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Evidence for phosphorylation and oligomeric assembly of presenilin 1

Seeger, Mary; Nordstedt, Christer; Petanceska, Suzana; Kovacs, Dora M.; Gouras, Gunnar K.; Hahne, Solveig; Fraser, Paul; Levesque, Lyne; Czernik, Andrew J.; George-Hyslop, Peter St; Sisodia, Sangram S.; Thinakaran, Gopal; Tanzi, Rudolph E.; Greengard, Pau
Fonte: The National Academy of Sciences of the USA Publicador: The National Academy of Sciences of the USA
Tipo: Artigo de Revista Científica
Publicado em 13/05/1997 EN
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Pathogenic mutations in presenilin 1 (PS1) are associated with ≈50% of early-onset familial Alzheimer disease. PS1 is endoproteolytically cleaved to yield a 30-kDa N-terminal fragment (NTF) and an 18-kDa C-terminal fragment (CTF). Using COS7 cells transfected with human PS1, we have found that phorbol 12,13-dibutyrate and forskolin increase the state of phosphorylation of serine residues of the human CTF. Phosphorylation of the human CTF resulted in a shift in electrophoretic mobility from a single major species of 18 kDa to a doublet of 20–23 kDa. This mobility shift was also observed with human PS1 that had been transfected into mouse neuroblastoma (N2a) cells. Treatment of the phosphorylated CTF doublet with phage λ protein phosphatase eliminated the 20- to 23-kDa doublet while enhancing the 18-kDa species, consistent with the interpretation that the electrophoretic mobility shift was due to the addition of phosphate to the 18-kDa species. The NTF and CTF eluted from a gel filtration column at an estimated mass of over 100 kDa, suggesting that these fragments exist as an oligomerized species. Upon phosphorylation of the PS1 CTF, the apparent mass of the NTF- or CTF-containing oligomers was unchanged. Thus, the association of PS1 fragments may be maintained during cycles of phosphorylation/dephosphorylation of the PS1 CTF.

Simian virus 40 T-antigen: identification of tryptic peptides in the C-terminal region and definition of the reading frame.

Denhardt, D T; Crawford, L V
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /05/1980 EN
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T-antigen (the simian virus 40 A cistron protein) was purified by immunoprecipitation and electrophoresis on polyacrylamide gels from monkey kidney CV-1 cells infected with simian virus S (SV-S), dl1263, or dl1265 and digested with trypsin. The tryptic peptides, labeled with [35S]methionine, [35S]cysteine, or [3H]proline, were fractionated either by chromatography on Chromobead-P resin or by two-dimensional electrophoresis and chromatography on cellulose thin layers. The T-antigen of SV-S was shown to give rise to a proline-rich (approximately 6 mol of proline) tryptic peptide which was absent in dl1265 T-antigen and hence, on the basis of DNA sequence data, must originate from the C-terminus of the SV-S protein. T-antigen from dl1265, but not SV-S, yielded a cysteine-rich terminal tryptic peptide. The presence of these cysteines caused the protein to be retarded during electrophoresis under the usual conditions in polyacrylamide gels. The T-antigen of dl1263 possessed the proline-rich tryptic peptide; the data are consistent with there being only one peptide altered by the deletion. Both deletion mutants produced a T-antigen that had a higher electrophoretic mobility than SV-S T-antigen but still a larger apparent molecular weight than was predicted by the DNA sequence. The major form of T-antigen found in several lines of 3T3 cells transformed by these mutants was indistinguishable from the T-antigen found in infected cells...

Interaction of Neomycin with Ribosomes and Ribosomal Ribonucleic Acid

Dahlberg, Albert E.; Horodyski, Frank; Keller, Patrick
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /02/1978 EN
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Neomycin binds ribosomes and ribosomal ribonucleic acid (rRNA) in vivo and in vitro producing changes detectable by increases in gel electrophoretic mobility. These changes were observed in gels that contain ethylenediaminetetraacetic acid or no added magnesium ion. The progressive increase in gel electrophoretic mobility with increasing antibiotic concentrations suggests that neomycin is binding at multiple sites on RNA. The binding was reversible but sufficiently stable to survive dialysis and electrophoresis. It is proposed that bound neomycin stabilizes the ribosome and RNA structures, restricting the unfolding of the particles during electrophoresis and thus allowing for a more rapid migration in the gel. Gentamicin produced an effect similar to that of neomycin. Paromomycin, differing from neomycin by only one amino group, had considerably less effect on ribosome and rRNA mobilities. The binding of neomycin to rRNA improved the linearity of the plot of log molecular weight versus mobility and thus may be of benefit in providing a more accurate estimation of molecular weights of large RNAs.

