Mutation of a Nopp140 gene dao-5 alters rDNA transcription and increases germ cell apoptosis in C. elegans.

Human diseases of impaired ribosome biogenesis resulting from disruption of rRNA biosynthesis or loss of ribosomal components are collectively described as 'ribosomopathies'. Treacher Collins syndrome (TCS), a representative human ribosomopathy with craniofacial abnormalities, is attributed to mutations in the tcof1 gene that has a homologous gene called nopp140. Previous studies demonstrated that the dao-5 (dauer and aged animal overexpression gene 5) of Caenorhabditis elegans is a member of nopp140 gene family and plays a role in nucleogenesis in the early embryo. Here, we established a C. elegans model for studying Nopp140-associated ribosomopathy. A null dao-5 mutant ok542 with a semi-infertile phenotype showed a delay in gonadogenesis, as well as a higher incidence of germline apoptosis. These phenotypes in dao-5(ok542) are likely resulted from inefficient rDNA transcription that was observed by run-on analyses and chromatin immunoprecipitation (ChIP) assays measuring the RNA Pol I occupancy on the rDNA promoter. ChIP assays further showed that the modifications of acetylated histone 4 (H4Ac) and dimethylation at the lysine 9 of histone 3 (H3K9me2) around the rDNA promoter were altered in dao-5 mutants compared with the N2 wild type. In addition, activated CEP-1 (a C. elegans p53 homolog) activity was also linked to the loss of DAO-5 in terms of the transcriptional upregulation of two CEP-1 downstream effectors, EGL-1 and CED-13. We propose that the dao-5 mutant of C. elegans can be a valuable model for studying human Nopp140-associated ribosomopathy at the cellular and molecular levels.

Human Nopp140, which interacts with RNA polymerase I: implications for rRNA gene transcription and nucleolar structural organization.

Nopp140 is thought to shuttle between nucleolus and cytoplasm. However, the predominant nucleolar localization of Nopp140 homologues from different species suggests that Nopp140 is also involved in events occurring within the nucleolus. In this study, we demonstrated that the largest subunit of RNA polymerase I, RPA194, was coimmunoprecipitated with the human Nopp140 (hNopp140). Such an interaction is mediated through amino acids 204 to 382 of hNopp140. By double immunofluorescence, hNopp140 was colocalized with RNA polymerase I at the rDNA (rRNA genes) transcription active foci in the nucleolus. These results suggest that Nopp140 can interact with RNA polymerase I in vivo. Transfected cells expressing the amino-terminal half of hNopp140, hNopp140N382 (amino acids 1 to 382), displayed altered nucleoli with crescent-shaped structures. This phenotype is reminiscent of the segregated nucleoli induced by actinomycin D treatment, which is known to inhibit rRNA synthesis. Consistently, the hNopp140N382 protein mislocalized the endogenous RNA polymerase I and shut off cellular rRNA gene transcription as revealed by an in situ run-on assay. These dominant negative effects of the mutant hNopp140N382 suggest that Nopp140 plays an essential role in rDNA transcription. Interestingly, ectopic expression of hNopp140 to a very high level caused the formation of a transcriptionally inactive spherical structure occupying the entire nucleolar area which trapped the RNA polymerase I, fibrillarin, and hNopp140 but excluded the nucleolin. The mislocalizations of these nucleolar proteins after hNopp140 overexpression imply that Nopp140 may also play roles in maintenance of nucleolar integrity.

Dynamic displacement changes of the bladder neck with the patient supine and standing.

