Paeciloquinones A, B, C, D, E and F: new potent inhibitors of protein tyrosine kinase produced by Paecilomyces carneus. II. Characterization and structure determination.

Paeciloquinones A to F and versiconol have been isolated as inhibitors of protein tyrosine kinases from the culture broth of the fungus Paecilomyces carneus P-177. The structures of the new anthraquinones were determined by spectroscopic methods, mainly 1H NMR and 13C NMR. The substitution pattern was established by investigation of the respective methylated derivatives.

Cells of the cellular immune and hemopoietic system of the mouse lack lamins A/C: distinction versus other somatic cells.

Almost all somatic cells in adult murine tissues express all three nuclear lamins (A, B, C). Here we demonstrate that cells of the hemopoietic system of the adult mouse are an exception in that they express only lamin B. Thus T and B lymphocytes as well as granulocytes and monocytic cells directly isolated from spleen, thymus, blood or bone marrow do not express lamin A/C but only lamin B. In agreement with this observation the murine hemopoietic cell lines EL4, BW5147, HK22, 70Z/3, SP2/0 and PAI express only lamin B. In immunoblotting experiments used to confirm the immunofluorescence data no lamin A/C expression was detected. However, we noticed that murine lamin B occurs in two isoforms, which can be distinguished immunologically. These results reinforce the idea that a functional nuclear lamina can be formed from lamin B alone. They also pose the question of whether cells lacking lamin A/C are more plastic in their developmental programs than those that express all three lamins.

B cell progenitors have different growth requirements before and after immunoglobulin heavy chain commitment.

Using the clonable pre-B cell assay, we have identified B cell progenitors that are not yet committed to the production of a particular H chain allele. These cells represent approximately 10-20% of clonable pre-B cells found in 15-d fetal liver. The clonable pre-B cell assay provides an environment adequate for the expansion and differentiation of these cells into mature, Ig-secreting, cells. Using the same methodology, we have also identified progenitors that are uncommitted to the production of a particular L chain isotype. Moreover, investigating the growth requirements for clonable pre-B cells has led to the discovery of selective growth conditions that distinguish cells before and after commitment to H chain allotype.

Detection of normal B-cell precursors that give rise to colonies producing both kappa and lambda light immunoglobulin chains.

The pre-B-cell cloning assay is an in vitro differentiation system in which B-lymphocyte precursors expand and generate colonies containing immunoglobulin-secreting cells. Analysis of surface characteristics, growth requirements, and kinetics suggested that these cells represent early stages of the B-cell differentiation pathway. Here we describe a modification of the assay, which allowed us to determine the differentiative potential of these clonable pre-B cells. Using a nitrocellulose protein-transfer technique, we studied immunoglobulin light chain expression in colonies derived from fetal mouse liver B-cell precursors; in particular, we explored whether the B-cell precursors are already committed to the expression of a particular light chain gene at the initiation of culture. Our results show that fetal liver-derived B-cell progenitors generate colonies in vitro that secrete kappa and lambda light chains at a ratio similar to that found in colonies derived from adult splenic B cells. Further, we document the existence of colonies that are derived from single cells and that simultaneously secrete both types of light chains. This indicates that the progenitors of (kappa + lambda)-producing colonies are light chain-uncommitted at the initiation of culture. These cells are able to rearrange their light chain genes in vitro and differentiate along the B-cell pathway to form colonies secreting both kappa and lambda chains.

In vivo suppression of perinatal multispecific B cells results in a distortion of the adult B cell repertoire.

The isolation of multispecific B cell hybridomas with a variety of anti-idiotype (anti-Id) activities from the lymphoid organs of fetal and neonatal BALB/c mice suggested that the development of the immune system may depend on Id interactions among autologous B cells. In vitro analysis of antibodies secreted by these hybridomas showed extensive sharing of an idiotope defined by the monoclonal antibody FD5-1. Early and timed administration of this antibody during the perinatal period results in a distortion of the phosphorylcholine (PC) and alpha (1----3)dextran (Dex)-specific B cell precursor compartment of the developing repertoire and is reflected by a drastic reduction of antibody responses to these antigens when challenged as adults. These observations provide strong evidence for the involvement of the early appearing multispecific B cells in Id interactions that bring about the uniform development of the normal adult B cell repertoire. Interference with these interactions at critical stages of developmental results in permanent deficiencies in the adult B cell repertoire.

Differentiation of murine B cell precursors in agar culture. II. Response of precursor-enriched populations to growth stimuli and demonstration that the clonable pre-B cell assay is limiting for the B cell precursor.

We established culture conditions under which fetal liver-derived B cell progenitors divide and differentiate in semisolid agar, forming colonies containing antibody-secreting cells. An important feature of this assay system is that, under certain conditions, it is limiting for only one component. This was revealed by determining the slope of the least-squares log-log regression line generated when the initial seeding density was varied. When support for the growth of the clonable pre-B cells was provided by fetal liver-derived adherent accessory cells, the slope of the regression line was 1, consistent with the interpretation that only one component, the pre-B cell, was limiting under these conditions. This interpretation was tested in the present report by positive selection for cells expressing B220, Lyb-2, or AA4.1, surface markers known to be present on cell lines of the B lineage. In all cases, an increased incidence of clonable pre-B cells was observed. Furthermore, regression analysis of titration experiments undertaken with these enriched populations revealed that the assay was still limiting for a single component. A second set of growth conditions have been defined in which the clonable pre-B assay appears to be limiting for more than one component. These conditions are obtained when the adherent accessory cells are replaced by one of three distinct hemopoietic growth factors, including CSF-1 (macrophage growth factor), GM-CSF (neutrophil/macrophage growth factor), or Multi-HGF (multi-lineage hemopoietic growth factor, also called BPA or IL 3). The most straightforward interpretation of these data is that a second cell type, distinct from the B cell precursor and dependent on the growth factors, was limiting under these conditions. In the present report, this hypothesis was confirmed because growth factor responsiveness was completely absent in B220 and Lyb-2-enriched populations. Factor responsiveness was found in unseparated fetal liver and in AA4-enriched populations. However, the AA4-enriched populations, in contrast to the B220- or Lyb-2-enriched populations, also contained a large number of factor-responsive neutrophil/macrophage colonies, raising the possibility that the effect of factors on AA4+ clonable pre-B cells was accessory cell-mediated.