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Results for “Chain Migration”

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At least 433 records · Page 24Linked to original sources

PubMed1993

Simultaneous determination of the migration coefficient of each base in heterogeneous oligo-DNA by gel filled capillary electrophoresis.

The direct determination of migration coefficients was achieved by analysing the migration time of heterogeneous oligo-DNA with a gel filled capillary using the Gauss least-squares method for the observation functions, assuming that the migration time of oligo-DNA is dependent on its base composition and chain length. By using the coefficients obtained, the migration time of oligo-DNA of any known sequence that does not have a secondary structure can be estimated with an accuracy of less than 0.5-mer of cytidine. In addition, from the deviation of the actual migration time from the calculated migration time in certain specially designed base sequences, the existence of a secondary structure such as a hairpin structure was strongly suggested even in the presence of 7 M urea. From the investigation of the effects of secondary structure on migration time, it was concluded that this approach will give qualitative information on secondary structure, which may be applicable in work such as single strand conformational polymorphism (SSCP) analysis or antisense DNA analysis, in which secondary structure plays an important role in accelerating or decelerating migration times. The results of the analysis also predict the apparent chain length reversal from short to long together with a reduction in the actual chain length in DNA sequencing using capillary electrophoresis.

Base Composition↗

PubMed2001

Breeding origin and migration pattern of dunlin (Calidris alpina) revealed by mitochondrial DNA analysis.

The large-scale migration of birds has been studied extensively by recoveries of ringed birds. However, there is very little ringing data from the arctic breeding grounds of waders. Here, the migration pattern of the dunlin, Calidris alpina, is studied with population genetic markers, using haplotype frequencies to estimate the breeding origin of migrating and wintering populations. Polymerase chain reaction (PCR) and restriction analysis of DNA from the mitochondrial control region was used to study the breeding origins of morphologically similar winter populations in the western Palaearctic, and to describe the population structure of the dunlin during winter. Also migrating dunlin from various stopover sites in Europe, Africa and Asia, were analysed with respect to their mitochondrial DNA (mtDNA) haplotypes. The genetic markers clearly show that the dunlin has a parallel migration system, with populations breeding in the western Palaearctic wintering mainly in the western part of the wintering range, and dunlin populations breeding further east wintering further east. The results also show that the distance between breeding and wintering area increases eastwards in this region.

Animals↗

PubMed1994

Characterization of the antigens recognized by two monoclonal antibodies reactive with basal-layer keratinocytes of human epidermis.

Two monoclonal antibodies, GR3 and GR4, reactive with the basal-layer keratinocytes of human epidermis, were derived by immunization of Balb/c mice with glycoproteins isolated from cultured keratinocytes by lectin-affinity chromatography. Immunoprecipitation of Triton X-100 extracts from human keratinocytes metabolically labelled with D-[1-14C]glucosamine revealed that GR3 recognized a major glycoprotein with migration properties identical with those of a glycoprotein (reduced form M(r) 126,000) which was previously shown to be implicated in intercellular adhesion [Roberts and Brunt (1985) Biochem J. 232, 67-70]. In their unreduced forms the antigens recognized by GR3 and GR4 both migrated as two bands with M(r) values of 118,000 and 147,000. Comparison of 125I-labelled glycoproteins immunoprecipitated by GR3, GR4 and integrin antibodies revealed that, under reducing conditions, the major band immunoprecipitated by both GR3 and GR4 co-migrated with the alpha 3 and beta 1 integrin chains. In addition the immunoprecipitate obtained with GR4 contained an additional band co-migrating with the alpha 2 integrin chain. Sequential immunoprecipitation studies with GR3, GR4 and integrin antibodies confirmed that GR3 is directed against the alpha 3 integrin chain, whereas GR4 is directed against the beta 1 chain. These studies also indicate that some of the alpha 2 integrin chains on keratinocytes may be associated with a beta-chain not recognized by the antisera against the beta 1 integrin chain used in this study.