PURPOSE: With the patient standing and supine we determine the differences in dynamic changes of the bladder neck and directions of dynamic bladder neck displacement. MATERIALS AND METHODS: To evaluate the dynamic movement of the bladder neck we recruited into the study 78 consecutive women 27 to 69 years old with various urogynecological complaints. The anatomical changes of the bladder neck from rest to maximal straining and from rest to holding were evaluated and compared with the patients supine and standing. RESULTS: Except for bladder neck rotational angle with the patient standing, all parameters were significantly different from corresponding measurements with the patient supine. Mean rotational angle of rest to maximal straining plus or minus standard deviation was 39.4 +/- 18.9 degrees when standing versus 39.8 +/- 23.4 degrees when supine (p > 0.05). The distances between the bladder neck and symphysis pubis at rest, and during maximal straining and holding the bladder neck in the supine position were significantly longer than those in the standing position. The direction of bladder neck displacement from rest to maximal straining was more caudad and ventral when standing. The bladder neck moved cephalad and ventral when the patient was standing, and cephalad and dorsal with the patient supine and holding the bladder neck. CONCLUSIONS: The anatomical locations and dynamic displacements of the bladder neck at rest, and during maximal straining and holding were significantly different in the supine and standing positions. While evaluating the dynamic motion of the bladder neck to determine bladder neck mobility, patient position must be considered and specified in accordance with diagnostic standards.

The nucleolar phosphoprotein P130 is a GTPase/ATPase with intrinsic property to form large complexes triggered by F- and Mg2+.

We have previously identified a human nucleolar phosphoprotein p130 whose alterations during mitosis are correlated well with the nucleolar disassembly and reassembly. Further studies found that p130 in the cell lysates or after being purified by immunoprecipitation was able to form large complexes triggered by F- and Mg2+. These sodium dodecyl sulfate-insoluble p130 molecules were readily dissociated by adding EDTA to the complexes. It is known that F- and Mg2+ act on many GTPases and ATPases through the induction of a conformational transition mimicking the nucleoside triphosphate-bound state. These initial observations led us to discover that p130 functions as a GTP/ATP binding protein with intrinsic GTPase/ATPase activities. The rate of GTP hydrolysis by purified p130 under our experimental conditions was 0.8 mol/min/mol of p130. These results imply that p130, a novel nucleolar GTPase/ATPase, may switch its conformation in a nucleotide-dependent manner.

Cell proliferation-dependent expression of two isoforms of the nucleolar phosphoprotein p130.

A highly phosphorylated human nucleolar protein p130 (130-kDa) was found to be expressed in synchrony with cell-growth activation. It was not detectable in the resting lymphocytes, but its expression was increased rapidly after mitogenic stimulation. During the terminal differentiation-coupled growth-arrest of HL60 cells, the mRNA and protein of p130 reduced significantly within 24 h after the induction. In addition to the previously identified form (now referred to as p130 alpha ), a novel isoform p 130 beta was found. It contains an insert of ten amino acids located within a region corresponding to the fourth proline-rich basic domain of p130 alpha. Both isoforms were coexpressed in cell lines of different origins, with the beta-transcript exhibiting much less compared to the alpha-transcript. Furthermore, both alpha- and beta-transcripts diminished when cells returned to the quiescent stage. cDNA transfection experiments demonstrated that the two p130 isoforms existed stably in the nucleoli and showed the same nucleolar distribution pattern. It implies that the ten-amino-acid-insert does not alter significantly the interactions of p130 with other nucleolar components in interphase cells.

Dynamic changes of NuMA during the cell cycle and possible appearance of a truncated form of NuMA during apoptosis.

