Identification and gene organization of three novel members of the IL-1 family on human chromosome 2.

Members of the IL-1 family of cytokines are important in mediating inflammatory responses. The genes encoding IL-1alpha, IL-beta, and the IL-1 receptor antagonist (IL-1Ra) are clustered within 450 kb on human chromosome 2q. By searching the EST databases and sequencing this region of chromosome 2, we have identified three novel genes that show homology to the IL-1 family, which we have named IL-1-related protein 1, 2, and 3 (IL-1RP1, IL-1RP2, and IL-1RP3). All three genes contain a signature motif common to the IL-1 family and appear to be more closely related to IL-1Ra. Similar to the intracellular form of IL-1Ra, these genes lack conventional hydrophobic signal sequences. The expression of these genes appears to be highly restricted to various epithelial cell populations. Our results demonstrate the existence of additional IL-1 gene family members within the previously defined IL-1 cluster and point to this region of chromosome 2 as an evolutionary hotspot for IL-1 gene duplication. These genes may prove to have an important role in inflammatory responses.

Functional and structural diversity of the human Dickkopf gene family.

Wnt proteins influence many aspects of embryonic development, and their activity is regulated by several secreted antagonists, including the Xenopus Dickkopf-1 (xDkk-1) protein. xDkk-1 inhibits Wnt activities in Xenopus embryos and may play a role in induction of head structures. Here, we characterize a family of human Dkk-related genes composed of Dkk-1, Dkk-2, Dkk-3, and Dkk-4, together with a unique Dkk-3 related protein termed Soggy (Sgy). hDkks 1-4 contain two distinct cysteine-rich domains in which the positions of 10 cysteine residues are highly conserved between family members. Sgy is a novel secreted protein related to Dkk-3 but which lacks the cysteine-rich domains. Members of the Dkk-related family display unique patterns of mRNA expression in human and mouse tissues, and are secreted when expressed in 293T cells. Furthermore, secreted hDkk-2 and hDkk-4 undergo proteolytic processing which results in cleavage of the second cysteine-rich domain from the full-length protein. Members of the human Dkk-related family differ not only in their structures and expression patterns, but also in their abilities to inhibit Wnt signaling. hDkk-1 and hDkk-4, but not hDkk-2, hDkk-3 or Sgy, suppress Wnt-induced secondary axis induction in Xenopus embryos. hDkk-1 and hDkk-4 do not block axis induction triggered either by Xenopus Dishevelled (Xdsh) or Xenopus Frizzled-8 (Xfz8), both of which function to transduce signals from Wnt ligands. Thus, hDkks 1 and 4 may inhibit Wnt activity by a mechanism upstream of Frizzled. Our findings highlight the structural and functional heterogeneity of human Dkk-related proteins.

The characterization, molecular cloning, and expression of a novel hematopoietic cell antigen from CD34+ human bone marrow cells.

The adhesion molecule BEN/SC1/DM-GRASP (BEN) is a marker in the developing chicken nervous system that is also expressed on the surface of embryonic and adult hematopoietic cells such as immature thymocytes, myeloid progenitors, and erythroid progenitors. F84.1 and KG-CAM, two monoclonal antibodies to rat neuronal glycoproteins with similarity to BEN, cross-react with an antigen on rat hematopoietic progenitors, but F84.1 only also recognizes human blood cell progenitors. We have defined the antigen recognized by F84.1 as the hematopoietic cell antigen (HCA). HCA expression was detected on 40% to 70% of CD34+ fetal and adult bone marrow cells and mobilized peripheral blood cells. Precursor cell activity for long-term in vitro bone marrow cell culture was confined to the subset of CD34+ cells that coexpress HCA. HCA is expressed by the most primitive subsets of CD34+ cells, including all rhodamine 123(lo), Thy-1+, and CD38(-/lo) CD34+ adult bone marrow cells. HCA was also detected on myeloid progenitors but not on early B-cell progenitors. We also describe here the cloning and characterization of cDNAs encoding two variants of the human HCA antigen (huHCA-1 and huHCA-2) and of a cDNA clone encoding rat HCA (raHCA). The deduced amino acid sequences of huHCA and raHCA are homologous to that of chicken BEN. Recombinant proteins produced from either human or rat HCA cDNAs were recognized by F84.1, whereas rat HCA but not human HCA was recognized by antirat KG-CAM. Expression of either form of huHCA in CHO cells conferred homophilic adhesion that could be competed with soluble recombinant huHCA-Fc. The molecular cloning of HCA and the availability of recombinant HCA should permit further evaluation of its role in human and rodent hematopoiesis.

