Divergent evolution may link human immunodeficiency virus GP41 to human CD4.

A local sequence similarity of HIV envelope proteins (gp120 and gp41) to immunoglobulins suggests that a mimicry phenomenon may form the basis of the HIV-cell membrane interaction and of HIV-induced autoimmune reaction. We explored the hypothesis of any deeper relationship between HIV env proteins and immunoglobulin family members. An overall DNA sequence similarity between gp41 coding region of env gene and the HIV-receptor CD4 gene was observed and a 14-base-long oligonucleotide, almost unique in the GenBank, was found in gp41 and CD4 genes. The alignment of env gene to CD4 gene and to 84 different sequences showed a significantly higher homology score and a nonrandom similarity in the CD4-env alignment. A significant similarity was also found between the env protein and the sequence encoded by an alternate reading frame of CD4 gene. Our observations suggest that gp41 coding region might have a different origin than the gp120 coding region of the env gene, and that a divergent evolution might link gp41 to CD4 or immunoglobulin family members. In this study the analysis of alternate-reading-frame products is also proposed as a novel approach to investigate evolutionary links and structure-function relationships.

Induction of nuclear lamins A/C during in vitro-induced differentiation of F9 and P19 embryonal carcinoma cells.

Lamin B is the major constituent of the nuclear lamina of undifferentiated mouse embryonal carcinoma cells. The full complement of the three major lamins A, B, and C, found in somatic mammalian cells, is acquired after induction of differentiation in vitro by certain drugs. In this study we have examined the time course of lamin A/C expression in the two embryonal carcinoma cell lines F9 and P19. We show here that lamins A/C are detectable in these cell lines, at the mRNA level and at the protein level, after 3 days of growth in media containing retinoic acid or retinoic acid + 3-isobutyl-1-methylxanthine. The data reported here indicate that the expression of lamins A/C is mainly regulated at the transcriptional level and occurs when the cells, by morphological and functional criteria, have differentiated along their developmental pathway.

Three-dimensional distribution of DNase I-sensitive chromatin regions in interphase nuclei of embryonal carcinoma cells.

In situ nick-translation allows the visualization of nuclease-sensitive chromatin regions in interphase nuclei. We have analyzed the three-dimensional (3-D) distribution of DNase I-sensitive regions of chromatin in nuclei from mouse P19 embryonal carcinoma cells by making optical sections using confocal scanning laser microscopy. In undifferentiated as well as embryonal carcinoma cells differentiated in vitro, DNase I-sensitive regions of chromatin are observed as discrete spots in the nucleus. These spots represent clusters of DNase I-sensitive sites. By optical sectioning, we show that these spots are preferentially, but not exclusively, localized at the nuclear periphery. No differences were observed in the spatial distribution of DNase I-sensitive sites in P19 EC cells or the differentiated P19 END-2 cells. Furthermore, we did not observe differences in the distribution of DNase I-sensitive chromatin regions during the cell cycle. These findings indicate, at least for P19 mouse embryonal carcinoma cells and their differentiated derivative END-2, that the compartmentalization of DNase I-sensitive chromatin regions is a general characteristic of the nucleus, independent of cell cycle stage or differentiation state. Since evidence has been presented that DNase I-sensitive sites are associated with actively transcribed chromatin, our results indicate that active transcribing chromatin is compartmentalized, preferentially in the periphery of the nucleus.

Role of protein synthesis in the accumulation of ferritin mRNA during exposure of cells to iron.

The iron-induced biosynthesis of ferritin is regulated at the translational level via multiple mechanism. A prolonged exposure of cells to iron leads to a marked increase in ferritin mRNA levels caused by stabilization of the message. Here we show that this stabilization requires the synthesis de novo of an iron-inducible protein factor.

The nuclear matrix from cells of different origin. Evidence for a common set of matrix proteins.

We compared the protein composition of the nuclear matrix isolated from several murine embryonal carcinoma cells and mature tissues by two-dimensional gel electrophoresis. Two nuclear matrix fractions were investigated: the "peripheral" nuclear matrix (matrix proteins that remain insoluble after reduction), and the "internal" nuclear matrix (matrix proteins released by reduction). The two subfractions have completely different protein compositions. Although numerous differences in nuclear matrix protein composition among different cell types were observed, a limited set of polypeptides common to all mouse cell types was identified. A majority of these common proteins was also present in cells from other mammalian species (i.e. rat and human). For this set of proteins, we coin the term "minimal matrix." As expected, lamin B, known to be expressed throughout differentiation, is part of the common set of peripheral nuclear matrix proteins. Lamins A and C are not because these proteins were absent from undifferentiated embryonal carcinoma cells. Since these common nuclear matrix proteins occur in all mammalian nuclear matrices analyzed so far, it is likely that they have a basic role in nuclear organization and function.