We have demonstrated that dynamic redistribution of nuclear-mitotic apparatus (NuMA) protein in the cell cycle is correlated temporally and spatially with its biochemical modifications. In interphase, NuMA behaves solely as a 220 kDa nuclear matrix-associated protein. After initiation of DNA condensation during mitosis, NuMA is phosphorylated by Cdc2 kinase into a 240 kDa form which is transported quickly to the centrosomal region. Once cells have passed the metaphase-anaphase transition, the 240 kDa form of NuMA either becomes a 180 kDa truncated form which is fated to be degraded completely before mitotic exit, or returns to the 220 kDa form that relocates to the daughter nuclei and remains throughout interphase. Apparently, a proteolytic enzyme is activated during the late stages of mitosis. After induction of a 180 kDa form of NuMA in interphase HeLa cells by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, nuclear apoptotic phenomena including chromatin condensation, DNA fragmentation, and micronucleation were observed. However, the same treatment did not induce apoptosis in mitotic phase-arrested HeLa cells. The 180 kDa form of NuMA was demonstrated to be a truncated product, at least lacking the tail domain. When HL60 cells were stimulated by diverse apoptosis inducers such as camptothecin, staurosporine, cycloheximide, and A23187, the extent of NuMA cleavage to produce a 180 kDa product was comparable with the degree of oligonucleosomal laddering. NuMA cleavage is likely to be a consequence of the onset of apoptosis. The intact 220 kDa NuMA functions in interphase cells to retain the nuclear structural integrity. Additionally, NuMA appears to act as a nuclear structural target for a death protease during apoptosis.

Cell-cycle-dependent alterations of a highly phosphorylated nucleolar protein p130 are associated with nucleologenesis.

We identified a novel human nucleolar phosphoprotein p130 (130 kDa) using a strategy for selecting monoclonal antibodies against nuclear proteins which oscillate in the cell cycle. p130 is localized in interphase nucleoli in a dotted manner. Complete extraction of p130 required a high concentration of salt (0.5 M NaCl) indicating that it binds firmly to the nucleolar components via ionic interaction. p130 is heavily phosphorylated, since alkaline phosphatase treatment converted the purified p130 into a 95 kDa product; this was further supported by the in vitro demonstration that cellular phosphatase and casein kinase II activities were responsible for the interchange of these two forms. Extracts of mitotic cells had lower concentrations of p130 compared to those of interphase cells suggesting that a proportion of p130 might be degraded during mitosis. Moreover, all the remaining p130 in mitotic cells was further phosphorylated, likely by a cdc2 kinase, resulting in increase in its solubility, and its dispersion throughout the entire cytoplasm. Thus, p130 in metaphase and anaphase cells was unable to be detected by immunofluorescence microscopy. At telophase, p130 reappeared and aggregated into a granular structure, resembling the prenucleolar bodies. These granules migrated from the nucleoplasm to the nucleoli in early G1-phase. Actinomycin D was able to induce segregation of p130-containing granules into the nucleoplasm, similar to the well-known behavior of the fibrillarin-containing granules, indicating that p130 is localized in the dense fibrillar component, a subnucleolar region for pre-rRNA synthesis and processing. The cDNA sequence of p130 revealed a remarkable feature, that a serine-rich stretch interspersed with acidic residues is repeated ten times. Such a characteristic is shared with a rat nucleolar phosphoprotein Nopp140, which is thought to shuttle between the nucleolus and the cytoplasm. Although p130 shows 74% identity to Nopp140, our observations suggest that during mitosis the functions of p130 are related to nucleologenesis.

Purification and characterization of nucleolin and its identification as a transcription repressor.

Expression of the acute-phase response genes, such as that for alpha-1 acid glycoprotein (AGP), involves both positive and negative transcription factors. A positive transcription factor, AGP/EBP, and a negative transcription factor, factor B, have been identified as the two most important factors responsible for the induction of the AGP gene. In this paper we report the purification, characterization, and identification of a B-motif-binding factor from the mouse hepatoma cell line 129p. The purified factor has been identified as nucleolin by amino acid sequence analysis. Biochemical and functional studies further established that nucleolin is a transcription repressor for regulation of AGP and possibly other acute-phase response genes. Thus, in addition to the many known functions of nucleolin, such as rRNA transcription, processing, ribosome biogenesis, and the shuttling of proteins between the cytoplasmic and nuclear compartments, it may also function as a transcriptional repressor.

The self-cleaving activity of nucleolin determines its molecular dynamics in relation to cell proliferation.

