band 1), 135,000 (band 2), and 120,000 (band 3). In two-dimensional gel electrophoresis, each subunit migrated as either a broad band or a series of spots at acidic isoelectric points. After treatment with neuraminidase, the spots became focused around pH 6.2 (band 1), pH 5.6 (band 2), and pH 5.3 (band 3). These three major bands were compared by two-dimensional peptide mapping in a series of pairwise combinations and were found to be distinct proteins. In sucrose gradients, these proteins co-migrated as a complex sedimenting at approximately 8.4 S either before or after affinity purification, whereas separated subunits migrated at 4.7 to 5.8 S. Amino acid analysis revealed no detectable hydroxyproline and a composition characterized by a substantial number of cysteine residues compared to the average protein. Our results suggest that a noncovalent complex of structurally distinct glycoproteins is involved in adhesive interactions of fibronectin with cells.

Amino Acids↗
PubMed1986

Expression of epidermal growth factor receptor and associated glycoprotein on cultured human brain tumor cells.

The expression of epidermal growth factor (EGF-R) in normal glial and glioma cells grown in culture was examined by using several independent assays. Immunoprecipitation with the monoclonal antibody R1 of extracts from metabolically labeled glial and glioma cells revealed a protein of Mr approximately 170,000, with a migration in sodium dodecyl sulfate-polyacrylamide gels identical to the EGR-R of A431 epidermal carcinoma cells. Furthermore, in the majority of glioma extracts, a protein of Mr approximately 190,000 was specifically immunoprecipitated by this antibody. Similar results were obtained by immunoblotting with a second antibody directed against a synthetic peptide in the sequence of the v-erb-B oncogene. In cell lines expressing both proteins, each was specifically phosphorylated on tyrosine in immune complex kinase assays. The majority of glioma cells bound between 40,000 to 80,000 125I-labeled epidermal growth factor molecules per cell. These results suggest that the expression of EGF-R is common in cultured human glioma cells. In addition, a structurally related protein, is expressed in some of these cells.

Brain Neoplasms↗
PubMed1991

Binding of glucocorticoid receptors to model DNA response elements.

DNA sequences were synthesized that contained the consensus 15-base pair glucocorticoid receptor binding site linked to flanking sequences of various lengths. Binding of these synthetic oligomers to glucocorticoid receptor, employing a reconstituted binding system with purified components, indicated that a minimal size of approximately 45 base pairs was necessary to bind the receptor optimally. Sequences containing multiple receptor binding sites competed more effectively for binding. These findings are consistent with recent demonstrations that multiple control elements act synergistically to affect transcriptional control by glucocorticoids and confirm that regions flanking the consensus GRE binding site are instrumental in optimizing binding interactions.

Animals↗
PubMed1994

Differential partition of anticoagulant heparan sulfate proteoglycans synthesized by endothelial and fibroblastic cell lines.

The heparan sulfate proteoglycans that bind and activate antithrombin III (aHSPGs) are synthesized by endothelial cells as well as other nonvascular cells. We determined the amounts of cell surface-associated and soluble aHSPGs generated by the rat fat pad endothelial (RFP) cell line and the fibroblast (LTA) cell line. The RFP cells exhibit higher levels of cell surface-associated aHSPGs as compared to LTA cells, whereas LTA cells release larger amounts of soluble aHSPGs as compared to RFP cells. After confluence RFP cells show an increase in both cell surface-associated and soluble aHSPGs. In contrast, postconfluent LTA cells maintain a constant level of cell surface-associated and soluble aHSPGs. These observations indicate that different cell types can preferentially accumulate aHSPGs as cell surface-associated or soluble forms which could reflect alternate biological functions.

Animals↗
PubMed1991

A filter affinity transfer method for the analysis of rheumatoid factors.

Transfer of serum proteins separated by thin layer agarose electrophoresis onto nitrocellulose sheets precoated with purified human polyclonal IgG followed by revelation with enzyme-coupled anti-mu or anti-alpha antisera resulted in the specific detection of rheumatoid factors (RF) belonging to the IgM or IgA classes. Mono- or polyclonality of such RF can be evaluated from the patterns of the blots (sharp bands). In addition, their light chain type can be determined using affinity filters coated with a gamma heavy chain disease protein or with IgG Fc fragments. This simple and rapid procedure allows an easy characterization of monoclonal RF, even if they are present in minute amount amongst polyclonal RF as in certain sera from rheumatoid arthritis patients.