Multiple post-transcriptional regulatory mechanisms in ferritin gene expression.

We have investigated the mechanisms involved in the regulation of ferritin biosynthesis in K562 human erythroleukemia cells during prolonged exposure to iron. We show that, upon addition of hemin (an efficient iron donor) to the cell culture, the rate of ferritin biosynthesis reaches a maximum after a few hours and then decreases. During a 24-hr incubation with the iron donor the concentrations of total ferritin heavy (H) and light (L) subunit mRNAs rise 2- to 5-fold and 2- to 3-fold, respectively, over the control values, while the amount of the protein increases 10- to 30-fold. The hemin-induced increment in ferritin subunit mRNA is not prevented by deferoxamine, suggesting that it is not directly mediated by chelatable iron. In vitro nuclear transcription analyses performed on nuclei isolated from control cells and cells grown in the presence of hemin indicate that the rates of synthesis of H- and L-subunit mRNAs remain constant. We conclude that iron-induced ferritin biosynthesis is governed by multiple post-transcriptional regulatory mechanisms. We propose that exposure of cells to iron leads to stabilization of ferritin mRNAs, in addition to activation and translation of stored H- and L-subunit mRNAs.

The protein composition of the nuclear matrix of murine P19 embryonal carcinoma cells is differentiation-stage dependent.

The protein composition of the nuclear matrix of murine P19 embryonal carcinoma (EC) cells was compared with that of clonal derivatives of P19 EC differentiated in vitro, and with that of P19 EC cells induced to differentiate with retinoic acid (RA). Several major differences in nuclear matrix protein composition were found between the cell lines tested. Some polypeptides were found to occur only in EC cells, whereas others proved to be restricted to one or more of the differentiated derivatives. During RA treatment of EC cells a transient expression of some matrix proteins was observed. Several new proteins appeared, and others disappeared. Our data indicate that the protein composition of the nuclear matrix is a sensitive gauge for the differentiation state of cells.

Regulation of intracellular iron distribution in K562 human erythroleukemia cells.

Following a pulse with 59Fe-transferrin, K562 erythroleukemia cells incorporate a significant amount of 59Fe into ferritin. Conditions or manipulations which alter the supply of iron to cells result in changes in the rate of ferritin biosynthesis with consequent variations in the size of the ferritin pool. Overnight exposure to iron donors such as diferric transferrin or hemin increases the ferritin level 2-4- or 6-8-fold above that of the control, respectively. Treatment with the anti-human transferrin receptor antibody, OKT9 (which reduces the iron uptake by decreasing the number of transferrin receptors) lowers the ferritin level by approximately 70-80% with respect to the control. The fraction of total cell-associated 59Fe (given as a pulse via transferrin) that becomes ferritin bound is proportional to the actual ferritin level and is independent of the instantaneous amount of iron taken up. This has allowed us to establish a curve that correlates different levels of intracellular ferritin with corresponding percentages of incoming iron delivered to ferritin. Iron released from transferrin appears to distribute to ferritin according to a partition function; the entering load going into ferritin is set for a given ferritin level over a wide range of actual amounts of iron delivered.

High-performance liquid chromatographic methods for antibodies, glycosidases and membrane proteins.

The broad range of applications of high-performance liquid chromatography (HPLC) in biochemistry and cell biology is demonstrated by the purification of antibodies, separation of glycosidases and isolation of a liver membrane protein with a molecular weight of 65 000-67 000 daltons. The advantage of HPLC over classical chromatographic methods is shown by the purification of the glycosidases from Streptococcus pneumoniae. These enzymes can be purified to a degree similar to what can be achieved by "classical" ion exchange, combined with affinity chromatography, but the time needed for the HPLC experiment is much shorter and the yield at least three to five times higher. Particular attention is directed to sample preparation before HPLC separation. For the best results, a combination of HPLC with other biochemical and immunochemical methods is necessary, as is also demonstrated.

Quantitative determination of intracellular, ferritin-associated radioactive iron by high-performance liquid chromatography and immunoprecipitation.

Antibodies raised against ferritin preparations of diverse origin provide an uncertain reagent for quantitation of the ferritin present in specific cell lysates. Utilizing K562 cells, a human leukemic cell line, techniques are described to resolve and to quantitate the ferritin-bound cytosolic iron. Processing the cell lysates by HPLC employing an anion-exchange or hydrophobic interaction column resulted in recovery of a single, ferritin-containing radioactive peak widely separated from the bulk of the non-ferritin-bound iron. Comparison of the yield obtained by chromatography with that by immunoprecipitation confirmed both the specificity and the quantitation of the antibody technique.

Immunological studies of an organic anion-binding protein isolated from rat liver cell plasma membrane.