Nucleolin (105 kDa) is the most abundant nucleolar phosphoprotein in exponentially growing cells. We have demonstrated that nucleolin has an intrinsic protease activity for autodegradation. This self-cleaving activity is markedly decreased when cells enter the proliferative stage, resulting in significant stabilization of the nucleolin molecule. The fusion protein derived from a partial nucleolin cDNA clone, which encodes the C-terminal two-thirds of nucleolin, maintained the self-cleaving activity similar to that of the nucleolin purified from nonproliferating cells. The enzymatic domain is therefore located within this region. An exogenous peptide, highly negatively charged, exerted an inhibitory effect, indicating that the enzymatic activity can be regulated. The cleavage kinetics of nucleolin isolated from proliferating cells was apparently concentration independent, suggesting that the autoproteolytic reaction of nucleolin in cells at dividing stage is an intramolecular event. There were many cleavage fragments, which were clustered at four major sites, with apparent molecular weights of about 100, 70, 60, and 50 kDa. These results imply that the nucleolin molecule has repeated motifs that serve as the cleavage sites. Since the detailed electrophoresis patterns, including location and intensity, of the in vitro self-cleaved products of nucleolin derived from nonproliferating and proliferating cells were not quite the same, preferential usage of the particular cutting sites according to cell stage was suggested. It is possible that post-translational modification of nucleolin in proliferating cells may cause these variations.

Cell cycle-dependent migration of the DNA-binding protein Ku80 into nucleoli.

The DNA-binding protein Ku (p70/p80) was originally discovered through the use of human autoimmune sera. In attempts to search out nucleolar proteins in relation to nucleolar dynamic changes, we developed monoclonal antibodies against nuclear proteins. One antibody, termed LL1, received particular attention since asynchronous cells exhibited tremendous differences in their nucleolar fluorescence intensities after immunostaining. The LL1 protein was proven to be the Ku subunit p80 (Ku80) by cDNA cloning and sequencing. Possible correlations between the heterogeneous distribution of Ku80 in nucleoli and the cell cycle were examined. HeLa cells were synchronized at M phase by arrest with nocodazole, or at the G1/S boundary by sequential treatments with thymidine and aphidicolin. These cells were then released by culturing in fresh medium to allow the cell cycle to progress synchronously. Immunofluorescent detection of Ku80 revealed that nucleoli of the cells at the G1/S boundary had very small amounts of Ku80, which was mainly present in the nucleoplasm. Ku80 was gradually accumulated in nucleoli during S phase and reached the maximum at late S or G2 phase. Immunoblotting experiments showed that cell extracts prepared from different phases of the cell cycle had virtually identical amounts of Ku80. These results suggest that Ku80 migrates from nucleoplasm to nucleoli in a cell cycle-dependent manner.

Increased stability of nucleolin in proliferating cells by inhibition of its self-cleaving activity.

Nucleolin is the major nucleolar phosphoprotein of exponentially growing eukaryotic cells and is presumably involved in pre-rRNA transcription and ribosome biogenesis. Monoclonal antibodies against nucleolin were selected by a differential dot-immunobinding assay. Nucleolin expression during T lymphocyte activation was monitored by the specific antibody. Results showed that nucleolin fluctuated in parallel to DNA synthesis. The intact 105-kDa nucleolin molecule was the major species in actively dividing cells, whereas the degraded forms were relatively abundant in nondividing cells. These results imply that stability of nucleolin molecule is cell proliferation-dependent. When affinity purified nucleolin containing undetectable contaminants was incubated at 37 degrees C, the majority of 105-kDa nucleolin was cleaved by 6 h and completely degraded within 24 h. This purified nucleolin was further separated from possible copurified protease, if any, on a reducing sodium dodecyl sulfate-polyacrylamide gel. After renaturation, the 105-kDa nucleolin immobilized in the gel was also cleaved at 37 degrees C. These data have confirmed that nucleolin protein autocatalyzes its own degradation. The self-cleaving activity of nucleolin was inhibited by nuclear extracts prepared from proliferating cells. Apparently, a proteolytic inhibitor(s) in the nuclei of proliferating cells stabilized the nucleolin molecule. It provides an unique regulatory mechanism for nucleolin expression.

