scholarly journals Molecular hybridization between rat liver deoxyribonucleic acid and complementary ribonucleic acid

1970 ◽  
Vol 120 (2) ◽  
pp. 225-235 ◽  
Author(s):  
Marialuisa Melli ◽  
J. O. Bishop
Keyword(s):  
Molecular Weight ◽  
Rat Liver ◽  
Sodium Citrate ◽  
Filter Method ◽  
Dna Molecules ◽  
Membrane Filters ◽  
Diminished Capacity ◽  
Cross Linkage ◽  
Total Dna

RNA (cRNA) was synthesized in vitro on a template of rat liver DNA and its hybridization with rat liver DNA was studied by using the nitrocellulose-filter method. Sonication of the DNA diminished its apparent capacity to hybridize with RNA by about 50%. This is not due to cross-linkage of DNA molecules, because it could be shown that less than 2% of the sonicated DNA was cross-linked. The effect is due instead to the small size of the sonicated DNA molecules. Below a single-stranded molecular weight of 5×105 the DNA showed a progressive loss of capacity to hybridize with decrease in molecular weight. Evidence is presented suggesting that the apparently diminished capacity of the DNA to hybridize is due to loss of hybridized DNA from the membrane filters. When cRNA at concentrations of up to 25μg/ml is annealed with sonicated total DNA, an apparent hybridization saturation value is found at which about 2.5% of the DNA is hybridized with RNA. Increasing the cRNA concentration tenfold brought about the hybridization of a second component of the DNA approximately equal in amount to the first. The renaturation of rat liver DNA was studied by measuring the fall in the extinction at 260nm and two different components of renaturation were observed within the reiterated fraction of DNA. By hybridizing cRNA with different fractions of rat DNA the two components of the hybridization curve are shown to correspond to the two components of the renaturation curve. The conclusion is drawn that at a cRNA concentration of 250μg/ml most of the reiterated fraction of rat liver DNA is hybridized after annealing for 16h under standard conditions (0.30m-sodium chloride–30mm-sodium citrate at 65°C). Even with such a high cRNA concentration little or no hybridization of the slowly renaturing DNA fraction occurs. It is suggested that the most highly reiterated DNA component is poorly transcribed in vitro.

Regulation of rat liver maturation in vitro by glucocorticoids

1988 ◽  
Vol 8 (1) ◽  
pp. 203-209
Author(s):  
J Y Chou ◽  
Y J Wan ◽  
T Sakiyama
Keyword(s):  
Molecular Weight ◽  
Rat Liver ◽  
Fetal Liver ◽  
Tyrosine Aminotransferase ◽  
Temperature Sensitive ◽  
Adult Rat ◽  
Normal Hepatocyte ◽  
Fetal Rat ◽  
Afp Mrna

The biochemistry of liver maturation was studied by using the RLA209-15 fetal rat hepatocyte line that is temperature sensitive for maintenance of the differentiated fetal liver phenotype. At 33 degrees C these cells were dedifferentiated; but at 40 degrees C they were phenotypically differentiated and, like normal fetal hepatocytes, synthesized moderate levels of albumin and transferrin, high levels of authentic (69,000 and 73,000 molecular weight) rat fetal alpha-fetoprotein (AFP), and low levels of a 65,000-molecular-weight variant AFP. Our results indicated that administration of glucocorticoid hormones to RLA209-15 cells at 40 degrees C induced a series of events associated with normal hepatocyte maturation; synthesis of fetal AFP was inhibited, whereas the synthesis of variant AFP, albumin, transferrin, tyrosine aminotransferase, and alpha 1-acid glycoprotein was induced. The variant AFP was produced by RLA209-15 cells at both temperatures and was encoded by an mRNA of 1.7 kilobases (kb). The fetal AFP was encoded by an mRNA of 2.2 kb. Normal adult rat liver contained three AFP mRNAs of 2.2 (minor), 1.7, and 1.5 kb. The 1.7-kb adult liver AFP mRNA comigrated with the RNA found in RLA209-15 cells, and both directed the synthesis of a 50,000-molecular-weight precursor polypeptide of the variant AFP. Administration of glucocorticoids to RLA209-15 cells grown at 33 degrees C stimulated synthesis of both the fetal and variant AFPs, but the levels of the 2.2-kb AFP mRNA were preferentially increased. RLA209-15 cells contained two glucocorticoid receptor mRNAs of 6.8 and 4.5 kb. The glucocorticoid-mediated maturation described above was blocked by the antiglucocorticoid RU486.

scholarly journals Regulation of rat liver maturation in vitro by glucocorticoids.