Antibodies, Monoclonal↗
PubMed1987

Coordinate expression of c-myc, c-myb, and histone H4 genes in reversibly differentiating HL 60 cells.

The expression of oncogenes c-myc and c-myb in human leukemic cells HL 60 was compared to the expression of histone H4 gene, known to be cell-cycle dependent. Steady-state levels of mRNA transcribed from these genes were determined by simultaneous hybridization of Northern transfers with four probes, and the rates of gene expression were measured by nuclear transcription ("run-on") assays. Expression of genes c-myc, c-myb and histone H4 varied coordinately and in parallel with the rates of DNA synthesis, while the rates of total and ribosomal RNA synthesis, the expression of gene c-Ha-ras, unrelated to proliferation of these cells, and gene p 72, a constitutively expressed human gene, were unchanged. Further, the levels of c-myc and c-myb mRNA but not p 72 mRNA were higher in cell populations enriched for S phase cells. Thus, transcription of genes c-myc and c-myb in HL 60 cells appears to be linked to DNA replication in a manner previously demonstrated for core histone gene expression.

Autoradiography↗
PubMed1987

Fusion-mediated microinjection of active amine and diamine oxidases into cultured cells: effect on protein and DNA synthesis in chick embryo fibroblasts and in glioma cells.

Serum amine oxidase and/or porcine kidney diamine oxidase were trapped within reconstituted Sendai virus envelopes, and retained their activity. The trapped enzymes that were detected by radioimmunoblots were microinjected into cultured cells by fusion. When diamine oxidase was microinjected into cultured fibroblasts of chick or rat embryos, a temporary arrest in protein and DNA synthesis was observed. The inhibitory effect was more significant when both serum amine oxidase and kidney diamine oxidase were microinjected into those cultured cells. Fibroblasts of either chick or rat embryos transformed by Rous sarcoma virus were more susceptible to the injected enzymes than the normal cultures, showing a complete arrest in protein and DNA synthesis within 4 hours. Similar results were obtained by microinjecting diamine oxidase into cultured glioma cells. The injected enzyme catalyzed the oxidation of intracellular polyamines. The resulting oxidation product (hydrogen peroxide and aminoaldehydes) apparently caused the arrest in the synthesis of macromolecules.

Amine Oxidase (Copper-Containing)↗
PubMed1987

Expression of granulocyte colony-stimulating factor by human cell lines.

We have isolated and expressed a cDNA clone that encodes a human granulocyte colony-stimulating factor from the MIA PaCa-2 cell line. A genomic clone of this factor has been isolated from the CHU-2 cell line and is reported to encode two alternative transcripts [The EMBO J. 5,575, 1986]; one transcript predicts an amino acid sequence identical to that predicted by our MIA PaCa-2 cDNA clone; the other transcript predicts a similar protein containing a three amino acid residue insertion. To investigate which types of this colony-stimulating factor are produced by other cell lines, we used specific oligonucleotides to determine which types of transcripts were present in MIA PaCa-2, 5637, and LD-1 cells, all of which have been reported to produce a factor that can stimulate the growth of predominantly granulocyte colonies in human bone marrow cell cultures. Northern analysis with these probes revealed MIA PaCa-2-like transcripts in all of these cell lines and failed to detect transcripts that would encode the colony-stimulating factor that contained the three-amino-acid-residue insertion.

Base Sequence↗
PubMed1995

Different patterns of deactivation of chemotaxis and haptotaxis of human peripheral blood mononuclear leukocytes by soluble and surface-bound attractants.