Animals↗

PubMed2006

High-sensitivity staining of proteins for one- and two-dimensional gel electrophoresis using post migration covalent staining with a ruthenium fluorophore.

This paper describes the use of a ruthenium complex ((bis(2,2'-bipyridine)-4'-methyl-4-carboxybipyridine-ruthenium-N-succidimyl ester-bis(hexafluorophosphate), abbreviated below as ASCQ_Ru) commercially available and chemically pure. This new ruthenium complex ASCQ_Ru brings an activated ester, allowing the selective acylation of amino acid side chain amines for the post migration staining of proteins separated in 1-DE and 2-DE. The protocol used is a simple three-step protocol fixing the proteins in the gel, staining and then washing, as no lengthy destaining step is required. First the critical staining step was optimized. Although in solution the best described pH for acylating proteins with this reagent is phosphate buffer at pH 7.0, we found that best medium for in-gel staining is unbuffered ACN/water solution (20/80 v/v). The two other steps are less critical and classical conditions are satisfactory: fixing with 7% acetic acid/10% ethanol solution and washing four times for 10 min with water. Sensitivity tests were performed using 1-DE on protein molecular weight markers. We obtained a higher sensitivity than SYPRO Ruby with a detection limit of 80 pg of protein per well. However, contrary to SYPRO Ruby, ASCQ_Ru exhibits a logarithmic dependency on the amount of protein. The dynamic range is similar to SYPRO Ruby and is estimated between three and four orders of magnitude. Finally, the efficiency of the post migration ASCQ_Ru staining for 2-D gel separation is demonstrated on the whole protein extract from human colon carcinoma cells lines HCT 116. ASCQ_Ru gave the highest number of spot detected compared to other common stains Colloidal CBB, SYPRO Ruby and Deep Purple.

Cell Line, Tumor↗

PubMed1995

Extragenic suppressors of a dynein mutation that blocks nuclear migration in Aspergillus nidulans.

Cytoplasmic dynein is a large molecular weight protein complex that functions as a microtubule-dependent, negative, end-directed "motor." Mutations in nudA, which encodes the heavy chain of cytoplasmic dynein, inhibit nuclear migration in Aspergillus nidulans. This paper describes the selection and characterization of extragenic suppressors of the nudA1 mutation preparatory to the identification of other proteins that interact directly or indirectly with the cytoplasmic dynein heavy chain. To facilitate future cloning of the suppressor genes, we have searched particularly for extragenic suppressor mutations that also convey a selectable phenotype, such as cold or dimethyl sulfoxide sensitivity. Genetic analysis of 16 revertants has defined at least five extragenic suppressors of nudA1 (snaA-E). All the sna mutations except one were recessive in diploids homozygous for nudA1 and heterozygous for sna mutations. To characterize the nuclear migration phenotype in the sna mutants, conidia of one representative of each complementation group were germinated, fixed and nuclei stained. The sna mutants display partial suppression of the nudA1 nuclear migration defect. Although conidiophores were produced in the sna mutants, they failed to develop normally and to produce spores. Examination of the nudA1,sna conidiophores under the microscope showed that nuclear migration into the metulae and phialides was defective.

Aspergillus nidulans↗

PubMed1990

J chain deficiency in human IgM monoclonal antibodies produced by Epstein-Barr virus-transformed B lymphocytes.

Six human IgM monoclonal antibodies against Pseudomonas aeruginosa were purified and characterized. On agarose-acrylamide sodium dodecyl sulfate (SDS) gels run under nonreducing conditions, IgM monoclonal antibodies showed variable amounts of a slower migrating form of IgM in addition to the one co-migrating with plasma IgM. Protein blotting with anti-J chain antibody showed that the slower migrating form did not contain J chain. Analysis of one of the monoclonal antibodies by sucrose gradient centrifugation showed that the J chain-deficient form sedimented faster than the complete IgM. It is known that IgM preparations lacking J chain sediment faster by sucrose gradient centrifugation analysis and tend to form hexamers. The slower migrating form of IgM we observed on SDS gels under nonreducing conditions could be hexameric IgM. Further evaluation of this monoclonal antibody demonstrated that both forms of IgM had the same antigen-binding activity. Glycosylation of the light chain was demonstrated in two of the monoclonal antibodies.