1988 ◽  
Vol 8 (1) ◽  
pp. 203-209 ◽  
Author(s):  
J Y Chou ◽  
Y J Wan ◽  
T Sakiyama
Keyword(s):  
Molecular Weight ◽  
Rat Liver ◽  
Fetal Liver ◽  
Tyrosine Aminotransferase ◽  
Temperature Sensitive ◽  
Adult Rat ◽  
Normal Hepatocyte ◽  
Fetal Rat ◽  
Afp Mrna

The biochemistry of liver maturation was studied by using the RLA209-15 fetal rat hepatocyte line that is temperature sensitive for maintenance of the differentiated fetal liver phenotype. At 33 degrees C these cells were dedifferentiated; but at 40 degrees C they were phenotypically differentiated and, like normal fetal hepatocytes, synthesized moderate levels of albumin and transferrin, high levels of authentic (69,000 and 73,000 molecular weight) rat fetal alpha-fetoprotein (AFP), and low levels of a 65,000-molecular-weight variant AFP. Our results indicated that administration of glucocorticoid hormones to RLA209-15 cells at 40 degrees C induced a series of events associated with normal hepatocyte maturation; synthesis of fetal AFP was inhibited, whereas the synthesis of variant AFP, albumin, transferrin, tyrosine aminotransferase, and alpha 1-acid glycoprotein was induced. The variant AFP was produced by RLA209-15 cells at both temperatures and was encoded by an mRNA of 1.7 kilobases (kb). The fetal AFP was encoded by an mRNA of 2.2 kb. Normal adult rat liver contained three AFP mRNAs of 2.2 (minor), 1.7, and 1.5 kb. The 1.7-kb adult liver AFP mRNA comigrated with the RNA found in RLA209-15 cells, and both directed the synthesis of a 50,000-molecular-weight precursor polypeptide of the variant AFP. Administration of glucocorticoids to RLA209-15 cells grown at 33 degrees C stimulated synthesis of both the fetal and variant AFPs, but the levels of the 2.2-kb AFP mRNA were preferentially increased. RLA209-15 cells contained two glucocorticoid receptor mRNAs of 6.8 and 4.5 kb. The glucocorticoid-mediated maturation described above was blocked by the antiglucocorticoid RU486.

Cell-free translation of mitochondrial nicotinamide nucleotide transhydrogenase

1985 ◽  
Vol 5 (6) ◽  
pp. 483-490 ◽  
Author(s):  
Elisabeth Carlenor ◽  
Vigg Joste ◽  
B. Dean Nelson ◽  
Jan Rydström
Keyword(s):  
Molecular Weight ◽  
Rat Liver ◽  
Liver Mitochondria ◽  
Cytochrome Bc1 Complex ◽  
Free System ◽  
Nicotinamide Nucleotide Transhydrogenase ◽  
Free Translation ◽  
A Cell ◽  
Matrix Fraction

Mammalian nicotinamide nucleotide transhydrogenase is translated as a 5000 daltons larger molecular weight precursor in a cell-free system programmed with rat liver polysomes. The mature rat liver enzyme had the same molecular weight as the purified beef heart enzyme, 115 000 daltons. The precursor was not processed in vitro by liver mitochondria or by a rat liver mitochondrial matrix fraction, nor did it appear to bind to mitochondria. In contrast, pre-FeS protein of the cytochrome bc1 complex was processed in the same samples by both mitochondria and matrix, suggesting an important difference in the processing mechanisms or in the efficiency of processing of the two precursors.

scholarly journals Effects of cytochalasin B on membrane-associated microfilaments in a cell-free system.