5-Azacytidine treatment of HA-A melanoma cells induces Sp1 activity and concomitant transforming growth factor alpha expression.

Shin, T H; Paterson, A J; Grant, J H; Meluch, A A; Kudlow, J E
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /09/1992 EN
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Evidence indicates DNA methylation as a part of the regulatory machinery controlling mammalian gene expression. The human melanoma cell line HA-A expresses low levels of transforming growth factor alpha (TGF-alpha). TGF-alpha mRNA accumulated, however, in response to DNA demethylation induced by a nucleoside analog, 5-azacytidine (5-azaC). The importance of DNA methylation in the TGF-alpha promoter region was examined by a transient transfection assay with luciferase reporter plasmids containing a portion of the TGF-alpha promoter. 5-azaC treatment of HA-A cells before the transfection caused a significant increase in the luciferase activity. Since input plasmids were confirmed to remain unmethylated, DNA demethylation of the TGF-alpha promoter itself does not account for the observed increase in TGF-alpha mRNA. Using an electrophoretic mobility shift assay, enhanced formation of protein-TGF-alpha promoter complex was detected in response to 5-azaC treatment. This 5-azaC-induced complex was shown to contain the transcription factor Sp1 by the following criteria: the protein-DNA complex formed on the TGF-alpha promoter contained immunoreactive Sp1; the mobility of the complex in an electrophoretic mobility shift assay was similar to that formed by recombinant Sp1; and DNase I footprinting analysis demonstrated that the 5-azaC-induced complex produced a footprint on the TGF-alpha promoter identical to that of authentic Sp1. These observations suggest that 5-azaC induces TGF-alpha expression by augmenting the Sp1 activity. However...

Heterologous expression and characterization of the human R-ras gene product.

Lowe, D G; Goeddel, D V
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /08/1987 EN
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We directly expressed human R-ras 23,000-dalton protein (p23) cDNA in Escherichia coli under the control of the trp promoter. GTP-dependent phosphorylation of a p23 threonine 85 substitution mutant was observed. This result is in direct analogy to the autokinase activity of H-ras and K-ras threonine 59 substitution mutants. Normal p23 protein was detected in the human fibrosarcoma cell line HT1080 by immunoprecipitation with rabbit antibodies raised against an E. coli-expressed R-ras fusion protein. The R-ras p23 protein was found to be 3H labeled in the presence of [9,10(n)-3H]palmitic acid and is associated with the P100 membrane fraction of HT1080 cells. These data suggest that human R-ras p23 has biochemical properties very similar to those of the p21 products of the H-, K-, and N-ras proto-oncogenes. We constructed an R-ras minigene and engineered the expression of normal and mutant alleles from the simian virus 40 early region promoter. Normal and mutant R-ras gene products were authenticated by transient expression in COS-7 cells and immunoprecipitation. The valine 38-substituted R-ras p23 displayed reduced electrophoretic mobility. R-ras p21-like proteins, made by eliminating the first 26 R-ras codons, displayed evident mobility differences between the pro form and mature form...

Electrophoretic Analysis of Ribosomal and Viral Ribonucleic Acids with a Simple Technique for Slicing Low-Concentration Polyacrylamide Gels

Schaffer, F. L.; Soergel, M. E.; Straube, D. C.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /10/1971 EN
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The electrophoretic mobilities of ribosomal ribonucleic acids (RNA) from cultured mammalian (HeLa, Vero, MDBK), avian (chick embryo), and bacterial (Escherichia coli) cells, and RNA species extracted from selected viruses (Sindbis, polio, tobacco mosaic, Sendai) were compared, employing a simple, inexpensive technique for slicing low-concentration polyacrylamide gels. The procedure provides for rapid fractionation of gels used for characterization of RNA, incorporating extrusion and serial sectioning of frozen gels. Among 28S ribosomal RNA species, Vero and MDBK were indistinguishable, whereas HeLA RNA had a slightly lower mobility (higher apparent molecular weight) and chick RNA had a higher mobility (lower apparent molecular weight). The 18S ribosomal RNA species of the three mammalian sources were indistinguishable, but chick 18S RNA had a slightly lower apparent molecular weight. The inverse relation between mobility and log-molecular weight among the ribosomal and viral RNA species, though not highly precise, demonstrates the applicability of the technique to the study of molecular weights of viral RNA species.

