Biological and structural properties of MIP-1 alpha expressed in yeast.

The murine macrophage inflammatory proteins-1 alpha (MIP-1 alpha) and MIP-1 beta are distinct but closely related cytokines. Partially purified mixtures of the two proteins affect neutrophil function and cause local inflammation and fever. The particular properties of MIP-1 alpha have not been well studied, although it has been identified as being identical to an inhibitor of haemopoietic stem cell growth. We have expressed MIP-1 alpha in yeast cells and purified it to sequence homogeneity. Structural analysis of this biologically active material by circular dichroism and fluorescence spectroscopy confirms that MIP-1 alpha has a very similar secondary and tertiary structure to platelet factor 4 and interleukin 8 with which it shares limited sequence homology. The in-vitro stem cell inhibitory properties have been confirmed using a range of murine progenitor cells including purified bone marrow progenitor cells (FACS-1), the FDCP-mix A4 cell line, and spleen colony forming unit (CFU-S) populations. Plateau levels of inhibition of stem cell growth were achieved using concentrations of 0.15 micrograms/ml MIP-1 alpha. We have also demonstrated that MIP-1 alpha is active in vivo: 5 micrograms of MIP-1 alpha per mouse given as a bolus injection, protects stem cells from subsequent in-vitro killing by tritiated thymidine. MIP-1 alpha was also shown to enhance the proliferation of more committed progenitor granulocyte macrophage-colony forming cells (GM-CFC) in response to granulocyte macrophage-colony stimulating factor (GM-CSF).     

Characterization and linkage mapping often sheep microsatellite markers derived from a sheep x hamster cell hybrid

A cosmid library has been constructed from a sheep x hamster cell hybrid containing sheep chromosome t₁, rob (6;24). Clones containing sheep DNA were identified by hybridizing to a total sheep genomic DNA probe. Small fragments (<500 bp) containing (AC)_n microsatellites were subcloned and sequenced. Ten microsatellite markers were characterized and six were mapped back to chromosomes 6 and 24. The remaining microsatellites mapped to chromosome 26, which was shown to be present in a small proportion of cells of the cell line.     

Formation of glycine and aminoacetone from L-threonine by rat liver mitochondria

Threonine is a precursor of glycine in the rat, but the metabolic pathway involved is unclear. To elucidate this pathway, the biosynthesis of glycine, and of aminoacetone, from L-threonine were studied in rat liver mitochondrial preparations of differing integrities. In the absence of added cofactors, intact mitochondria formed glycine and aminoacetone in approximately equal amounts from 20 mM L-threonine, but exogenous NAD+ decreased and CoA increased the ratio of glycine to aminoacetone formed. In intact and freeze-thawed mitochondria, the ratio of glycine to aminoacetone formed was markedly sensitive to the concentration of L-threonine, glycine being the major product at low L-threonine concentrations. Disruption of mitochondrial integrity by sonication (1 min) decreased the ratio of glycine to aminoacetone formed, and in 20000 X g supernatant fractions from sonicated (3 min) mitochondria, aminoacetone was the major product. The main non-nitogenous two-carbon compound detected when intact mitochondria catabolized L-threonine to glycine was acetate, which was probably derived from deacylation of acetyl-CoA. These results suggest that glycine formation from L-threonine in rat liver mitochondria occurred primarily by the coupled activities of threonine dehydrogenase and 2-amino-3-oxobutyrate CoA-ligase, the extent of coupling between the enzymes being dependent upon a close physical relationship and upon the flux through the dehydrogenase reaction. In vivo glycine synthesis would predominate, and aminoacetone would be a minor product.     