Rapid identification of monoclonal antibodies to differential antigens by dot immunobinding assay.

A simple and highly sensitive method, dot immunobinding assay, has been developed to select of monoclonal antibodies recognizing differential antigens. Antigen as low as picogram level can be detected by monoclonal antibody at concentrations even lower than 0.1 microgram/ml. This technique has successfully identified six differential antigens which are either increased (three) or decreased (three) during T cell activation. The differential properties were further confirmed by direct visualization of the resting and activated T cells with immunofluorescent staining or by indirect evidence when resting and activated T cell extracts were prepared and analysed with immunoblot assay. CC-98 (105 KD) was found in the nuclei, principally associated with the nucleoli. Activated T cells expressed a highly intense immunofluorescent pattern at their nucleolar localization compared to that of resting T cells. SH-1 antigen with a diffuse molecular weight range of 90-145 KD was expressed mainly in resting T cells and rapidly decreased, especially the high molecular weight species (120-145 KD), to a very low level in the activated T cells. SH-1 was identified as an intracellular molecule. NY-16 and CC-262 are identical with characteristics ascribed to those of the major histocompatibility class I antigen. Cell surface molecules CC-80 (135 KD) and CC-86 (135 KD) were decreased, while CC-196 was increased during T cell activation. These six molecules, with their differentiating properties, may have very important functions related to cell activation.

Induction and upregulation by interleukin 2 of high-affinity interleukin 2 receptors on thymocytes and T cells.

We show that purified recombinant interleukin 2 (rIL-2) alone induces the expression of high- and low-affinity interleukin 2 (IL-2) receptors in vitro on human T cells and thymocytes that have not been activated previously by lectins or other inducing agents. IL-2 receptors are expressed after 24 hr, as determined by the binding of 125I-labeled monoclonal anti-IL-2 receptor antibody 2A3, which binds equally to high- and low-affinity receptors. High-affinity receptors were distinguished from low-affinity receptors by the binding of 125I-labeled IL-2 to T cells and by the proliferative response of thymocytes to IL-2, in concentrations that selectively interact with the high-affinity class of IL-2 receptors. The IL-2-induced proliferation of thymocytes in vitro induced by IL-2 alone is dependent upon the concentration of IL-2 and is inhibited by monoclonal anti-Tac antibody, indicating that the proliferative response is mediated by the binding of IL-2 to the receptors. In addition, we demonstrate that IL-2 augments the number of high-affinity receptors on concanavalin A-activated thymocytes. These results document that IL-2 acts as a hormone that induces the activation of thymocytes and T cells, as evidenced by the de novo induction of biologically active, high-affinity IL-2 receptors. IL-2 also upregulates the expression of high-affinity IL-2 receptors on activated thymocytes. These observations illustrate the biologic importance of the regulatory role of IL-2 in the immune response.

Modulation of the expression of interleukin 2 receptors of human thymocytes by recombinant interleukin 2.

The effect of purified recombinant interleukin 2 on the expression of the receptors for interleukin 2 by human thymocytes was examined. Interleukin 2 augmented the expression of interleukin 2 receptors and interferon-gamma synthesis by thymocytes activated with concanavalin A, and it was required to maintain the growth of thymocytes in vitro and the expression of interleukin 2 receptors. The increase observed in the number of receptor bearing thymocytes and in the density of receptors due to interleukin 2 occurred within the first 2 days of culture. Dexamethasone inhibited the expression of interleukin 2 receptors, the synthesis of interferon-gamma, and the early proliferation and protein synthesis of lectin-activated thymocytes during the first 2 days of culture. The inhibitory effect of dexamethasone on the expression of interleukin 2 receptors and on the synthesis of interferon-gamma was reversed by interleukin 2, whereas its effect on proliferation and on protein synthesis during the first two days of culture was not reversed by interleukin 2. Interleukin 2 induced the proliferation of thymocytes in vitro, even in the absence of activation by lectin; however, the number of cells displaying receptors which could be detected with anti-Tac remained low throughout the first week of culture and interferon-gamma synthesis was not observed. Nonetheless, interleukin 2-induced proliferation was inhibited by anti-Tac on a dose dependent manner. The results of the study document that recombinant interleukin 2, like purified natural interleukin 2, is required for the expression of interleukin 2 receptors, for interferon-gamma synthesis, and for the growth of thymocytes in vitro.