Leukemia inhibitory factor upregulates cytokine expression by a murine stromal cell line enabling the maintenance of highly enriched competitive repopulating stem cells.

Attempts to maintain or expand primitive hematopoietic stem cells in vitro without the concomitant loss of their differentiative and proliferative potential in vivo have largely been unsuccessful. To investigate this problem, we compared the ability of three cloned bone marrow (BM) stromal cell lines to support the growth of primitive Thy-1lo Sca-1+H-2Khi cells isolated by fluorescence-activated cell sorting from the BM of Ly-5.2 mice treated 1 day previously with 5-fluo- rouracil. Sorted cells were highly enriched in cobblestone area-forming cells (CAFC), but their frequency was dependent on the stromal cell lines used in this assay (1 per 45 cells on SyS-1; 1 per 97 cells on PA6). In the presence of recombinant leukemia inhibitory factor (LIF), CAFC cloning efficiency was increased to 1 per 8 cells on SyS-1 and 1 per 11 cells on PA6, thus showing the high clonogenicity of this primitive stem cell population. More primitive stem cells with competitive repopulating potential were measured by injecting the sorted cells into lethally irradiated Ly-5.1 mice together with 10(5) radioprotective Ly-5.1 BM cells whose long-term repopulating ability has been "compromised" by two previous cycles of marrow transplantation and regeneration. Donor-derived lymphocytes and granulocytes were detected in 66% of animals injected with 50 sorted cells. To quantitate the maintenance of competitive repopulating units (CRU) by stromal cells, sorted cells were transplanted at limiting dilution before and after being cultured for 2 weeks on adherent layers of SyS-1, PA6, or S17 cells. CRU represented 1 per 55 freshly sorted cells. CRU could be recovered from cocultures supported by all three stromal cell lines, but their numbers were approximately-sevenfold less than on day 0. In contrast, the addition of LIF to stromal cultures improved CRU survival by 2.5-fold on S17 and PA6 cells (approximately two-fold to threefold decline), and enabled their maintenance on SyS-1. LIF appeared to act indirectly, because alone it did not support the proliferation of Thy-1lo Sca-1+H-2Khi cells in stroma-free cultures. Polymerase chain reaction (RT-PCR) analysis revealed that Interleukin-1beta (IL-1 beta) IL-2, IL-6, granulocyte-colony stimulating factor, granulocyte macrophage-colony stimulating factor, transforming growth factors, LIF, and Steel Factor (SLF) mRNAs were upregulated in SyS-1 within 1 to 6 hours of LIF-stimulation. To determine if increased expression of SLF by LIF-stimulated SyS-1 cells could account for their capacity to support stem cells, sorted calls were cocultured on simian CV-E cells that were transfected with an expression vector encoding membrane-bound SLF, or supplemented with soluble SLF. In both cases, SLF synergized with IL-6 produced endogenously by CV-E cells enabling CAFC growth equivalent to that on LIF-stimulated SyS-1. CAFC development on LIF-stimulated SyS-1 could also be completely abrogated by an anti-SLF antibody. These data provide evidence for a role of LIF in the support of long-term repopulating stem cells by indirectly promoting cytokine expression by BM stroma. Furthermore, we have used quantitative assays to show a maintenance of CRU numbers, with retention of in vivo function following ex vivo culture.

