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Biomedical subjects

G Krystal

Publications and source records attributed to G Krystal.

122 records · Page 7Linked to original sources
PubMed1981

Properties of a methotrexate-insensitive variant of dihydrofolate reductase derived from methotrexate-resistant L5178Y cells.

We have established the presence of two distinct forms of dihydrofolate reductase in methotrexate-resistant L5178Y lymphoma cells. It was previously found that one of the reductases (Form II) was approximately 100,000-fold more resistant to methotrexate inhibition than Form I. In this report, the two forms of dihydrofolate reductase have been partially purified and their properties have been compared. The two forms were found to differ markedly in their heat stability, isoelectric points, and sensitivity to p-chloromercuriphenyl sulfonate. There were, in addition, minor differences in their Km and Vmax for folic acid, their pH optima, and their molecular weights. The loss of sensitivity to methotrexate inhibition by the Form II reductase is associated with significant alteration in the physical and chemical properties of the enzyme.

Animals↗
PubMed1981

A quantitative bioassay for erythropoietin, using mouse bone marrow.

A new in vitro assay for epo using the suspension culture-59Fe incorporation technique has been devised. The assay is based on the use of readily available adult mouse marrow cells. Particular attention has been given to defining conditions that would permit this assay to be applied to the evaluation of epo levels in a wide variety of test samples, including human sera, which contain factors in addition to epo that influence both the level of hemoglobin synthesis obtained and the extent of 59Fe incorporation. To eliminate variability in 59Fe uptake due to uncontrolled additions of transferrin or unlabeled iron from test samples, the assay has been split into two parts separated by a centrifugation and cell wash step. Optimal conditions during the initial 24 hr when cells were exposed to epo were found to include the use of 20% FCS (preselected for its ability to support erythroid colony formation in vitro), 0.1 mM beta-mercaptoethanol, and 2 X 10(6) marrow cells in a final culture volume of 0.5 ml. Optimal labeling during the subsequent 3 hr exposure to [59Fe]ferrous citrate in serum-free medium required the addition of 50 microgram/ml transferrin. Substantial diminution of 59Fe incorporation was observed below 10 and above 150 microgram/ml. Optimal labeling iron concentrations were provided during the labeling period solely by 1 muCi of [59Fe]ferrous citrate (10,000 to 25,000 muCi/mg of Fe). Thus no addition of unlabeled iron was necessary, and reduction of the specific activity of the added 59Fe by exogenous Fe from FCS or test samples was avoided. Careful choice of an appropriate prelabel incubation period (23 to 25 hr) made it possible to measure epo levels from 400 down to 1 mU per 0.5 ml culture, with a corresponding variation in counts of 12-fold. By comparison, the minimum epo level detectable with fetal liver cells under the same conditions was 10 mU/0.5 ml culture, and the maximum range in counts was less than fourfold.

Animals↗
PubMed1981

Determination of normal human serum erythropoietin levels, using mouse bone marrow.

A simple, rapid, and economic method for measuring epo bioactivity in small samples of normal human serum has been developed. The method requires an initial fractionation of the test sample on WG-Sepharose beads. The material eluted by 1 mM N,N'-diacetylchitobiose is then assayed for its ability to stimulate the uptake of 59Fe into heme by mouse marrow cells under conditions previously optimized for the stimulation of epo-dependent hemoglobin synthesis. Potential secondary effects on 59Fe uptake, due to variations in the iron-transferrin content of different serum samples, were eliminated by the introduction of a centrifugation and cell-wash step prior to the addition of 59Fe. Preliminary chromatography of test samples on WG was necessary because different samples of untreated human serum were still found to give variable secondary effects on 59Fe uptake when assayed in the presence of increasing amounts of an epo standard. These were more clearly revealed when human serum was chromatographed on Sephadex G150, and the presence of a number of "inhibitors" and "stimulators" (ranging from 8000 to more than 150,000 daltons) were demonstrated. In contrast, only material that was additive with epo was demonstrable in the WG column eluate, and this activity coeluted with epo on Sephadex G150. With the WG fractionation procedure, sera from 20 normal volunteers between the ages of 20 and 40 were assayed for epo bioactivity. Epo levels ranging from 0.3 to 1.3 U/ml (average 0.8) were obtained. Values for men and women were not significantly different. Comparison of the extent of 59Fe incorporation obtained before and after WG fractionation showed no correlation, indicating significant natural variation in the relative amounts of non-epo stimulators and inhibitors in the sera of different normal individuals.

Animals↗
PubMed1978

Selective digestion of mouse metaphase chromosomes.

