Bone and the hematopoietic and immune systems: a report of the proceedings of a scientific workshop.

Recent observations underscore the linkage between endochondral bone formation and the establishment of hematopoietic marrow and suggest that interactions among bone, marrow, and the immune system persist in the mature skeleton. A workshop was held at the National Institutes of Health, Bethesda, Maryland, to discuss recent work on these interactions and to identify new areas of research. Marrow stromal cells include the precursors of the osteochondrogenic lineage, exert important influences on osteoclastogenesis and lymphopoiesis, and mediate the effects of some systemic factors on bone turnover. Recent evidence indicates that hematopoietic cells can influence the differentiation of osteogenic cells and suggests that mature lymphocytes can influence osteoclastic and osteoblastic functions. However, interpretation of experiments may be confounded by the potential for stage-specific responses within a cell lineage, the likelihood that divergent pathways compete for limited pools of precursor cells, and the possibility that important cells or factors are still unidentified. Further, in vitro models may be limited by species and anatomical site specificities, the absence of intermediary or accessory cells, and the absence of normal marrow spatial organization and cellular interactions with the extracellular matrix. Nevertheless, current approaches hold the potential for significant advances in our understanding of the relationships between bone and the hematopoietic and immune systems. Refinements of in vitro systems, the use of genetically manipulated mice, and the examination of clinical syndromes promise important insights. Collaborations among bone biologists, hematologists, and immunologists, and between basic scientists and clinical investigators, will be crucial for continued progress.

Sequence of lamprey vitellogenin. Implications for the lipovitellin crystal structure.

The amino acid sequence of lamprey vitellogenin has been predicted from the nucleotide sequence of cloned cDNA. The sites of proteolytic cleavage that produce the lipovitellin complex from the vitellogenin have been located by comparing the N-terminal sequences of two lamprey lipovitellin polypeptides with the predicted sequence. These results also confirm that the vitellogenin sequence derived here corresponds to the lipovitellin complex for which the crystal structure has been solved previously. Predictions of secondary structure indicate that the region most likely to correspond to the large alpha-helical domain of the crystallographic model consists of vitellogenin residues 300 to 600. Similar to the lipovitellins of Xenopus laevis, lamprey lipovitellin appears to lack approximately 200 C-terminal residues that are present in vitellogenin. However, the lamprey lipovitellin differs from those of Xenopus and chicken in two respects. First, most of the serine-rich domain that is present as the phosvitin polypeptide in the lipovitellins of the higher vertebrates appears to be lost in the maturation of lamprey vitellogenin to lipovitellin. Second, the domains that constitute the large lipovitellin-1 polypeptide in Xenopus and chicken are present in two polypeptides in lamprey, owing to an additional proteolytic processing event.

Two distinct yolk lipoprotein complexes from Caenorhabditis elegans.

The four yolk polypeptides of the nematode Caenorhabditis elegans are found in two types of lipoprotein particle: 12 S particles with Mr estimated at 450,000 and 8 S particles with Mr estimated at 250,000. Both types of particle contain approximately 8% phospholipids, 3% triglycerides, and 3% other lipids by mass. All four C. elegans yolk polypeptides can be found in either 12 or 8 S particles, depending upon the conditions of isolation. While the properties of the 12 and 8 S lipoprotein particles are consistent with a dimermonomer relationship, the asymmetric distribution of the yolk polypeptides between 12 and 8 S fractions suggests that at least two different oligomeric lipoprotein complexes are present in C. elegans embryos. In order to clarify the subunit composition of the C. elegans yolk lipoproteins, the patterns of polypeptides retained in immunoaffinity binding procedures by immunoglobulins of different antigenic specificities have been compared. When immunoaffinity binding is performed in the absence of sodium dodecyl sulfate, three C. elegans yolk proteins (yp170A, yp115, and yp88) are retained together by polyclonal immunoglobulins directed against either yp115 or yp88. A monoclonal immunoglobulin also retains these three proteins together. In contrast, a second monoclonal immunoglobulin retains only the fourth yolk protein (yp170B). Aggregate species, evidently reflecting the spontaneous formation of interchain disulfide bonds, indicate that yp170A and yp88 are physically associated, whereas yp170B self-associates in dimers. It is concluded that there are two distinct lipoprotein complexes in C. elegans: the A complex, which consists of yp170A, yp115, and yp88 and is essentially heterodimeric and the B dimer, a simple dimer of yp170B.

