Immunochemical analysis of heat-shock protein synthesis in maize (Zea mays L.)

1986 ◽  
Vol 28 (6) ◽  
pp. 1076-1087 ◽  
Author(s):  
Chris L. Baszczynski
Keyword(s):  
Molecular Weight ◽  
Heat Shock ◽  
Molecular Mass ◽  
High Molecular Weight ◽  
Polyclonal Antibodies ◽  
Cross Reactivity ◽  
Antigenic Determinants ◽  
Genetic Stocks ◽  
Young Leaves ◽  
Shock Proteins

Polyclonal antibodies to 18-kilodalton (kDa) heat-shock proteins (HSPs) and to the high molecular weight (73 000 – 89 000) HSPs from 5- day-old maize plumules have been produced in rabbits. The antisera to high molecular weight HSPs show minor cross-reactivity to proteins of similar molecular mass in not heat-shocked tissues, while antisera to 18-kDa HSPs react only with this 18-kDa HSP class. HSPs of similar molecular mass and isoelectric points in maize plumules, mesocotyls, radicles, and young leaves also have similar antigenic determinants based on positive reactions with antisera to plumule HSPs. Among 13 maize inbreds and genetic stocks tested, differences were noted in the amount of immunoprecipitable 18-kDa HSPs. Antisera to maize plumule HSPs also showed cross-reactivity with similar-sized HSPs from pea epicotyls and soybean hypocotyls but not with HSPs from several animal tissues.Key words: polyclonal antibodies, maize, heat-shock.

scholarly journals Durable synthesis of high molecular weight heat shock proteins in G0 cells of the yeast and other eucaryotes.

1984 ◽  
Vol 99 (1) ◽  
pp. 199-207 ◽  
Author(s):  
H Iida ◽  
I Yahara
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Weight ◽  
Heat Shock ◽  
Heat Shock Proteins ◽  
High Molecular Weight ◽  
Resting State ◽  
Specific Class ◽  
Embryonic Fibroblasts ◽  
Long Period ◽  
Shock Proteins

We report that eucaryotic cells were induced to synthesize a specific class of heat shock proteins (hsps) when they entered the resting state, G0. This finding was originally made with Saccharomyces cerevisiae cells by taking advantage of the system in which we can distinguish between G1 arrests leading to G0 and those that do not result in G0 (Iida, H., and I. Yahara, 1984, J. Cell Biol. 98:1185-1193). Similar observations were subsequently made with higher eucaryotic cells including chick embryonic fibroblasts (CEF), mouse T lymphocytes, and Drosophila GM1 cells. The induction of hsps in G0 cells was distinct from that in heat-shocked cells in two respects. First, hsps with molecular weight around 25,000 were not induced in G0 cells, whereas most, if not all, high molecular weight (HMW) hsps were commonly induced both in G0 cells and in heat-shocked cells. Second, in contrast to the transient synthesis of hsps in heat-shocked cells, G0 cells continued to synthesize hsps at the stimulated rate for a relatively long period. These results suggest the possibility that high molecular weight hsps might function in a transition from the proliferating state to G0 or in maintaining G0 in the eucaryote.

Identification of mRNAs encoding low molecular mass heat-shock proteins in maize (Zea mays L.)

1986 ◽  
Vol 28 (6) ◽  
pp. 1106-1114 ◽  
Author(s):  
C. A. B. Rees ◽  
N. C. Hogan ◽  
D. B. Walden ◽  
B. G. Atkinson
Keyword(s):  
Heat Shock ◽  
Heat Shock Proteins ◽  
Molecular Mass ◽  
Polyclonal Antibodies ◽  
Relative Molecular Mass ◽  
Temperature Heat ◽  
Free Translation ◽  
Shock Proteins

Subjecting 5-day-old maize seedlings to a rapid elevation in growth temperature (heat shock; 25–42 °C) results in a shift in the pattern of protein synthesis in maize plumules from the production of a broad spectrum of proteins to the new and (or) enhanced synthesis of a small number of heat-shock proteins (HSPs). The low relative molecular mass (Mr) HSPs, and more specifically an 18-kDa HSP with four major isoelectric variants, represent the majority of HSP synthesis following cell-free translation of total cellular poly (A)+ RNAs and polyribosomal RNAs extracted from heat-shocked plumules. Immunochemical studies, using polyclonal antibodies raised against the 18-kDa HSPs, show that the 18-kDa HSPs synthesized in vitro share immunochemical properties with HSPs of the same Mr synthesized in vivo by heat-shocked plumules. Furthermore, size fractionation and translation analyses of total cellular poly(A)+ RNAs extracted from heat-shocked plumules demonstrate that poly(A)+ RNAs encoding an 18-kDa HSP(s) have an estimated size of 0.6–0.95 kilobases. The observation that 18-kDa HSPs are absent among the translation products and immunoprecipitates of proteins synthesized in vitro by RNAs extracted from control plumules (25 °C) suggests that the mRNAs encoding 18-kDa HSPs are heat-shock induced.Key words: mRNA, maize, heat shock.

