Analysis of alpha-fetoprotein by a procedure combining crossed immunoaffinoelectrophoresis and immunoblotting.

A newly developed procedure combining crossed immunoaffinoelectrophoresis and an immunoblotting assay (CIAE-IBA) has been used to discriminate between and precisely quantitate lectin-bound and lectin-free forms of alpha-fetoprotein (AFP) derived from human serum at levels of AFP as low as 5.6 ng/ml (adult reference range, 0 to 15). Twenty-two serum specimens with elevated levels of AFP were examined by a reference CIAE method, using either of two lectins, concanavalin A (Con A) or Lens culinaris agglutinin (LCA); aliquots of the specimens were then diluted in AFP-free serum to normal or near-normal levels of AFP and examined by CIAE-IBA, which included a final protein transfer to nitrocellulose. The quantitative results produced by the two methods were essentially the same. Overall, the sensitivity of the CIAE-IBA procedure surpasses that of other CIAE methods of AFP fractionation. The CIAE-IBA procedure may be used for study of the heterogeneity of AFP and may be applicable as well to the study of other proteins.

Analysis of alpha-fetoprotein heterogeneity by an improved crossed immunoaffinoelectrophoretic technique.

The crossed immunoaffinoelectrophoretic procedure described here for the analysis of alpha-fetoprotein heterogeneity includes three major modifications of a procedure previously used by others: impregnation of the second dimension gel with methylated sugar derivatives; rigorous gel washing at 37 degrees C with the same derivatives; and an immunostaining technique. These simple changes have resulted in clear, sharp electrophoretic patterns at levels of total alpha-fetoprotein as low as 50 ng/ml, a sensitivity not previously approached with the method. Individual peaks representing as little as 7.5 ng/ml can be identified.

Differential effect of coformycin on the cell cycle traverse of normal and simian virus 40-transformed human fibroblasts.

Normal and simian virus 40-transformed human embryonic lung fibroblasts in culture were exposed to 3.5 microM coformycin (CF). This treatment resulted in almost complete inhibition of adenosine deaminase activity in both cell lines and retarded the progress of the fibroblasts through the cell cycle, as measured by the rate of cell proliferation. There was a marked difference, however, in the effect of CF on the traverse of different segments of the cell cycle. In normal fibroblasts, CF rapidly but transiently inhibited cell entry into visible stages of mitosis and delayed the progress through S and G2. In transformed fibroblasts, the effects of CF on the cell cycle included an early acceleration of the cell entry into visible mitosis and prolongation of this phase of the cell cycle. These results indicate that inhibition of adenosine deaminase can have different effects on the traverse of the critical segments of the cell cycle in normal and transformed cells.

Basis for the differential action of aminonucleoside on normal and transformed human fibroblasts.

Acid-soluble extracts of normal human fibroblasts (IMR 90 cells) exposed to [3H]aminonucleoside of puromycin ([3H]AMS) contained larger amounts of unchanged AMS than did similar extracts of their transformed counterparts (AG 2804 cells). The radioactive compounds present in IMR 90 cells were further analyzed by sequential high-voltage paper electrophoresis, enzyme digestion, and paper chromatography. In addition to unchanged [3H]AMS, only [3H]adenosine, and [3H]inosine, and [3H]adenosine monophosphate could be detected, apparently derived from [3H]adenosine present in the [3H]AMS samples added to the cultures. Consistent with the absence of metabolism of AMS in IMR 90 cells was the failure to find AMS derivatives in the RNA or DNA of these cells. The content of ribonucleoside triphosphates (rNTPs) in acid-soluble extracts in IMR 90 cells was significantly reduced by AMS treatment, and nuclei or broken cell preparations obtained from AMS-treated IMR 90 cells incorporated [3H]UTP into macromolecules at approximately control rates, when supplemented with rNTPs. These findings indicated that the reduced level of rNTPs may be responsible for the AMS-induced inhibition of RNA synthesis in normal cells.

Nascent RNA chain termination and ultrastructural changes in nucleoli isolated from SV40-transformed fibroblasts treated with aminonucleoside of puromycin.

The mechanism for the selective inhibition of preribosomal RNA synthesis by puromycin aminonucleoside (AMS) was investigated in cultured AG 2804 cells, which are human embryonic lung fibroblasts transformed by SV40 virus. Sequential high voltage electrophoresis and paper chromatography of the RNA of fractionated nuclei isolated from fibroblasts exposed to tritiated AMS (340 microM, 5 microCi. per ml., for 18 hours) demonstrated that the inhibitor is demethylated to 3'-amino-3'-deoxyadenosine and incorporated into terminal positions in both nucleolar and nonnucleolar nuclear RNA chains in the same relative amounts. 3'-Amino-3'-deoxyadenosine was not detected in the RNA obtained from the cytoplasm of AMS-treated fibroblasts. Electron microscopic observation of isolated nucleoli showed that neighboring intranucleolar fibrils have a tendency to coalesce after AMS treatment. It is suggested that, when the modified AMS molecule has reached the growing ends of nascent polyribonucleotide chains elongation stops, the chain remains attached to its template, and the strong positive charge of the aminosugar portion of AMS interacts with negatively charged components (e.g., phosphate groups) of adjacent nascent RNA chains. The high density of the components of the nucleolus produces frequent interactions of this type and leads to secondary interference with nucleolar RNA synthesis, thus accounting for the selective effect of AMS on preribosomal RNA synthesis.

Selective inhibition of preribosomal RNA synthesis by puromycin aminonucleoside in transformed human fibroblasts: studies of the nature of the inhibition in isolated nuclei and nucleoli.

Puromycin aminonucleoside selectively inhibits the synthesis of ribosomal RNA in human lung fibroblasts transformed by the oncogenic virus SV40. The mechanism of this inhibition was studied utilizing nuclei and nucleoli isolated from cells treated for 18 hours with 100 micrograms/ml of this compound. It was established that for a limited period of time nuclei and nucleoli isolated from the fibroblasts continue synthesis of RNA of size classes seen in intact cells, and that the inhibitory effect of aminonucleoside persists after isolation of these organelles. The inhibition was shown to be directed primarily to the activity of RNA polymerase I. Studies of the mechanism of this inhibition have indicated that the decreased rate of the polymerase reaction is not due to the impairment of the template function of nucleolar chromatin, and that unbound, as well as chromatin-bound, RNA polymerase I is present in both control and treated nucleoli. Analysis of the size distribution of the products of cell-free RNA synthesis showed that aminonucleoside pretreatment results in marked reduction in the synthesis of preribosomal 45S RNA, abnormal accumulation of 32S RNA, and reduced formation of mature ribosomal RNA species in the in vitro system. The data suggest that the inhibitory effect of aminonucleoside on ribosomal synthesis is due in part to a lower rate of transcription by RNA polymerase I of preribosomal RNA, and in part to its impaired maturation.