Regulation by interleukin 2 of interleukin 2 receptors and gamma-interferon synthesis by human thymocytes: augmentation of interleukin 2 receptors by interleukin 2.

The role of interleukin 2 (IL 2) on the expression of IL 2 receptors and on the synthesis of gamma-interferon (gamma-IFN) by human thymocytes was investigated. Human thymocytes isolated from specimens obtained during cardiac surgery of infants and children were induced with one or all of the following agents: IL 2, concanavalin A (Con A), and 12-O-tetradecanoylphorbol 13-acetate (TPA). The expression of IL 2 receptors and gamma-IFN titers were determined. The results indicate that thymocytes cultured in complete medium do not express receptors for IL 2, nor did IL 2 by itself induce the expression of IL 2 receptors. Con A induced the expression of IL 2 receptors by a moderate number of the thymocyte population and induced the synthesis of low amounts of gamma-IFN. Preincubation of thymocytes with TPA increased the response to Con A; both the number of thymocytes expressing receptors and the synthesis of gamma-IFN were increased. Addition of IL 2 to these cultures further augmented the expression of IL 2 receptors and gamma-IFN synthesis and resulted in the optimal expression of IL 2 receptors and maximal gamma-IFN synthesis. The expression of IL 2 receptors could be detected within 24 hr and preceded the induction of proliferation; it was therefore probably not due to the clonal expansion of a population of receptor-bearing thymocytes. Conversely, inhibition of IL 2 synthesis with dexamethasone (Dex) by thymocytes activated with Con A, or inhibition of the function of IL 2 receptors by anti-Tac, resulted in a decrease in the number of IL 2 receptor-bearing thymocytes activated with Con A, or inhibition of the function of IL 2 receptors by anti-Tac, resulted in a decrease in the number of IL 2 receptor-bearing thymocytes and of gamma-IFN synthesis. Thymocytes activated with TPA and Con A were more resistant to the inhibitory effects of Dex on the expression of IL 2 receptors than thymocytes activated with Con A alone. Maximal inhibition of the expression of IL 2 receptors and of gamma-IFN synthesis was achieved as a result of the synergistic effect of anti-Tac with Dex. Therefore, when IL 2 was prevented from binding to the receptors, and IL 2 synthesis was inhibited, the number of thymocytes expressing IL 2 receptors was sharply reduced and gamma-IFN synthesis was markedly inhibited.(ABSTRACT TRUNCATED AT 400 WORDS)

Interleukin 2 regulates expression of its receptor and synthesis of gamma interferon by human T lymphocytes.

Interleukin 2 (IL-2) has an important role in the regulation of the expression of IL-2 receptors and the synthesis of gamma interferon (IFN-gamma) by T lymphocytes. IL-2 is required for the optimum expression of IL-2 receptors on activated T lymphocytes and for maximum synthesis of IFN-gamma in vitro. Dexamethasone, an immunosuppressant drug that inhibits IL-2 synthesis, diminished the expression of IL-2 receptors and the synthesis of IFN-gamma. Anti-Tac, a monoclonal antibody known to prevent the binding of IL-2 to its receptor without inhibiting IL-2 synthesis, down-regulated the expression of the receptor and partially inhibited synthesis of IFN-gamma. In a population of T lymphocytes prevented from synthesizing IL-2 by dexamethasone and incapable of using IL-2 as a result of blockage of IL-2 receptors by anti-Tac, the number of receptor-bearing cells and receptor density were diminished. Anti-Tac in combination with dexamethasone also exerted a synergistic effect on IFN-gamma synthesis, inhibiting it almost completely. The inhibitory effect of dexamethasone IFN-gamma synthesis may be of clinical importance, since IFN-gamma activates macrophages and thereby triggers one of the defense mechanisms against bacterial infections.