Antibodies, Monoclonal↗

PubMed1978

Embryonic neural retina collagen: in vitro synthesis of high molecular weight forms of type II plus a new genetic type.

Chicken neural retina cells from 6- to 7-day embryos were labeled with [(3)H]proline for 24 and 72 hr and the collagenous proteins of the medium were analyzed. Ninety percent of the collagenous proteins eluted from DEAE-cellulose columns as a peak near the middle of the gradient. Upon sodium dodecyl sulfate/polyacrylamide gel electrophoresis, this material consisted predominantly of a slowly migrating procollagen, a smaller amount of an intermediate migrating form, and some alpha-chain material. Limited enzymatic digestion of this DEAE peak material plus mapping of its CNBr peptides identified this material as a type II precursor. The remaining 10% of the collagenous proteins eluted from DEAE-cellulose at the end of the gradient. Electrophoresis of this fraction showed four major bands. Two migrated near the type II precursors, a third migrated somewhat more slowly, and the fourth was near beta-chain dimers. In addition, there were two minor bands. Limited pepsin digestion of this DEAE peak material produced two bands: one migrated slightly more slowly than alpha1, and the second, considerably more slowly. The CNBr peptide pattern of this material appears to be different from any previously described. Thus, neural retina cells in culture synthesize at least two genetically distinct classes of collagenous proteins. One represents precursors to type II. The second is composed of multiple, very high molecular weight forms which may represent precursors (procollagens) of a new genetic type(s) of collagen.

Animals↗

PubMed1994

Recombinant rat protein C: comparative studies of structure, function and synthesis with plasma protein C.

In order to elucidate the role of protein C (PC) in the rat, we expressed, purified, and characterized recombinant rat PC. The purified recombinant rat PC was 70-90% two-chain (41 kDa heavy chain; 22 and 23 kDa light chain) and 10-30% single-chain (61 kDa). Amino acid analysis confirmed the presence of 10 moles of gamma-carboxyglutamic acid residues per mol of protein. For comparison, plasma rat PC was purified from a barium citrate precipitate using similar method. Plasma rat PC was a two-chain form (41 kDa heavy chain; 22 kDa light chain) with no detectable single-chain nor 23 kDa light chain. For determination of the in vitro secreted species, primary cultured rat hepatocytes were incubated for 6 h with methionine-free MEM containing vitamin K1, aprotinin, and [35S]methionine. The supernatant was immunoprecipitated and analyzed by SDS-PAGE followed by autoradiography. Approximately 90% of the PC radioactivity migrated as a two-chain molecule. These results indicate that rat PC is secreted mainly as a two-chain molecule from the liver. PROTAC-activated forms of recombinant rat PC, plasma rat PC, and plasma human PC hydrolyzed the S-2366 chromogenic substrate at the same rate. Recombinant rat PC was also activated by the thrombin-thrombomodulin complex at a rate similar to plasma rat PC. The anticoagulant activities of the three activated PCs were examined in rat plasma. Both recombinant and plasma rat PC prolonged the activated partial thromboplastin time in a dose-dependent manner, but plasma human PC was less effective.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1980

Pig cardiac myosin isoenzymes.

Pig heart myosins isolated from the free wall of the right ventricle and the free wall of the left ventricle were compared with respect to structural and enzymatic properties. The following parameters were studied (1) activation of myosin ATase by Ca2+ and K+j(2) molecular weight of the heavy and light chains of myosins as determined by electrophoretic migration in polyacrylamide sodium dodecyl sulfate (SDS) gels; (3) ability of the heavy chains to form aggregates at low ionic strength as revealed by electron microscopy; (4) sensitivity to the action of chymotrypsin. Differences were observed between left and right ventricular myosins (L-myosin and R-myosin) for all these parameters except for the molecular weight of heavy and light chains. The existence of large amounts of short synthetic filaments for R-myosin compared with L-myosin as revealed by the length repartition of the filaments, and the production of smaller quantities of HMM-S by chymotryptic digestion for R-myosin, strongly suggest the presence of different cardiac myosin heavy chain species.