Amylases in Developing Barley Seeds 1

Bilderback, D. E.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /09/1971 EN
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282.04807%
The amylases of developing barley seeds (Hordeum vulgare L. cv. Himalaya) were investigated by colorimetric and electrophoretic methods. Maxima of amylolytic activity appeared in the aleurone layers and starchy endosperm at 5 and 20 days after anthesis. Amylase from 5-day-old aleurone layers could be separated into four rapidly moving bands with α-amylase activity. By 20 days the four bands had been replaced by seven bands of medium mobility. These seven bands of amylase were electrophoretically identical to those observed when mature aleurone layers are treated with gibberellic acid. Immature aleurone layers failed to respond to exogenous gibberellic acid. In the starchy endosperm the seven bands of medium mobility were also present. Calcium-dependent alterations in the electrophoretic mobility and activity of particular bands occurred during the maturation of the starchy endosperm. Treatment of the immature starchy endosperm with papain yielded four forms of β-amylase.

Studies on human thyroxine-binding globulin (TBG). IX. Some physical, chemical, and biological properties of radioiodinated TBG and partially desialylated TBG.

Refetoff, S; Fang, V S; Marshall, J S
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /07/1975 EN
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Thyroxine-binding globulin (TBG) and partially desialylated or slow TBG (STBG) were purified from human serum by affinity chromatography. Purified TBG was identical to TBG present in serum by the criteria of electrophoretic mobility, affinity for thyroxine (T4), and heat-inactivation response. Purified STBG had slower electrophoretic mobility and lower affinity for T4. Both bound T4 in an equimolar ratio, were immunoprecipitable, and had similar inactivation t1/2 at 61 degrees C. TBG and STBG were iodinated by the chloramine-T-catalyzed reaction. An average of from 0.02 to 6 atoms I could be incorporated per molecule of the protein by adjusting the conditions of the reaction (time, protein and iodide concentrations). 125-I, 131-I, and 127-I were used. Iodination increased the anodal mobility of TBG but did not affect the reversible T4-binding, precipitation by antiserum, or the heat-inactivation properties. "Heavily" and "lightly" iodinated TBG had identical disappearance half-times from serum in the rabbit. 15 min after the intravenous administration of [131-I]-STBG and [125-I]TBG mixture to rats, more than 90% of the injected 131-I dose was in the liver, and the liver 131-I/125-I ratio was 32-fold that of serum. Selective uptake of STBG by the liver was also observed in the rabbit and in man. The serum [125-I]STBG/[131-I]TBG ratio declined from 1 to 0.2 in 10 min in the intact rabbit but remained unchanged for 1 h in the acutely hepatectomized animal. In the rabbit...

K-ras point mutations in routinely processed tissues: non-radioactive screening by single strand conformational polymorphism analysis.

Korn, S H; Moerkerk, P T; de Goeij, A F
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /07/1993 EN
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AIMS--To develop a non-radioactive method to screen routinely fixed, paraffin wax embedded specimens for the occurrence of point mutations; to evaluate the single strand conformational polymorphism (SSCP) analysis technique for the detection of K-ras point mutations as a result of electrophoretic mobility shifts. METHODS--DNA was extracted from archival specimens of colon cancer and from established colon cancer cell lines with known point mutations. A K-ras gene fragment containing codons 12 and 13 of exon 1 was amplified with the polymerase chain reaction (PCR). Denatured DNA fragments were run on 10% polyacrylamide gels under non-denaturing conditions. After electrophoresis DNA was blotted and the single stranded DNA was detected using a digoxigenin labelled ras probe. The nature of the detected point mutations was identified and confirmed by sequencing and hybridisation with oligonucleotides using 32P labelling. RESULTS--Wild type and aberrant alleles were detected caused by mobility shifts after electrophoresis of the PCR products. Commonly occurring mutations in the K-ras gene--in the first two positions of codon 12--could easily be detected in DNA from archival paraffin wax embedded colon cancer tissue. In all the colon tumour samples studied wild type gene alleles were also found...

The transport of vitamin D in the serum of primates.