Modulation of the expression of Tac antigen (T-cell growth factor response) on human thymocytes.

This study shows that anti-Tac antibody does not bind to human thymocytes unless they are activated. Human thymocytes could be induced to express Tac antigen (TCGF receptor) on their cell surface by Concanavalin A. B lymphoblastoid cells or 12-O-tetradecanoylphorbol 13-acetate alone did not induce TCGF receptors, but they did exert a very marked synergistic effect with Concanavalin A. This observation is consistent with our earlier finding that B lymphoblastoid cells secrete a factor which exerts a synergistic effect on the induction of lymphokine secretion by thymocytes and T-cells. The early expression of Tac antigen was independent of thymocyte proliferation. Anti-Tac antibody (10(-3)) inhibited the expression of TCGF receptors and late proliferation of thymocytes. TCGF did not reverse this inhibition nor did it prevent the binding of Tac antibody, but it enhanced the expression of Tac antigen.

T cells and the anti-trinitrophenyl antibody response to fetal calf serum and 2-mercaptoethanol.

Unprimed murine lymphocytes maintained in culture medium containing fetal calf serum (FCS) and 2-mercaptoethanol (2-ME) developed very high levels of anti-trinitrophenyl (TNP) plaque forming cells (PFC). Both FCS and 2-ME contributed to the response. The development of anti-TNP PFC during culture was accompanied by a 10-fold expansion in the number of immunoglobulin-secreting cells, indicating polyclonal stimulation. However, the number of anti-TNP PFC was disproportionately high and not accompanied by a similar increase in plaques specific for sheep red blood cells. The TNP-specific plaques were not artifacts of the plaque assay since they were 98% inhibited by specific antigen. The in vitro induction of anti-TNP PFC by FCS and 2-ME was common to a number of mouse strains, although some genetic variation occurred. Nylon-wool-separated B cells, nude mouse spleen cells, and bone marrow cells all produced high levels of anti-TNP after culture with medium containing FCS and 2-ME. The addition of T cells to B-cell cultures increased the numbers of anti-TNP PFC by 1.5- to 2.5-fold. The presence of a TNP-cross-reacting antigen in FCS probably contributed to the unexpectedly high anti-TNP response. The response to the antigen in FCS was potentiated by the enhancing activity of 2-ME.

Fetal calf serum and 2-mercaptoethanol induce anti-trinitrophenyl antibody production: II. Preferential stimulation of B cells in culture.

Fetal calf serum (FCS) and 2-mercaptoethanol (2-ME) are commonly used in culture media because they enhance lymphocyte responsiveness. We had previously reported that FCS and 2-ME generate high antitrinitrophenyl (TNP) antibody plaque-forming cell responses in unprimed murine spleen cells without added antigen. This was due in part to a component in FCS, which was antigenically cross-reactive with TNP. In the present report, we studied the mitogenic activity of FCS and 2-ME, which also contributed to the high anti-TNP response. Such mitogenic activity could not be replaced by B- or T-cell mitogens. Both FCS and 2-ME were required for the mitogenic activity, which was directed primarily at B cells. Purified T cells and spleen cells from B-cell-depleted mice were unresponsive to FCS and 2-ME stimulation. The mitogenic activity was not due to lipopolysaccharide (LPS) contamination of FCS, since cells of the LPS-unresponsive mouse strain, C3H/HeJ, were stimulated by FCS and 2-ME.