Temperature-stress response in maize: a comparison of several cultivars

1986 ◽  
Vol 28 (6) ◽  
pp. 1125-1131 ◽  
Author(s):  
Reza K. Yacoob ◽  
W. Gary Filion
Keyword(s):  
Molecular Weight ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Cold Tolerance ◽  
Cold Shock ◽  
Polyclonal Antibodies ◽  
Cross Reactivity ◽  
Species Variation ◽  
Cold Shock Response ◽  
Shock Response

The protein synthetic response to a heat (28–41 °C) and a cold (28–4 °C) shock was studied in seedlings from 10 cultivars of maize with varying levels of cold tolerance. This response was compared by fluorography of one-dimensional polyacrylamide gels and immunoblot analysis. We utilized polyclonal antibodies against the 18 000 dalton (Da) heat-shock protein and the 73 000–89 000 Da heat-shock protein complex from Oh43 maize seedlings to ascertain antigenic similarity of these polypeptides. The heat-shock response varied in the numbers and relative molecular masses of the heat-shock proteins. Only three polypeptides appeared to be conserved across cultivars: a 93 000, 71 000, and 18 000 Da polypeptide. The cold-shock response varied from none to a dramatically altered pattern in a few cultivars. Thus, the heat- and cold-shock responses in these cultivars of corn differ in the types of polypeptides that are induced. All cultivars showed varying degrees of cross-reactivity when probed with the anti 18 000 Da heat-shock protein antibody. The inbred lines appeared to respond more to a cold shock than the hybrid lines but there was little relationship between the cold tolerance and the induction of a cold-shock response. Two of the cultivars demonstrated unique binding to a higher molecular weight polypeptide under control (28 °C) conditions. These data suggest that within species variation in both number and relative molecular weight of thermal stress polypeptides (heat and cold) is a function of genotype.Key words: heat shock, cold shock, cold tolerance, maize, gene expression.

scholarly journals Purification of a high molecular weight actin filament gelation protein from Acanthamoeba that shares antigenic determinants with vertebrate spectrins.

1984 ◽  
Vol 99 (6) ◽  
pp. 1970-1980 ◽  
Author(s):  
T D Pollard
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Actin Filament ◽  
Fluorescent Antibody ◽  
Deae Cellulose ◽  
Cross Reactivity ◽  
Actin Binding ◽  
Antigenic Determinants ◽  
Selective Precipitation ◽  
Pig Brain

I have purified a high molecular weight actin filament gelation protein (GP-260) from Acanthamoeba castellanii, and found by immunological cross-reactivity that it is related to vertebrate spectrins, but not to two other high molecular weight actin-binding proteins, filamin or the microtubule-associated protein, MAP-2. GP-260 was purified by chromatography on DEAE-cellulose, selective precipitation with actin and myosin-II, chromatography on hydroxylapatite in 0.6 M Kl, and selective precipitation at low ionic strength. The yield was 1-2 micrograms/g cells. GP-260 had the same electrophoretic mobility in SDS as the 260,000-mol-wt alpha-chain of spectrin from pig erythrocytes and brain. Electron micrographs of GP-260 shadowed on mica showed slender rod-shaped particles 80-110 nm long. GP-260 raised the low shear apparent viscosity of solutions of Acanthamoeba actin filaments and, at 100 micrograms/ml, formed a gel with a 8 microM actin. Purified antibodies to GP-260 reacted with both 260,000- and 240,000-mol-wt polypeptides in samples of whole ameba proteins separated by gel electrophoresis in SDS, but only the 260,000-mol-wt polypeptide was extracted from the cell with 0.34 M sucrose and purified in this study. These antibodies to GP-260 also reacted with purified spectrin from pig brain and erythrocytes, and antibodies to human erythrocyte spectrin bound to GP-260 and the 240,000-mol-wt polypeptide present in the whole ameba. The antibodies to GP-260 did not bind to chicken gizzard filamin or pig brain MAP-2, but they did react with high molecular weight polypeptides from man, a marsupial, a fish, a clam, a myxomycete, and two other amebas. Fluorescent antibody staining with purified antibodies to GP-260 showed that it is concentrated near the plasma membrane in the ameba.