Bouillon, R; van Baelen, H; de Moor, P
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/12/1976 EN
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"Transcalciferin" (the serum transport protein for cholecalciferol and related substances) of two New World monkeys, Cebus apella and Cebus albifrons, was found to be immunologically identical with the transcalciferin of other monkeys and partially with that of man. In contrast with the alpha-globulin mobility of the transcalciferin of other primates, the transcalciferin of cebus monkey has the electrophoretic mobility of albumin. Most of the serum 25-hydroxycholecalciferol was precipitable with isolated monospecific anti-(human transcalciferin) gamma-globulins but not with anti-(human albumin) gamma-globulins. These results indicate that the transport of 25-hydroxycholecalciferol in the cebus monkey is not due to albumin itself but to transcalciferin with the electrophoretic mobility of albumin. Similar variants of transcalciferin also exist in man.

Characterization of the serum lipoproteins and their apoproteins in hypercholesterolaemic guinea pigs.

Chapman, M J; Mills, G L
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/10/1977 EN
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1. Hypercholesterolaemia was induced in male guinea pigs after 6 days on a chow diet supplemented with 1.6% (w/w) cholesterol and 15% (w/w) corn oil. Both the VLD (very-low-density) and LD (low-density) lipoproteins were increased in cholesterol-fed animals, although the low concentrations of HD (high-density) lipoproteins remained essentially unchanged. LD lipoproteins of d 1.019-1.100 were the major class, accounting for 74% of the total substances of d less than 1.100. 2. Both VLD and LD lipoproteins exhibited alterations in their chemical composition, physical properties and apolipoprotein content. The VLD lipoproteins in cholesterolaemic animals were rich in cholesterol (25.9%), deficient in protein (4.9%) and exhibited electrophoretic mobility greater than that of beta-globulin; their average particle size (64.5 nm) was larger than that in controls (46.3 nm). The LD lipoproteins in animals fed on the experimental diet were also richer in cholesterol (53.1%) and of larger diameter (24.3 nm) than in the control group (41.1% and 21.4 nm respectively). 3. The apolipoprotein-B content of both VLD and LD lipoproteins was elevated in cholesterolaemic animals, particularly in the VLD class, where it represented 74.8% of the total protein moiety. 4. Apo-VLD lipoprotein exhibited an increase from 6 to 19% in its complement of tetramethylurea-soluble apolipoproteins with low electrophoretic mobility (relative mobility less than 0.29); this was primarily accounted for by apolipoproteins characterized by high arginine (7.2 and 6.4% respectively) and glutamic acid (20.1 and 20.0% respectively) contents. 5. By contrast...

T and B lymphocytes in pituitary dwarf Snell-Bagg mice.

Dumont, F; Robert, F; Bischoff, P
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /09/1979 EN
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The lymphocyte composition of the thymus and spleen from weaned (4 month old) hypopituitary dwarf Snell-Bagg mice were compared to those of their phenotypically normal littermates and of hormone (somatotropic hormone plus thyroxine)-treated individuals. Detection of cells bearing receptors for peanut agglutinin, physical analysis and measurement of in vitro reactivities to phytohaemagglutinin and concanavae intra-thymic lymphocyte population of dwarf mice. Examination of spleen-cell suspensions demonstrated a slightly higher frequency of T lymphocytes (Thy 1-2+ alpha-Naphthyl esterase+, high electrophoretic mobility) and lower frequency of B lymphocytes (surface immunoglobulin+, low electrophoretic mobility) in dwarf mice than in control mice. The degree of splenocyte responsiveness to T- and B-cell mitogens, however was similar in the two mouse types. High mobility (T) splenic cells were found to exhibit a smaller modal volume in dwarf mice (110 micron3) than in control mice (122 micron3) but this difference was not corrected by hormone administration. More pronounced were the quantitative differences between the spleens of hormone-deficient and normal mice. Thus, when expressed as a function of body weight, the numbers of splenic T and B lymphocytes in untreated dwarf mice were about half the corresponding values in hormone-reconstituted or normal littermates. These data suggested that in adult life...