Adenosine Triphosphatases↗

PubMed2002

Construction and electrophoretic migration of single-stranded DNA knots and catenanes.

In recent years there has been growing interest in the question of how the particular topology of polymeric chains affects their overall dimensions and physical behavior. The majority of relevant studies are based on numerical simulation methods or analytical treatment; however, both these approaches depend on various assumptions and simplifications. Experimental verification is clearly needed but was hampered by practical difficulties in obtaining preparative amounts of knotted or catenated polymers with predefined topology and precisely set chain length. We introduce here an efficient method of production of various single-stranded DNA knots and catenanes that have the same global chain length. We also characterize electrophoretic migration of the produced single-stranded DNA knots and catenanes with increasing complexity.

DNA, Single-Stranded↗

PubMed2004

Electrophoretic variants of cardiac myosin heavy chain-alpha in Sprague Dawley rats.

Analysis of cardiac myosin revealed differences in gel electrophoretic migration patterns of the alpha-isoform of myosin heavy chain, but not the beta-isoform, in Sprague Dawley rats. No differences in the migration patterns of the alpha-or beta-isoforms were observed in other rat strains. Three electrophoretic migration patterns of the alpha-isoforms were observed in individual rats: a slower migrating isoform alone (4% of all rats tested), a faster migrating isoform alone (55%), and both isoforms (41%). The isoform expression pattern was identical in all myocardial regions in each rat. Frequency of expression patterns suggests multiple gene sequences for alpha-cardiac myosin heavy chain in Sprague Dawley rats. Sequence analysis of amplified regions of the Sprague Dawley and Brown Norway rat alpha-myosin genes, specifically the 5'-untranslated region, exons 1-3, and associated introns, showed numerous single nucleotide polymorphisms in coding and noncoding regions, including putative regulatory sites in Sprague Dawley rats, but not in Brown Norway rats. All Sprague Dawley rats varied from Brown Norway rats and no heterogeneity was observed in Brown Norway rats. Several deletions and dimorphic positions were also observed. Dimorphic positions were evident on automated sequencing comparisons. The data indicate that at least two alpha-myosin heavy chain isoforms exist in Sprague Dawley rats and these rats exhibit sequence diversity within that portion of the alpha-myosin heavy chain gene reported in this study.

Animals↗

PubMed1995

Myogenic cell migration from somites is induced by tissue contact with medial region of the presumptive limb mesoderm in chick embryos.

It is known that myogenic cells in limb buds are derived from somites. In order to examine the potential of the limb primordium (presumptive limb somatopleure) to induce myogenic cell migration, we transplanted chick presumptive limb somatopleure to the flank region of an embryo, a region that does not normally contribute myogenic cells to the limb. Somitic cell migration was examined using a vital labeling technique. When the presumptive limb somatopleure was transplanted and was in contact with the host flank somite, somitic-cell migration toward the graft was observed. The labeled somitic cells within the graft were identified as myogenic cells in two ways: first, we found that N-cadherin-expressing cells appeared in the graft. Second, after 3 further days of incubation, the somitic cells formed dorsal and ventral masses and expressed sarcomeric myosin heavy chain within the graft. Cell migration occurred only when the somite was in contact with the medial region of the presumptive limb somatopleure. When the somite was not in contact with the limb somatopleure, or when the somite was in contact with the lateral region of the limb somatopleure, migration did not occur. These observations indicate that the potential to induce myogenic cell migration is restricted to the medial region of the presumptive limb somatopleure and that tissue contact is required.

Animals↗

PubMed1992

Diffuse aggressive B-cell lymphomas of the gastrointestinal tract. An immunophenotypic and gene rearrangement analysis of 22 cases.