The mechanism of organic anion uptake by hepatocytes has kinetics that suggest facilitated diffusion, and carrier-mediated membrane transport has been postulated. In previous studies, we purified a 55,000-mol wt organic anion-binding protein (OABP) by affinity chromatography on sulfobromophthalein (BSP)-Sepharose of deoxycholate solubilized liver cell plasma membrane preparations. Using specific goat and rabbit antibodies to OABP, we have now investigated the distribution of this protein in liver fractions and other tissues by an enzyme-linked immunosorbent assay and by the immunoblot (Western blot) procedure. These studies indicated that OABP is present in significant amounts in all tissues examined except for blood. Although OABP has not as yet been isolated from each of these tissues and characterized, OABP in heart retained the ability to bind organic anions, and was purified by affinity chromatography on BSP-sepharose. In liver, OABP was membrane bound and remained so after extraction with 0.9 M NaCl, which suggests that it is an intrinsic membrane protein. OABP did not have a ubiquitous subcellular distribution within the hepatocyte. Preparation of subfractions of liver cell plasma membrane revealed that OABP is present in the sinusoidal and absent from the canalicular membrane. Immunofluorescence studies performed in short-term cultured hepatocytes suggest that OABP is associated with the surface of these cells and does not have a significant intracellular distribution.

Rapid internalization of the transferrin receptor in K562 cells is triggered by ligand binding or treatment with a phorbol ester.

Treatment of human K562 cells with 4 beta-phorbol 12-myristate 13-acetate (PMA) resulted in an approximately 50% reduction in cell surface transferrin receptors within 30-45 min as judged by binding of both ligand and anti-receptor antibody. The affinity of the remaining surface receptors for diferric transferrin appeared to be unaltered. The time-dependent loss in transferrin receptors was also dependent upon PMA concentration, with a half-maximal effect observed at approximately 1 nM. The kinetic parameters for the binding, internalization, intracellular residency, and recycling of 125I-labeled transferrin were unchanged by PMA treatment, as were the rate and extent of internalization of anti-receptor antibody. Moreover, despite the decrease in surface receptors, uptake of 59Fe from transferrin proceeded at a rate comparable to that seen in untreated cells. Accounting for this observation was the fact that ligand induced a reduction in surface receptors in untreated but not PMA-treated cells. Quantitative immunoprecipitation of transferrin receptors from surface-iodinated K562 cells revealed that little receptor internalization occurred in untreated cells in the absence of ligand, but internalization of ligand-occupied receptors in these cells was readily detected. In contrast, PMA treatment resulted in the rapid internalization of surface receptors irrespective of occupancy. Thus, binding of ligand appeared to trigger the internalization of receptors that were relatively static in their unoccupied state, and a signal for receptor internalization was also provided by PMA treatment. The possibility that this signal involves phosphorylation of the transferrin receptor is discussed.

Failure to release iron from transferrin in a Chinese hamster ovary cell mutant pleiotropically defective in endocytosis.

A Chinese hamster ovary cell mutant defective in the receptor-mediated endocytosis of several unrelated ligands (Robbins, A. R., S. S. Peng, and J. L. Marshall, 1983, J. Cell Biol., 96:1064-1071) failed to accumulate iron provided in the form of diferric transferrin. Analysis of the steps of the transferrin cycle indicated that binding and internalization of transferrin proceeded normally in mutant cells. However, the mutant appeared unable to dissociate iron from transferrin, as evidenced by release of diferric transferrin from the mutant versus apotransferrin from the parent. Uptake of ferric ions from the growth medium was enhanced in the mutant.

Prediction of the three-dimensional structure of the transforming region of the EJ/T24 human bladder oncogene product and its normal cellular homologue.

The three-dimensional structures of the transforming region of the product of the EJ/T24 human bladder oncogene and of the c-Ha ras-1 gene product have been calculated by using conformational energy calculations. These two genes, representing a transforming oncogene and its normal cellular homologue, encode 21,000-dalton peptides that differ by one amino acid at position 12. We therefore examined the energetically allowed conformations of the hydrophobic decapeptide surrounding this substitution site. The calculations show that the most favorable form of the c-Ha ras-1 gene product exists when glycine-12 is in a left-handed bend conformation. No other amino acid can adopt this conformation and thus the bladder oncogene peptide containing valine at position 12 has a markedly different three-dimensional structure. A simple model is proposed to account for the consequences of a position 12 mutation.

Separation of Fe+3 from transferrin in endocytosis. Role of the acidic endosome.

NH4Cl and monensin, two agents which neutralize intracellular acidic compartments, block the segregation of iron from transferrin after endocytosis, while neither of these reagents affects internalization of diferric transferrin into the cell. In conclusion the molecular separation of iron from transferrin inside the cell requires a non-lysosomal acidic compartment.