Reliable molecular weight measurements of RNA molecules as large as 4.0 X 10(6) dalton can be made on agarose gels containing 2.2 M formaldehyde as denaturant (Lehrach et al., 1977). Both eucaryotic and procaryotic ribosomal RNAs have generally been used as molecular weight markers. However, Maniatis et al. (1982) have suggested the use of restriction fragments of DNA as convenient molecular weight markers for RNA samples run in formaldehyde/agarose gels. This communication compares RNA and DNA molecular weight markers run under identical conditions. 相似文献
Summary The artificial sweetener saccharin inhibits binding of epidermal growth factor (EGF) to cultured rat pituitary tumor cells
(GH4C1 cells). Saccharin also causes morphological alterations in these cells, resulting in pronounced elongation, stretching, and
firmer attachment of cells to the culture dishes. These alterations in cell shape are similar to those observed after treatment
of GH4C1 cells with EGF and with thyrotropin-releasing hormone (TRH), both of which enhance prolactin (PRL) production in these cells.
After assaying for PRL in saccharin-treated cultures, it was observed that this sweetener is also capable of stimulating PRL
production two-to sixfold in a dose-dependent manner. Enhancement of PRL production can be observed at 0.5 mM saccharin, yet this is 10 times less than the saccharin concentration required to alter cell shape. These effects of saccharin
on cell morphology and on PRL production are reversible in GH4C1 cell cultures. When added to cultures along with maximal concentrations of EGF or TRH, the effects of saccharin on PRL production
are additive, suggesting that the actions of saccharin are mediated by a somewhat different pathway from that of the peptide
hormones. Pulse labeling studies indicate that the enhancement of PRL production is highly specific inasmuch as saccharin
was found to decrease the overall rate of protein synthesis in these cells. Saccharin also causes a decrease in the rate of
DNA synthesis under these treatment conditions. Mitomycin C, which similarly inhibited DNA synthesis, had no effect on cell
morphology or PRL production.
This investigation was supported by a Faculty Research Grant from Wheaton College 相似文献
The role of circadian rhythmicity in the photoperiodic time measuring processes regulating antifreeze protein production in the beetle Dendroides canadensis was further investigated. Using “T” experiments larvae were exposed to environmental light cycle periods close to the period length of the endogenous circadian oscillator. The following light cycles were employed: light/dark 8/13, 8/14, 8/16, 8/18 and 8/19 corresponding to period lengths of 21, 22, 24, 26 and 27 h. Larvae maintained in cycles equal to or less than 24 h displayed a characteristic short-day response, showing significantly (P < 0.01) greater antifreeze protein activity than did those measured on the day of collection in late summer. In contrast, a long-day response was observed in larvae maintained under a 26- or 27-h light cycle in that antifreeze protein activity did not differ from that measured on the initial collection date.
The role of photoperiod and temperature in influencing the photoperiodic timing processes were examined with a series of resonance experiments. The first group consisted of a 24, 36, 48, 60 or 72-h light cycle, each with an 8-h photophase at temperatures of 20 or 17°C. Rhythmic increases in antifreeze protein levels at intervals of 24 h occurred under both temperatures. However, the lower temperature displaced the resonance curve in the vertical direction (i.e. increasing % population response) and reduced the difference between peaks and troughs on the resonance curve. Resonance experiments incorporating a 14-h photophase resulted in low antifreeze protein activity under all conditions except a 36-h light cycle in which a 67% induction was observed.
Eight hour resonance experiments were also conducted with D. canadensis collected in early spring to determine whether the circadian system participates in the photoperiodic timing processes influencing the spring termination of antifreeze protein production. Positive resonance results were obtained in that only larvae maintained in cycles of 36 and 60 h displayed significantly (P < 0.01) lower antifreeze activity when compared to animals on the initial collection date.