Metaphase chromosomes prepared from colcemid-treated mouse L929 cells by non-ionic detergent lysis exhibit distinct heterochromatic centromere regions and associated kinetochores when viewed by whole mount electron microscopy. Deoxyribonuclease I treatment of these chromosomes results in the preferential digestion of the chromosomal arms leaving the centromeric heterochromatin and kinetochores apparently intact. Enrichment in centromere material after DNase I digestion was quantitated by examining the increase in 10,000 X g pellets of the 1.691 g/cc satellite DNA relative to main band DNA. This satellite species has been localized at the centromeres of mouse chromosomes by in situ hybridization. From our analysis it was determined that DNase I digestion results in a five to six-fold increase in centromeric material. In contrast to the effect of DNase I, micrococcal nuclease was found to be less selective in its action. Digestion with this enzyme solubilized both chromosome arms and centromeres leaving only a small amount of chromatin and intact kinetochores.

Animals↗
PubMed1978

Partial purification and characterization of the intercellular bridge from cultured mouse cells.

At the completion of mitosis, the two daughter cells are connected by a channel of membrane-bound cytoplasm, the intercellular bridge. This structure contains parallel arrays of spindle microtubules which are associated, at the bridge midline, with a metallophilic band called the midbody. In an effort to characterize midbody components, intercellular bridges were partially purified. The purification consisted of brief sonication of telophase populations of mouse L929 cells in order to shear intercellular bridges from daughter cells, digestion of chromatin by an excess of micrococcal nuclease, and differential centrifugation to enrich for intercellular bridges. Electron microscopy of these preparations substantiated the identity of the bulk of material as intercellular bridges. After solubilization with sodium dodecyl sulfate, the protein components of these preparations were iodinated with Na(125)I and separated by two-dimensional gel electrophoresis. From these analyses, the major polypeptide components of intercellular bridges appear to be tubulin, varying amounts of plasma membrane proteins, and a polypeptide with an apparent molecular weight of 42,000. Time-course studies reveal that this polypeptide is first detectable in a pelletable form approximately 30 min after cells are released from metaphase block, reaches maximal spot intensity in late telophase, and is no longer detectable in G1 populations. We interpret these data to suggest that the 42,000-dalton polypeptide is a component of the midbody.

Cells, Cultured↗
PubMed1976

Regulated transcription of the genomes of defective virions and temperature-sensitive mutants of reovirus.

Defective reovirus, which lacks the largest (L1) of the 10 double-stranded (ds) RNA genomic segments, attaches to L cells and is uncoated in the same way as reovirus. The defective genome does not replicate in the cells, but it is transcribed. During the first 5 h after infection, three of the genomic segments, M3, S3, and S4, are more frequently transcribed than the remaining six segments. During the succeeding 5 h, there is a transition to a situation in which all nine segments are transcribed at the same relative frequencies. Since the class C ts mutation has been allocated to the L1 segment (Spandidos and Graham, 1975) the transcription of the C mutant genome was investigated in cells infected with it at the nonpermissive temperature, at which the parental genome does not replicate. Genomic segments L1, M3, S3, and S4 are predominantly transcribed at early times, and later all 10 segments are transcribed with the same relative frequencies. Transcription of the defective viral genome and the C mutant genome is therefore regulated in the same way as previously found for wild-type virus (Nonoyama, Millward, and Graham, 1974), and the regulation is independent of genome replication. Apparently the L1 segment function is involved in dsRNA synthesis but not in regulating the early to late transcription. It is suggested that a cellular repressor may be involved in this regulation and that derepression might be effected by one of the early viral gene products. Virion transcriptase activity was studied in vitro with cores prepared by chymotrypsin digestion of purified defective and standard virions. For both genomes the relative frequencies of transcription of the dsRNA segments are inversely proportional to their molecular weights. These results can be accounted for in a model that postulates each segment to be transcribed independently of the other. The same model with certain restrictions can describe the in vivo transcription of the viral genome.

Cell-Free System↗
PubMed1971

Multiple forms of uridine kinase in normal and neoplastic rat liver.

Two species of uridine kinase with molecular weights of approximately 120000 (I) and 30000 (II) have been identified in the rat liver system. Species I predominates in the 7-day postnatal and adult rat liver and increases in the regenerating remnant of the latter after partial hepatectomy; the concentration of species II is low in these tissues. Species I also predominates in the slow-growing hepatomas 5123D and 7800. In contrast, II is the predominant form in the foetal rat liver and accounts for 40% of the total activity in the rapidly growing Novikoff ascites hepatoma. In contrast to species II, which was stable, species I was inactivated by preincubation for 30min at 37 degrees C, before assay at 23 degrees C.

Animals↗