Twenty-two diffuse aggressive B-cell lymphomas of the gastrointestinal tract were studied using light microscopic examination, immunohistochemical methods, and Southern blot analysis. The results suggest that diffuse aggressive B-cell gastrointestinal tract lymphomas may be divided into two groups: large cell lymphomas and small noncleaved cell lymphomas. Large cell lymphomas often involve the stomach; commonly express the lymphocyte adhesion molecules CD44, LFA-1 (CD11a and CD18), and CD54; and may express monotypic cytoplasmic immunoglobulin in approximately one third of cases. Southern blot analysis demonstrates rearrangements of the c-myc gene that do not co-migrate with rearrangements of the immunoglobulin heavy chain gene, as detected with a JH probe in approximately one half of the cases. Small noncleaved cell lymphomas typically involve the ileocecal region. In these lesions, monotypic cytoplasmic immunoglobulin is not detected, and the CD44 and LFA-1 molecules usually are not expressed, particularly in small noncleaved cell lymphomas of the Burkitt type. The CD54 antigen is positive in fewer than one half of cases. Southern blot studies often demonstrate rearrangements of the c-myc gene that co-migrate with immunoglobulin heavy chain gene rearrangements indicative of the t(8;14) chromosomal translocation, with the c-myc region translocated into the immunoglobulin heavy chain gene joining region. Thus, immunohistochemical and genotypic results, in accordance with the site of involvement and histologic findings, suggest a different pathogenesis for large cell lymphomas and small noncleaved cell lymphomas. The findings in large cell immunoblastic lymphomas are more akin to those of the large cell group. In addition, immunophenotypic and molecular data may be helpful in improving histologic classification when the morphologic findings are equivocal.

Adult↗

PubMed2001

Expression of CD44 and its isoforms in the fetal neuroblasts.

CD44 is a polymorphic transmembrane glycoprotein that exists as multiple isoforms resulting from alternative splicing and posttranslational modifications. Enhanced expression of CD44 has been correlated to the tumorigenicity and metastatic behavior in different malignant tumors. In contrast, human neuroblastomas exhibit an inverse correlation between CD44 expression and tumor malignancy. To determine whether there is a CD44 silencing in sympatho-adrenal precursors as a possible explanation for the down-regulation of CD44 in neuroblastomas, the expression of standard CD44H and v6, v7, v7v8, or v10 isoforms was analyzed by immunohistochemistry in human adrenal glands of 14- to 20-week-old gestational age fetuses. All of the fetal neuroblasts localized in the adrenal gland parenchyma and migrating from the sympathetic nerve chain into the fetal adrenal cortex strongly expressed CD44H but none of the CD44 isoforms could be detected in these cells. In contrast, a strong expression of CD44v7 and v6 was detected in the fetal adrenal cells. These results indicate that, as for many other cell types, the CD44H expressed by fetal neuroblasts may contribute to controlling their migration into the adrenal medulla and that the down-regulation of CD44H in neuroblastoma cells should be explained by mechanisms other than the fetal suppression of CD44H expression in their normal counterparts.

Adrenal Glands↗

PubMed2001

Light-induced proteolysis of myosin heavy chain by Rose Bengal-conjugated antibody complexes.

The specific light-induced, non-enzymatic digestion of chicken skeletal muscle myosin heavy chain by xanthene dye-conjugated antibodies is reported. The xanthene dye Rose Bengal was conjugated to either a mouse monoclonal anti-myosin primary specific antibody or to goat anti-mouse IgG secondary antibodies. Under our experimental conditions, visible light induced the non-enzymatic breakdown of myosin heavy chains when chicken skeletal muscle myosin either directly formed a complex with Rose Bengal-conjugated anti-myosin antibodies or indirectly formed a complex with anti-myosin antibody-Rose Bengal-conjugated secondary antibodies. The rate of the photochemical reaction depended on irradiation time and temperature. Although SDS-PAGE and immunoblot analyses showed that fragments migrating below the myosin heavy chain polypeptide predominated, these analyses also showed higher molecular mass polypeptides were generated.