Recombinant human acetylcholine receptor alpha-subunit induces chronic experimental autoimmune myasthenia gravis.

A synthetic gene encoding the 210 N-terminal residues of the alpha-subunit of the nicotinic acetylcholine receptor (AChR) of human skeletal muscle was cloned into an inducible expression plasmid to produce a fusion protein in high yield in Escherichia coli. Like native human AChR, the recombinant human alpha 1-210 protein induced AChR-binding, AChR-modulating, and AChR-blocking autoantibodies in rats when injected once intradermally as an emulsion in CFA, with Bordetella pertussis vaccine as supplementary adjuvant. The minimum dose of recombinant protein required to induce biochemical signs of experimental autoimmune myasthenia gravis (EAMG) with 100% incidence was 2.2 micrograms. With 6.6 to 22 micrograms, serum levels of autoantibodies were persistent, and clinically apparent EAMG lasted more than a month. Clinical, electrophysiological, and biochemical indices of EAMG induced by doses of 66 micrograms or more were more uniformly severe and persistent, with 33% fatality. Rats receiving a control extract of E. coli containing plasmid without the alpha 1-210 codon insert, with adjuvants, did not develop autoantibodies or signs of EAMG. This highly reproducible new model of EAMG induced by a recombinant human autoantigen should be valuable for testing Ag-specific immunotherapeutic strategies that might be applicable to treating acquired myasthenia gravis in humans.

Primary structure of bovine conglutinin, a member of the C-type animal lectin family.

We report the complete amino acid sequence of bovine conglutinin obtained by structural characterization of peptides derived from the protein by various chemical and enzymatic fragmentation methods. The protein consists of 351 amino acid residues including 55 apparent Gly-X-Y repeats with two interruptions. This 171-residue-long collagenous domain separates a short noncollagenous NH2-terminal region of 25 residues from the 155-residue-long globular COOH terminus revealing the structural relation of conglutinin with mannose-binding proteins, pulmonary surfactant-associated proteins, and a complement component C1q. Eight hydroxylysine residues were found in the collagenous domain. All of these hydroxylysine residues which occupy a Y position in a Gly-X-Y triplet are possible glycosylation sites since no phenylthiohydantoin amino acid was identified in automated Edman degradation cycles corresponding to these sites. The noncollagenous COOH domain of conglutinin, on the other hand, contains a carbohydrate recognition domain which shares substantial sequence homology with C-type animal lectins. Conglutinin has the greatest sequence similarity with mannose-binding proteins and pulmonary surfactant-associated proteins.

Cloning and expression in Escherichia coli of a synthetic gene encoding the extracellular domain of the human muscle acetylcholine receptor alpha-subunit.

To better define the antigenic sites on the human muscle acetylcholine receptor (AChR) that are involved in stimulating the production of pathogenic antibodies in myasthenia gravis (MG), the nucleotide sequence encoding the major extracellular domain of the AChR alpha subunit was chemically synthesized. The gene cassettes encoding amino acids (aa) 1-85 (AChR-I) and 86-210 (AChR-II), were cloned individually, and the coding sequence representing the complete major extracellular domain (aa 1-210; AChR-C) was obtained by subsequent fusion of cassettes encoding AChR-I and AChR-II. The genes were inserted into the inducible expression plasmid, pKK-223-3, and expressed in vitro and in vivo in Escherichia coli. Biological activity was demonstrated by immunoprecipitation of in vitro-synthesized AChR-C by sera from MG patients and by the alpha-bungarotoxin-binding activity of E. coli-synthesized AChR-II and AChR-C. The availability of the recombinant AChR polypeptides should facilitate studies on the molecular basis of the autoimmune response in MG.

Purification of Drosophila acidic ribosomal proteins.