Immunological analyses of selected eukaryotic RNA polymerases II

1985 ◽  
Vol 63 (12) ◽  
pp. 1217-1230 ◽  
Author(s):  
Michael F. Bettiol ◽  
Randall T. Irvin ◽  
Paul A. Horgen
Keyword(s):  
Molecular Weight ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
High Molecular Weight ◽  
Wheat Germ ◽  
Polyclonal Antibodies ◽  
Sodium Dodecyl ◽  
Cross Reactivity ◽  
Rna Polymerases ◽  
Antibody Preparations

Polyclonal antibodies to native RNA polymerase II of Achlya ambisexualis and Agaricus bisporus were produced in rabbits and in mice. Monoclonal antibodies were produced against the α-amanitin resistant RNA polymerase II of the mushroom A. bisporus. These antibodies were used in comparative cross-reactivity studies with five purified RNA polymerases II (A. bisporus, A. ambisexualis, Saccharomyces cerevisiae, wheat germ, and calf thymus). A method for quantitatively comparing cross-reactivity was developed utilizing an enzyme-linked immunosorbant assay (ELISA). ELIS A comparisons indicated that the two filamentous fungi cross-reacted effectively with one another and depending upon the preparation reacted less effectively with yeast and wheat germ RNA polymerases II. Cross-reactivity measurements were also made by immunoblotting sodium dodecyl sulfate – polyacrylamide separated RNA polymerases II. The mouse anti-A. bisporus RNA polymerase II immunoglobulin G (IgG) and the monoclonal antibody preparations did not react with high molecular subunits of A. bisporus RNA polymerase II. The sera did, however, cross-react with high molecular weight subunits of A. ambisexualis. Similarily, rabbit anti-A. ambisexualis RNA polymerase II IgG reacted only with low molecular weight subunits of A. bisporus RNA polymerase II, but reacted with high molecular weight subunits of A. ambisexualis and wheat germ. Our results indicate differences in the cross-reactivity of native and denatured RNA polymerases II and suggest differences in the tertiary and quaternary organization of the enzymes examined.

scholarly journals Production of monoclonal antibodies that detect Hodgkin's high molecular weight transforming growth factor-beta

Blood ◽  
1990 ◽  
Vol 75 (12) ◽  
pp. 2434-2437
Author(s):  
SR Newcom ◽  
LH Muth ◽  
ET Parker
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibodies ◽  
Growth Factor ◽  
High Molecular Weight ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Antigenic Determinants ◽  
Enzyme Linked Immunosorbent Assay ◽  
Tgf Beta ◽  
Growth Factor Beta

High molecular weight transforming growth factor-beta (TGF beta) is a physiologically active TGF secreted by nodular sclerosing Reed- Sternberg cells. Five monoclonal murine antibodies were prepared that distinguished Hodgkin's TGF beta from platelet-derived TGF beta using an enzyme-linked immunosorbent assay, neutralization of biologic activity, and Western blotting. These monoclonal antibodies directed at unique antigenic determinants (epitopes) of Hodgkin's TGF beta will allow further characterization of the role of Hodgkin's TGF beta in Hodgkin's disease and related entities.

HUMAN HEPATOCYTES CONTAIN HIGH MOLECULAR WEIGHT POLYPEPTIDES OF FACTOR VIII

1987 ◽  
Author(s):  
J Ingerslev ◽  
B Sloth Christiansen ◽  
A Bukh ◽  
S Stenbjerg ◽  
T Munck Jørgensen ◽  
...  
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Polyclonal Antibodies ◽  
Plasma Source ◽  
Isolated Hepatocytes ◽  
Human Hepatocytes ◽  
Isolated Cells ◽  
Left Liver Lobe ◽  
Viable Cells ◽  
A Cell

Human hepatocytes were isolated by the two-step collagenase technique applied on distal left liver lobe. Homogenous and large cells were isolated revealing hepatocyte characteristics by light-microscopy. Hepatocytes were washed repeatedly in albumine buffer (5%), resuspended in the same buffer and sonicated using a cell density of 0.75 × 106 cells/ml. In some cases cells were separated from non-viable cells by flotation on a linear Percoll gradient. Supernate material after sonication was subjected to ELISA for VIII:Ag using human antibodies and vWf:Ag by polyclonal antibodies. Freshly isolated cells contained at least 0.25 IU/ 0.75 × 106 hepatocytes, whereas the vWf:Ag was below 0.01 IU/ 0.75 × 106 cells. The material obtained from sonication was further studied using fast protein liquid chromatography by Mono-Q HR 5/5 revealing a single peak of VIII: Ag eluting in the same position as the high molecular weight polypeptides of VIII :Ag of high purity FVIII derived from the plasma source. Isolated hepatocytes also were cultivated at 37°C in medium RPMI 1640 supplemented with Ultroser G (4%), glutamine and antibiotics. Cells secreted increasing quantities of albumin, fitrinogpn and protease-inhibitors. The supernatants also contained VIII: Ag in quantities ranging from 0.04 - 0.17 IU/ml after 24 hours, but no further secretion was observed. No vWf: Ag could be detected. Cells harvested and sonicated after 30 hours of culture only contained 0.04 IU/ 0.75 × 106 cells. Our results shows, that VIII :Ag is present in freshly isolated human hepatocytes and that only traces of vWf:Ag is found. A hepatocyte site of production of VIII is speculated. These very preliminary findings do not permit conclusions concerning active synthesis of VIII in hepatocytes. Further studies are underway.

Sign in / Sign up

Export Citation Format

Share Document