Animals↗

PubMed1991

Purification and characterization of rabbit factor IX and its existence as a two-chain factor IX alpha in circulating plasma.

The long term objective of this study is to immunodeplete rabbits of factor IX as a means of developing a rabbit model for hemophilia B for use in studies of tissue factor-dependent blood coagulation. As a first step, we have purified rabbit factor IX by basically two different methods: (1) conventional chromatography utilizing DEAE-Sephadex and heparin-agarose column chromatography (2) immunoaffinity chromatography on monoclonal anti-rabbit factor IX IgG column. Purified rabbit factor IX migrated as a single band with an apparent molecular mass of 76 kD on nonreduced SDS-PAGE. On reduced SDS-PAGE the majority of factor IX migrated as a two-chain molecule (molecular masses 51 and 28 kD) and a faint band corresponding to 78 kD. We have shown that the purification of rabbit factor IX as a two-chain molecule is not due to the partial proteolysis of factor IX during its purification from commercially obtained rabbit plasma. Analysis of 3H-labelled rabbit factor IXa on SDS-PAGE revealed that, in contrast to human factor IXa, carbohydrate was found associated with the heavy chain of activated factor IX (H beta) after release of the activation peptide. Further understanding of the molecular properties of rabbit factor IX and the generation of neutralizing monoclonal and polyclonal antibodies will facilitate the development of a rabbit model for hemophilia B.

Amino Acid Sequence↗

PubMed1992

Requirement for VLA-4 and VLA-5 integrins in lymphoma cells binding to and migration beneath stromal cells in culture.

Physical interaction between human lymphomas and murine bone marrow derived stromal cells were studied. Nalm-6 pre-B cells adhered to BMS2 stromal cells and subsequently migrated beneath them, while Ramos Burkitt lymphoma cells, adhered but did not migrate. Four mAbs were established against Nalm-6 cells, which were able to block initial adhesion of Nalm-6 cells. Two of them were directed against the alpha 4 chain of VLA-4, and other two recognized the beta 1 chain of VLA integrins. Therefore, the initial adhesion of Ramos and Nalm-6 cells to BMS2 was largely mediated by the VLA-4 integrin expressed on lymphocytes. The corresponding ligand on stromal cells appears to be VCAM-1, because antibodies against murine VCAM-1 blocked the adhesion. However, antibodies against the alpha chain of VLA-4 were not capable of blocking subsequent migration beneath stromal cells. In contrast, antibodies against the beta chain of VLA integrins blocked the migration beneath stromal cells as well as the initial adhesion. Because a common beta chain can be shared among integrins, the role of other VLA integrins in Nalm-6 cells migration was investigated. VLA-5 and VLA-6 as well as VLA-4 were expressed on Nalm-6 cells, but not on Ramos cells. Additional blocking experiments revealed that VLA-4 and VLA-5 are likely to work in concert to mediate the migration of Nalm-6 cells beneath stromal cells. Thus, particular VLA integrins appear to be responsible not only for lymphocyte adhesion but also for migration with respect to stromal cells. These findings may have implications for cell-cell interactions and directed migration of lymphocytes in bone marrow and other tissues.

Animals↗

PubMed1978

Structural properties of frog muscle myosin.

Frog myosin is a labile molecule, undergoing irreversible aggregation and rapid loss of ATPase; however, a procedure is described which provides highly purified myosin, with stable solubility and enzymatic properties, from skeletal muscle of Rana catesbeiana. Frog myosin contains heavy chains and light chains 1, 2, and 3. Light chain 3 is present in excess over light chain 1, and light chain 2 may occur as either, or both, of 2 closely migrating bands. On two-dimensional electrophoresis, light chain 1 generates an isoelectric component with pK 5.60; light chain 2 generates a complex pattern with 3 or 4 major components; and light chain 3 generates 2 major components with pK 5.00 and 4.92. The same subunit composition is obtained for frogs acclimated at 25 and 5 degrees C; however, proteolytic artifacts may occur in myosin preparations purified in the absence of protease inhibitors, especially in warm-acclimated frogs.

Acclimatization↗