Cleavage of two yolk proteins from a precursor in Caenorhabditis elegans.

Four yolk proteins have been identified previously in the nematode Caenorhabditis elegans. However, only two of these proteins ( yp170A and yp170B ) are found among the products of in vitro translation of nematode RNA. The other two yolk proteins ( yp115 and yp88 ) are apparently cleaved from a precursor polypeptide of approximately 180,000 Mr. This precursor has been identified as an in vitro translation product and as a metabolically unstable polypeptide in vivo. It is bound by immunoglobulin G (IgG) specific for yp115 and by IgG specific for yp88 . The immunoadsorbed material yields the same pattern of fragments on partial digestion with Staphylococcus aureus V8 protease regardless of whether anti- yp115 or anti- yp88 IgG is used in the adsorption. Like the yp170 polypeptides, the yp115 / yp88 precursor is synthesized by the intestine and secreted intact. The precursor is evidently cleaved to yield yp115 and yp88 after secretion from the intestine but independent of the presence of the gonad. Thus, cleavage probably occurs in the body cavity of the nematode.

Yolk proteins of Caenorhabditis elegans.

A group of proteins judged on several criteria to be yolk proteins have been isolated from a homogenate of the nematode Caenorhabditis elegans. Comparison of partial proteolysis fragments indicates that the two bands of a 170,000-dalton doublet (yp170) are closely related; bands observed at 115,000 daltons (yp115) and 88,000 daltons (yp88) appear to be structurally distinct. All three yolk protein species are glycoproteins, as judged by binding of the lectin concanavalin A. The yp170 doublet has been purified by gel filtration in the presence of sodium dodecyl sulfate. An antiserum obtained by immunization with the purified yp170 doublet does not bind either of the two smaller proteins. Staining of C. elegans eggs by indirect immunofluorescence with the anti-yp170 serum indicates a dispersed cytoplasmic location for the antigen throughout embryogenesis, with apparent segregation to the intestine immediately prior to hatching.

Tissue-specific synthesis of yolk proteins in Caenorhabditis elegans.

The primary site of yolk protein synthesis in the nematode, Caenorhabditis elegans, has been determined. In animals containing no gonadal cells (obtained by laser ablation of the gonadal precursor cells early in development), yolk proteins are present in abundance. This demonstrates that yolk proteins are made outside the gonad. An examination of proteins present in tissues isolated by dissection, and a comparison of proteins synthesized by isolated tissues incubated in vitro have identified the intestine as the major site of yolk protein synthesis. We propose that yolk proteins are synthesized in the intestine, secreted from the intestine into the body cavity, and taken up from the body cavity by the gonad to reach oocytes. The site of yolk protein synthesis has also been examined in four mutants that have largely male somatic tissues, but a hermaphrodite germ line. Here again, yolk proteins are produced by intestines in a hermaphrodite-specific manner. This suggests that sex determination is coordinately regulated in intestinal and germ line tissues.

Specificity of bacterial ribosomes and messenger ribonucleic acids in protein synthesis reactions in vitro.

Ribosomes from two Gram-negative bacteria translated f2 RNA, T4 early mRNA, mRNA from three Gran-negative bacteria, and mRNA from six Gram-positive bacteria; ribosomes from three Gram-positive bacteria translated mRNA from the Gram-positive strains, but did not translate the other mRNAs. Ribosomes from the Gram-negative bacterium Escherichia coli translated synthetic poly(U,G) but ribosomes from the Gram-positive bacterium Clostridium pasteurianum translated poly(U,G) very poorly, mRNA from Gram-negative bacteria was translated only in the presence of a high salt ribosomal wash containing initiation factors. mRNA from Gram-positive bacteria and synthetic poly(U,G) were translated much more efficiently when wash components were present, but were also translated to a small, but significant, extent in the absence of wash components. The translation specificity of each type of ribosome was independent of the source of ribosomal wash components. When the radioactively labeled products of in vitro protein synthesis were analyzed by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and autoradiography, it was found that each different bacterial and phage RNA preparation directed the synthesis of a unique set of polypeptide products of discrete sizes. Three different types of ribosomes were used to translate each of several Gram-positive bacterial messenger preparations; the overall patterns of products obtained with a given mRNA are similar, but some differences in the products formed or the relative amounts of the various products synthesized can be detected.