The combined results emphasize the involvement of the circadian system in the photoperiodic control of antifreeze protein production by D. canadensis during the fall and spring. Furthermore, the induction of antifreeze protein production is a function of light cycle and its waveform (photoperiod). Temperature appears to modify the photoperiodic response in some manner involving the photoperiodic time measuring processes. It is concluded that the photoperiodic response of antifreeze protein production by D. canadensis is dependent upon the entrainment of the circadian system by the light cycle. 相似文献
The incorporation of [methyl-H]thymidine into three macromolecular fractions, designated as DNA, RNA, and protein, by bacteria from Hartbeespoort Dam, South Africa, was measured over 1 year by acid-base hydrolysis procedures. Samples were collected at 10 m, which was at least 5 m beneath the euphotic zone. On four occasions, samples were concurrently collected at the surface. Approximately 80% of the label was incorporated into bacterial DNA in surface samples. At 10 m, total incorporation of label into bacterial macromolecules was correlated to bacterial utilization of glucose (r = 0.913, n = 13, P < 0.001). The labeling of DNA, which ranged between 0 and 78% of total macromolecule incorporation, was inversely related to glucose uptake (r = -0.823), total thymidine incorporation (r = -0.737), and euphotic zone algal production (r = -0.732, n = 13, P < 0.005). With decreased DNA labeling, increasing proportions of label were found in the RNA fraction and proteins. Enzymatic digestion followed by chromatographic separation of macromolecule fragments indicated that DNA and proteins were labeled while RNA was not. The RNA fraction may represent labeled lipids or other macromolecules or both. The data demonstrated a close coupling between phytoplankton production and heterotrophic bacterial activity in this hypertrophic lake but also confirmed the need for the routine extraction and purification of DNA during [methyl-H]thymidine studies of aquatic bacterial production. 相似文献
The glucose transporter in the plasma membrane of rat skeletal muscle has been identified by two approaches. In one, the transporter was detected as the polypeptide that was differentially labeled by photolysis with [3H]cytochalasin B in the presence of l- and d-glucose. [3H]Cytochalasin B is a high-affinity ligand for the transporter that is displaced by d-glucose. In the other, the transporter was detected by means of its reaction with rabbit antibodies against the purified glucose transporter from human erythrocytes. By both procedures, the transporter was found to be a polypeptide with a mobility corresponding to a molecular weight of 45,000–50,000 upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 相似文献
An ideal medium for metabolic studies would maintain cultured vascular smooth muscle cells in a quiescent, viable state, as they are in normal arteries in vivo, and would be chemically defined so that the concentrations of hormones and nutrients could be manipulated precisely. In unsupplemented serum-free media these cultures lose protein and DNA, indicating impaired viability. Addition of maximally effective concentrations of insulin (10?6 M) and transferrin (5 m?g/ml) prevents loss of DNA and produces near neutral protein balance. Further addition of ascorbic acid (10?4 M) actually promotes net gain of protein with little or no increase in DNA. Ascorbate consistently increased noncollagen protein synthesis by cultured aortic smooth muscle cells. This novel action of the vitamin did not require insulin but was additive to the effect of this hormone, and was produced by isoascorbate, but not by a variety of other reducing agents. Thus, vascular smooth muscle cells can be maintained in a quiescent but noncatabolic state in simple chemically defined culture media. This finding should facilitate studies of the effects of nutrients and hormones on the metabolism of these cells under conditions that resemble those in the normal artery in vivo. Such an approach may also prove valuable for culture of other differentiated cell types that do not usually divide in the intact organism. 相似文献
Summary The major membrane protein of the bovine lens fiber cell is a 26-kilodalton (kD) protein (MP26), which appears to be a component
of the extensive junctional specializations found in these cells. To examine the arrangement of MP26 within the junctional
membranes, various proteases were incubated with fiber cell membranes that had been isolated with or without urea and/or detergents.
These membranes were analyzed with electron microscopy and SDS-PAGE to determine whether the junctional specializations or
the proteins were altered by proteolysis. Microscopy revealed no obvious structural changes. Electrophoresis showed that chymotrypsin,
papain, and trypsin degraded MP26 to 21–22 kD species. A variety of protease treatments, including overnight digestions, failed
to generate additional proteolysis. Regions on MP26 which were sensitive to these three proteases overlapped. Smaller peptides
were cleaved from MP26 with V8 protease and carboxypptidases A and B. Protein domains cleaved by these proteases also overlapped
with regions sensitive to chymotrypsin, papain, and trypsin. Specific inhibition of the carboxypeptidases suggested that cleavage
obtained with these preparations was not likely due to contaminating endoproteases. Since antibodies are not thought to readily
penetrate the 2–3 nm extracellular gap in the fiber cell junctions, antibodies to MP26 were used to analyze the location of
the protease-sensitive domains. Antisera were applied to control (26 kD) and proteolyzed (22 kD) membranes, with binding being
evaluated by means of ELISA reactions on intact membranes. Antibody labeling was also done following SDS-PAGE and transfer
to derivatized paper. Both assays showed a significant decrease in binding following proteolysis, with the 22 kD product showing
no reaction with the anti-MP26 sera. These investigations suggest that MP26 is arranged with approximately fourfifths of the
primary sequence “protected” by the lipid bilayer and the narrow extracellular gap. One-fifth of the molecule, including the
C-terminus, appears to be exposed on the cytoplasmic side of the membrane. 相似文献
Photoinactivation of ammonia oxidation in cells of Nitrosomonas was shown to follow first-order kinetics with a rate constant proportional to incident light intensity. The action spectrum for photoinactivation consisted of a broad peak in the ultraviolet range, where both hydroxylamine and ammonia oxidation were affected, and a shoulder at approximately 410 nm where only ammonia oxidation was affected. In photoinactivated cells, hydroxylamine but not ammonia was oxidized to nitrite and hydroxylamine but not ammonia caused reduction of cytochromes in vivo. The amount per cell of the following constituents was not measurably altered by photoinactivation: cytochromes b, c, a, and P460; ubiquinone; phospholipid; free amino acids; hydroxylamine-dependent nitrite synthetase; nitrite reductase; p-phenylenediamine oxidase; and cytochrome c oxidase. Malonaldehyde or lipid peroxides were not detected in photoinactivated cells. Photoinactivation was prevented (i) under anaerobic conditions, (ii) in the presence of methanol, allylthiourea, thiosemicarbazide, hydroxylamine, ethylxanthate, or CO at concentrations wich caused 100% inhibition of ammonia oxidation, and (iii) at concentrations of ammonia or hydroxylamine which gave a rapid rate of nitrite production. Recovery of ammonia oxidation activity in 90% inactivated cells took place in 6 h, required an energy and/or nitrogen source, and was inhibited by 400 mug of chloramphenicol per ml. 相似文献