Soluble mediators and inducible cell-surface and matrix-bound molecules coordinate the cascade of events giving rise to leukocyte emigration. Knowledge of the specific mechanisms underlying the attraction of cells into a local site, however, remains sketchy. In particular, it is unclear how chemoattractants cause rapidly moving immune cells to adhere to the blood vessel wall and to enter tissues. Here we show that the neuroendocrine human growth hormone, a chemoattractant for monocytes and lymphocytes in vitro, promotes haptotaxis, the migration of the cells induced by surface-bound gradients. Combination of soluble growth hormone with soluble attractants, RANTES or formyl peptide, deactivates the migratory responses, as do combinations of surface-bound growth hormone with surface-bound RANTES or formyl peptide. In contrast, exposure of mononuclear leukocytes to combinations of soluble chemotactic with surface-bound haptotactic gradients of attractants does not deactivate migration. The findings suggest that growth hormone may act as haptotactic agent, on the one hand, and that soluble attractants do not appear to affect haptotaxis when acting in concert with a surface-bound attractant, on the other. This observation may have implications for the differential regulation of leukocyte accumulation in the vessel wall at systemic and local sites.

Amino Acid Sequence↗
PubMed1986

Hepatitis B virus DNA in leukocytes of patients with hepatitis B virus-associated liver diseases.

In order to determine the relationship between hepatitis B virus (HBV) infection of human white blood cells and different forms of HBV-associated liver diseases, we tested for HBV DNA in the sera and leukocytes of 11 healthy individuals without any serological markers of HBV infection and 91 patients with HBV infection and other gastrointestinal and urinary diseases by dot and Southern blot hybridization. HBV DNA was found in leukocytes of chronic HBV carriers, in acute and chronic hepatitis, and in patients with liver cirrhosis and hepatocellular carcinoma. Between 27 and 50% of individuals in different categories of patients examined were positive for leukocyte HBV DNA. HBV DNA was also detected in the sera of some of these patients but was absent in others. Serum HBV DNA-positive rates seemed to be highest in hepatitis B e antigen-positive asymptomatic carriers (8/10, 80%), and tended to drop to lower levels as the disease progressed to liver cirrhosis (0/8) while leukocyte HBV DNA-positive rates were highest in patients with cirrhosis (4/8, 50%). The results also show that in individuals who were serologically negative for hepatitis B surface antigen (HBsAg) and positive for antibodies to HBsAg and/or HBcAg, HBV DNA was absent in most of the sera (27/28, 96%) but it was present in leukocytes of some of these patients (7/28, 25%). In control experiments with 11 healthy individual, HBV DNA was not detected in either sera or leukocytes. In all the cases with leukocyte HBV DNA, the HBV DNA molecules were present in free forms with discrete sizes. The exceptions were a case of liver cirrhosis and a case of chronic hepatitis with possible HBV sequence integration into high molecular weight cellular DNA. Since HBV does infect human leukocytes, it may perhaps interfere with the immunological functions of the white blood cells, and thus play an important role in the pathogenesis of HBV-induced liver disease.

Carcinoma, Hepatocellular↗
PubMed1988

Characterization of BK virus-specific antibodies in human sera by Western immunoblotting: use of a zwitterionic detergent for restoring the antibody-binding capacity of electroblotted proteins.

After initial problems related to denaturation of antigenic epitopes, we developed a Western immunoblotting method for the characterization of antibodies reacting with BK virus (BKV) structural polypeptides. When a zwitterionic detergent, Empigen BB, was added to the running buffer during electroblotting, the antibody-binding capacity of electrophoretically separated BKV polypeptides was partially restored. Antibodies reacting with different BKV antigens were detected and visualized by biotinylated anti-species-specific antibodies, peroxidase-conjugated streptavidine, and diaminobenzidine staining. Human sera containing anti-BKV antibodies reacted with VP1, but a serum containing antinuclear antibodies also reacted with VP4, -5 and -6 (histones). Serum from a rabbit inoculated with purified BKV reacted with VP1, and also with VP4, indicating that BKV inoculation may imply production of antibodies against histones.

Antibodies, Viral↗
PubMed1985

Identification of a neuroregulated phosphoprotein in skeletal muscle as the regulatory subunit of cyclic AMP-dependent protein kinase II.