The relatively acidic proteins (group A80) of Drosophila melanogaster ribosomes were separated by ion-exchange chromatography. Fractions containing one or more acidic proteins were combined into thirteen pools. The criterion for the combination was the migration pattern in one-dimensional polyacrylamide gels containing sodium dodecyl sulphate. Five proteins (7/8, S25/S27, S14, L1/L2 and L5/L6) required no further purification. The others were further purified as follows: proteins S7, and S9 by preparative gel electrophoresis: and protein 13 (to newly identified protein) by adsorption with conconavalin-A--agarose. Four proteins had no detectable contamination, and in each of the others the impurities were no greater than 3%. The amount of purified protein recovered from a starting amount of 2.63 g total 80-S ribosomal protein and a starting amount of 105 mg group A80 varied from 0.4 mg to 8.8 mg. The molecular weight of the proteins was estimated by polyacrylamide gel electrophoresis in sodium dodecyl sulphate. The amino acid composition of the individual purified proteins was determined. Several phosphorylated proteins were identified. Proteins 13b and 13c are phosphorylated derivatives of 13a; 7b/8b and 7c/8c are phosphorylated derivatives of 7a and/or 8a. Proteins 7/8 and 13 are distinct proteins but are very similar in amino acid composition.

An electron microscopic method for localization of ribosomal proteins during transcription of ribosomal DNA: a method for studying protein assembly.

We describe a method for the localization of ribosomal proteins on electron microscopic spreads of active rRNA genes. The method consists of raising antibodies against Drosophila melanogaster proteins and allowing these antibodies to react with lysates of D. melanogaster egg chambers. The locations of the bound IgGs in the active transcripts are detected with goat anti-rabbit IgGs that have been labeled with electron-dense polymethacrylate spheres. By statistical analysis we can generate a confidence interval for the initial point of protein assembly for a particular protein. The first point of protein assembly for S14 is located near the 5' end of the pre-18S rRNA. In contrast, the first point of protein assembly for L4 is at 0.38 unit from the initiation point (a unit being the length of a ribosomal transcription unit). The binding patterns of S14 and L4 are consistent with the 5' proximal and the 5' distal orientations of the pre-18S and the pre-28S rRNAs. The method described here provides an approach to the elucidation of the assembly of eukaryotic ribosomal proteins in vivo.

The in vivo expression of pseudo ribosomal RNA genes in Drosophila melanogaster.

Electron microscopic examination of chromatin has indicated that there are two major classes of ribosomal transcription units (rTUs) in Drosophila melanogaster. (a) a standard class which is about 8 kb in length and (b) a longer class (up to 15 kb) which has been hypothesized as having an intron in the 28S region of the gene. On rare occasions, we have observed a third class of TUs, which we term pseudo rTUs (or pseudo ribosomal RNA genes), distributed among the standard rTUs. Pseudo rTUs are composed of short fiber arrays with clear gradients in their RNP fiber lengths. The pseudo rTUs observed in egg chambers and blastoderm embryos are 4.1 +/- 0.58 kb in length and are found in tandem with non-transcribed spacer regions. The lengths of the non-transcribed spacer regions are found in 2 classes: one at 4.94 +/- 1.78 kb and the other at 19.28 +/- 2.11 kb. The present information suggests that these pseudo rTU fiber arrays are ribosomal in origin as their RNP fibers cross-react with antibodies raised against Drosophila ribosomal proteins. The pattern of distribution of D. melanogaster ribosomal protein L4 on RNP fibers in standard rTUs is compared with that in pseudo rTUs. This was determined by a novel approach involving the use of electron microscopic spreads of rTUs to which had been added IgGs labelled with polymethacrylate spheres. Pseudo ribosomal RNA genes are present in both the X (6%) and in the Y (6%) ribosomal chromatin as is indicated by their existence in nurse cells of both Oregon-R females, and females of the genotype sc4sc8/sc4sc8/y+Y.