1981 ◽  
Vol 91 (2) ◽  
pp. 524-530 ◽  
Author(s):  
M J Phillips ◽  
M Oda ◽  
I M Yousef ◽  
K Funatsu
Keyword(s):  
Molecular Weight ◽  
Plasma Membrane ◽  
Rat Liver ◽  
Mode Of Action ◽  
Actin Filaments ◽  
Cytochalasin B ◽  
Bile Canaliculus ◽  
Free System ◽  
A Cell

The mode of action of cytochalasin B was examined in vitro using bile canaliculus-enriched plasma membrane fractions isolated from rat liver. The pericanalicular microfilaments, which are mainly actin filaments and which are normally attached to the canalicular membranes, were dissociated from the membranes by cytochalasin B treatment. A microfilamentous network was found in the supernate of the cytochalasin B treatment. A microfilamentous network was found in the supernate of the cytochalasin-treated specimens and a number of polypeptides, of which a polypeptide corresponding in molecular weight to actin was a notable member. These results suggest that actin filaments become detached from the canaliculus membranes by cytochalasin B.

scholarly journals The degradation of intravenously injected chondroitin 4-sulphate in the rat

1973 ◽  
Vol 134 (4) ◽  
pp. 1009-1013 ◽  
Author(s):  
Keith M. Wood ◽  
Frederick S. Wusteman ◽  
C. Gerald Curtis
Keyword(s):  
Molecular Weight ◽  
Rat Liver ◽  
Degradation Products ◽  
Chondroitin Sulphate ◽  
Whole Body ◽  
Low Molecular Weight ◽  
Inorganic Sulphate ◽  
Liver Lysosomes ◽  
Isolated Liver

The degradation of chondroitin 4-[35S]sulphate isolated from chick-embryo cartilage was studied in the rat by experiments on free-range animals, on wholly anaesthetized animals with ureter cannulae, by perfusion of isolated liver, by whole-body radioautography and by isolation of liver lysosomes. After injection into rats 68% of the radioactivity was recovered in the urine after 24h, approximately one-half of this being in the form of low-molecular-weight material, chiefly inorganic sulphate. Cannulation experiments demonstrated that the proportion of low-molecular-weight components excreted in the urine increased with time until, after 12h, virtually all was inorganic sulphate. Whole-body radioautography identified the liver as the major site of radioisotope accumulation after injection of labelled polysaccharide. Perfusion through isolated liver indicated that this organ has the ability to metabolize the polymer with the release of low-molecular-weight products, principally inorganic sulphate. Incubation of a lysosomal fraction prepared from rat liver after injection of chondroitin 4-[35S]sulphate gave rise to degradation products of low molecular weight, and experiments in vitro with rat liver lysosomes confirmed that these organelles are capable of the entire degradative process from chondroitin sulphate to free inorganic sulphate.

Kinetic study of the interaction between rat haptoglobin and rat liver cathepsin B

1980 ◽  
Vol 58 (5) ◽  
pp. 410-417 ◽  
Author(s):  
M. Pagano ◽  
R. Engler ◽  
M. Gelin ◽  
M. F. Jayle
Keyword(s):  
Molecular Weight ◽  
Kinetic Study ◽  
Rat Liver ◽  
Cathepsin B ◽  
Michaelis Constant ◽  
Rat Serum ◽  
Acute Phase Reactants ◽  
Serum Haptoglobin ◽  
Lysosomal Proteinase

One of the roles of the acute phase reactants (APR), according to Koj, is to regulate the action of tissue proteinases released during the inflammatory reaction. To study this phenomenon in vitro, rat liver cathepsin B (EC 3.4.22.1), a lysosomal proteinase, and a typical APR, rat serum haptoglobin, were purified. By kinetic study, haptoglobin was found to inhibit cathepsin B and the inhibitory activity of a competitive type was increased with the molecular weight of the substrate. When 125I-labelled denatured bovine serum albumin (BSA) was used as a substrate, the apparent Michaelis constant (Km app.) for cathepsin B was 5 ± 0.4 × 10−6 M. When haptoglobin was added, the apparent inhibition constant (Ki app.) was 3.5 ± 1.5 × 10−8 M. Native haptoglobin was not catabolized by cathepsin B while under the same conditions denatured haptoglobin was degraded by the enzyme.

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