When soluble proteins in cytosolic fractions of rat soleus muscles are 32P-phosphorylated in vitro by an ATP:protein phosphotransferase reaction, the major substrate is a 56-kilodalton (56K) protein. As we have also reported previously, the onset and development of increased 32P-phosphorylation of this 56K protein, which are observed after the soleus is denervated, temporally correlate with the denervation period and length of the distal nerve stump [Held et al, 1983]. Conclusive evidence which identifies this neuroregulated muscle protein as the regulatory subunit of cyclic AMP-dependent protein kinase type II (R-II) is presented in this paper. The 56K soleus protein and purified bovine heart R-II were 32P-phosphorylated and subjected to limited proteolysis with bovine pancreas trypsin. After resolution of the generated 32P-phosphopeptides by SDS slab PAGE and visualization by autoradiography, no tryptic products were observed from the 56K soleus protein which were not also produced by proteolysis of the purified R-II. These tryptic phosphopeptides included 39, 16.5, and 12K fragments which retained the autophosphorylation site of R-II. After denervation, the 32P-phosphorylation of the 56K soleus protein and of the 39K tryptic peptide product were comparably increased. The identification of the neuroregulated 56K soleus protein as R-II was also confirmed by Western blotting with a specific anti-R-II sera. Taken together, our results demonstrate that the previously observed neuroregulation of the 32P-phosphorylation of the 56K soleus protein is identifiable with some alteration which affects the intramolecular 32P-autophosphorylation of R-II.

Animals↗
PubMed1986

Characterization of antibodies against major fish CNS myelin proteins: immunoblot analysis and immunohistochemical localization of 36K and IP2 proteins in trout nerve tissue.

Antisera against the trout CNS myelin proteins 36K and IP2 were prepared in rabbits and characterized by immunoblot analysis and immunohistochemistry. The anti-36K antiserum exclusively stained its corresponding antigen from trout CNS myelin but failed to recognize any myelin polypeptide from either trout PNS or mammalian CNS and PNS. Antibodies against the IP2 glycoprotein specifically cross-reacted with related intermediate proteins (IP) of both CNS and PNS myelin from trout but only faintly labeled the PO protein of mouse peripheral nerve. Immunohistochemical localization of both antigens in the CNS of young trout was confined to the myelin sheath, except that anti-36K antiserum also stained oligodendrocytes. Nodes of Ranvier, neuronal cell bodies, and dendrites, as well as other glial elements, were negative. Specificity of the immunofluorescent reaction was established by crossed immunoadsorption experiments. Whereas on adjacent sections through trout brain both antigens exhibited a nearly identical distribution pattern, immunostaining in peripheral nerves was seen only with anti-IP2 antibodies.

Animals↗
PubMed1986

Mu-type calcium-activated neutral protease in the rat peripheral nerve.

Previously we reported results of an incubation experiment with neurofilaments that supported the existence of a mu-type of Ca2+-activated neutral protease in the rat peripheral nerve; it was active with microM order Ca2+ (mu-CANP). This time, we partially purified the mu-CANP from a crude CANP fraction of rat peripheral nerve by using a DE52 column and a Phenyl-Sepharose column followed again by DE52 column chromatography. The presence of mu-CANP was verified by an immunoblotting technique. The mu-CANP degraded the neurofilament triplet as previously reported; i.e., among the neurofilament triplet, the 160K component was most sensitive, and in the order of the 160K, 68K, and 200K components, respectively.

Animals↗
PubMed1986

Astrocyte culture on nitrocellulose membranes and plastic: detection of cytoskeletal proteins and mRNAs by immunocytochemistry and in situ hybridization.

Neonatal rat brain astrocyte secondary cultures were established on nitrocellulose membrane filters (13-mm diameter Millipore disk) and on plastic coverslips in serum-supplemented medium. On these substrata, cultured astrocytes changed their shape from flat and polygonal to stellate in the absence of hormones or growth factor supplements. Cultures became confluent after 4 days, and astrocytes on nitrocellulose filters continued to differentiate morphologically and biochemically, as evidenced by extensive cytoplasmic process formation and glial fibrillary acid protein (GFAP) accumulation. Cultures were immunostained for GFAP and vimentin. mRNAs to GFAP, vimentin, alpha and beta tubulin, and actin also were detected by in situ hybridization with biotinylated cDNA probes. The astrocyte culture method on nitrocellulose provides a simple, versatile means of comparing undifferentiated, morphologically mature, reactive, and neoplastic astrocytes in vitro.

Animals↗