Species-specific tissue antigens. II. A human species-specific antigen possessing esterase activity*

Bartholomew, W. R.; Bartholomew, Eleanor A.; Therrien, Georgirene D.; Rose, N. R.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /09/1972 EN
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An esterase that is limited in species distribution to human and monkey tissues was demonstrated by enzymoimmunoelectrophoresis and enzymoimmunodiffusion. The monkey esterase exhibited a slightly faster electrophoretic mobility than the human tissue esterase. An antigenically identical esterase found in concentrated human urine had a mobility slightly more anodal than the human tissue esterase. Despite the differences in electrophoretic mobility among the human tissue esterase, urinary esterase and monkey tissue esterase, they all reacted in enzymoimmunodiffusion with antiserum to cellular or urinary esterase to produce lines of immunologic identity. Tissues of the other species tested did not exhibit the human cathodal esterase.

The detection by cellular electrophoresis of surface antibodies in human lymphocytes

Bert, G.; Di Cossano, Donatella Lajolo; Pecco, Paola
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /12/1969 EN
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The electrophoretic mobility of peripheral blood lymphocytes from human subjects with positive Mantoux tests was measured before and after treatment with purified protein derivative. Lymphocytes from subjects with negative Mantoux reactions were used as controls. A large number of the first group lymphocytes showed a reduction of electrophoretic mobility and hence of the surface electrical charge. The mobility of the control cells submitted to the same treatment was unchanged. The possible presence of cytophilic antibodies or of cellular specific receptors on the lymphocyte surface is discussed.

NUCLEAR MEMBRANES OF CULTURED MAMMALIAN CELLS IN THE PERIOD PRECEDING DNA SYNTHESIS

Kishimoto, Susumu; Lieberman, Irving
Fonte: The Rockefeller University Press Publicador: The Rockefeller University Press
Tipo: Artigo de Revista Científica
Publicado em 01/04/1965 EN
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282.04807%
When kidney cells are cultured directly from the rabbit, the nuclear membranes undergo a change that can be measured as an increase in electrophoretic mobility. The change appears to begin immediately upon culture and is maximal 2 hours later, after which the mobility remains constant at the elevated level. Actinomycin D and p-fluorophenylalanine, but not EDTA or ionizing radiation, suppress the increase in nuclear electrophoretic mobility. With synchronously growing L cells, no change was detected in nuclei from cells taken during various parts of the division cycle.

Purification of calmodulin from Chlamydomonas: calmodulin occurs in cell bodies and flagella

Fonte: The Rockefeller University Press Publicador: The Rockefeller University Press
Tipo: Artigo de Revista Científica
Publicado em 01/12/1980 EN
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282.04807%
Calmodulin has been purified from cell bodies of the green alga Chlamydomonas by Ca++-dependent affinity chromatography on fluphenazine- Sepharose 4B. Calmodulin from this primitive organism closely resembles that from bovine brain in a number of properties, including (a) binding to fluphenazine in a Ca++-dependent, reversible manner, (b) functioning as a heat-stable, Ca++-dependent activator of cyclic nucleotide phosphodiesterase, and (c) electrophoretic mobility in SDS- polyacrylamide gels in both the presence and absence of Ca++, which causes a shift in the relative mobility of calmodulin. Calmodulin has also been identified by the criteria of phosphodiesterase activation and electrophoretic mobility in both the detergent soluble "membrane plus matrix" and the axoneme fractions of Chlamydomonas flagella. Calmodulin is not associated with the partially purified 12S or 18S dynein ATPases of Chlamydomonas. The presence of calmodulin in the flagellum suggests that it is involved in one or more of the Ca++- dependent activities of this organelle.

The Surface Charge of Isolated Toad Bladder Epithelial Cells : Mobility, effect of pH and divalent ions

Lipman, Kenneth M.; Dodelson, Robert; Hays, Richard M.
Fonte: The Rockefeller University Press Publicador: The Rockefeller University Press
Tipo: Artigo de Revista Científica
Publicado em 01/01/1966 EN
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282.04807%
The surface charge of epithelial cells isolated from the toad bladder has been determined by the microscope method of cell electrophoresis. The cells possess a net negative charge, and a net surface charge density of 3.6 x 104 electronic charges per square micron at pH 7.3. Estimates of net surface charge over the alkaline pH range indicate (a) that an average distance of the order of 40 A separates the negatively charged groups, and (b) that amino as well as acid groups are present at the electrophoretic surface of shear. A significant increase in mobility following cyanate treatment of the cells suggests that a large proportion of the amino groups are the ε-amino groups of lysine. In view of the known effects of calcium and other divalent ions on cell permeability and cell adhesion, the extent of binding of calcium and magnesium to the cell surface was determined by the electrophoretic technique. Mobility was significantly decreased in the presence of calcium or magnesium, indicating that these ions are bound by surface groups. When the pH was lowered from 7.3 to 5.2, calcium binding was markedly decreased, an observation consistent with competition between calcium and hydrogen ions for a common receptor site.

New molecular forms of human liver alcohol dehydrogenase: isolation and characterization of ADHIndianapolis.

Bosron, W F; Li, T K; Vallee, B L
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /10/1980 EN
Relevância na Pesquisa
283.04373%
The biochemical determinants of alcoholism and genetic correlates for the variability in man's response to alcohol have remained obscure until recently. The identification of genetically determined isoenzymes of alcohol dehydrogenase with different catalytic properties may bear importantly upon this problem. New molecular forms of human liver alcohol dehydrogenase (ADH; alcohol:NAD+ oxidoreductase, EC 1.1.1.1) have recently been identified in 16% of the liver specimens from an urban population from Indianapolis, Indiana [Bosron, W. F., Li, T.-K. & Vallee, B. L. (1979) Biochem. Biophys. Res. Commun. 91, 1549-1555]. The distinguishing features of these specimens were (i) they showed activity optima for ethanol oxidation at both pH 7.0 and 10.0 and (ii) they formed electrophoretic bands cathodic to the beta beta isoenzyme. From such livers, three new ADH forms have now been isolated, one of which has a single pH optimum at 7.0 and two of which have dual optima at pH 7.0 and 10.0. These new forms were designated ADHIndianapolis forms 1,2, and 3, respectively. They can be differentiated from previously described ADH isoenzymes, including the so-called "atypical" isoenzyme, by their electrophoretic mobility, pH optima, and Km for ethanol (approximately 60 mM at pH 7.5). Based upon the electrophoretic pattern of livers containing ADHIndianapolis and the mobility of the three isolated molecular forms...

Effects of two major activating lesions on the structure and conformation of human ras oncogene products.

Srivastava, S K; Yuasa, Y; Reynolds, S H; Aaronson, S A
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /01/1985 EN
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283.04373%
ras oncogenes are frequently activated in human tumors by mutations at codon 12 or 61 in their coding sequences. To investigate how these subtle alterations exert such profound effects on the biologic activities of these genes, we studied structural and conformational properties of human ras-oncogene-encoded 21-kDa proteins (p21s). We observed striking differences in the electrophoretic mobilities of the proteins under reducing and nonreducing conditions. These findings imply that intramolecular disulfide bonds affect native p21 conformation. The two activating lesions were shown to induce distinctly different alterations in p21 electrophoretic mobility that were unmasked only under reducing conditions. These results suggest that regions of the molecule containing such alterations are either not exposed or under conformational constraints in the native p21 molecule. We confirmed the opposing effects on protein mobility induced by the two activating lesions by using a recombinant gene containing both lesions. The recombinant gene's high-titer transforming activity further established that the two lesions do not negatively complement one another with respect to transforming-gene function. Our findings of distinct alterations in electrophoretic mobilities of position-12- and position-61-altered p21 molecules should be applicable to the rapid immunologic diagnosis of ras oncogenes in human malignancies.

DNA electrophoresis in designed channels

Sakaue, Takahiro
Fonte: Universidade Cornell Publicador: Universidade Cornell
Tipo: Artigo de Revista Científica
Relevância na Pesquisa
283.04373%
We present a simple description on the electrophoretic dynamics of polyelectrolytes going through designed channels with narrow constrictions of slit geometry. By analyzing rheological behaviours of the stuck chain, which is coupled to the effect of solvent flow, three critical electric fields (permeation field $E^{(per)} \sim N^{-1}$, deformation field $E^{(def)} \sim N^{-3/5}$ and injection field $E^{(inj)} \simeq N^0$, with $N$ polymerization index) are clarified. Between $E^{(per)}$ and $E^{(inj)}$, the chain migration is dictated by the driven activation process. In particular, at $E>E^{(def)}$, the stuck chain at the slit entrance is strongly deformed, which enhances the rate of the permeation. From these observations, electrophoretic mobility at a given electric field is deduced, which shows non-monotonic dependence on $N$. For long enough chains, mobility increases with $N$, in good agreement with experiments. An abrupt change in the electrophoretic flow at a threshold electric field is formally regarded as a nonequilibrium phase transition.; Comment: 11 pages, 8 figures