Metabolically inactive 3T3 cells can substitute for marrow stromal cells to promote the proliferation and development of multipotent haemopoietic stem cells

When highly enriched multipotential spleen colony forming cells (CFU-S) obtained following fluorescence activated cell sorting (FACS-CFU-S) are cultured on marrow stromal cells, they undergo proliferation and development to produce mature haemopoietic cells (Spooncer et al., Nature, 316:62-64, 1985). We now show that FACS-CFU-S behave in a similar way when cultured on monolayers of 3T3 cells, indicating that the 3T3 cells can supply at least part of the environment which is representative of marrow stromal cells and provide, therefore, a system for studying stromal cell: haemopoietic cell interactions. We also demonstrate that IL-3-dependent multipotential stem cell lines (FDCP-Mix), but not a variety of other "committed" IL-3-dependent cell lines, resemble FACS-CFU-S in terms of their ability to proliferate and differentiate when cultured on 3T3 cells in the absence of IL-3. In this system, attachment of the FDCP-Mix to the 3T3 cells is critical for the subsequent maintenance of viability and stimulation of development of the cells. When the FDCP-Mix cells are physically separated from the 3T3 cells, they die and their death cannot be prevented by using 3T3-cell-conditioned medium. The extracellular matrix generated by 3T3 cells is not sufficient for promoting attachment or viability of the FDCP-Mix cells, indicating the importance of integral membrane components. However, attachment and development of FDCP-Mix cells occurs on 3T3 cells that have been lightly fixed with glutaraldehyde indicating that active metabolism is not essential for the effects promoted by the 3T3 cells. We suggest that the ability of FACS-CFU-S and FDCP-Mix cells to respond to 3T3 cells involves specific ligand/receptor interactions.     

W3/25+ T cells mediate the induction of immunologic unresponsiveness in enhanced rat recipients of cardiac allografts

Cellular suppressor mechanisms developing during the induction of immunologic enhancement were studied in LEW rats immunized actively with BN spleen cells and passively with LEW anti-BN hyperimmune serum 11 and 10 days before receiving a (LEW X BN)F1 cardiac allograft, respectively. With this regimen, graft survival is prolonged from 7.4 +/- 0.5 days in unmodified, acutely rejecting hosts to 25 +/- 12 days in enhanced recipients, with one-third of the grafts surviving indefinitely. To test for suppressor capacities, 60 X 10(6) splenic T helper/inducer (W3/25+) and T suppressor/cytotoxic (OX8+) cells were adoptively transferred 7 and 14 days after transplantation either into unmodified syngeneic LEW animals that received (LEW X BN)F1 test grafts 24 hr later or into T cell-deprived B rats with indefinitely functioning heart transplants that were reconstituted with sensitized lymph node cells (100 X 10(6). W3/25+ T cells harvested on days 7 and 14 from enhanced recipients prolonged test graft survival in unmodified hosts (13.1 +/- 2.3 and 13.3 +/- 1.3 days, respectively, p less than 0.001) and delayed rejection in reconstituted B rats from 6.7 +/- 0.5 to 18.2 +/- 6.5 and 23.3 +/- 5.8 days, respectively (p less than 0.001). OX8+ and B lymphocytes had no suppressor activity. However, enzymatic stripping of the surfaces of W3/25+ cells abrogated suppressor function. Similarly, after i.p. treatment with cyclophosphamide, W3/25+ T cells lost their suppressor properties. Lack of donor specific but not third party alloaggressiveness was demonstrated by the profoundly diminished ability of W3/25+ lymphocytes from enhanced hosts to recreate rejection in nonreconstituted B rats, even when exogenous interleukin 2 was administered. After pronase treatment, however, W3/25+ T cells were capable of inducing immunoresponsiveness at a tempo similar to naive T helper cells (31.5 +/- 12.5 vs 32.8 +/- 7.9). Thus, the present studies provide evidence for the development of a specific W3/25+ suppressor cell in the induction of active and passive enhancement. Coincident abrogation of specific T effector alloaggressiveness is apparently mediated by surface-blocking factors.     

Population genetics of coagulant factor IX: frequencies of two DNA polymorphisms in five ethnic groups.

Two frequently used restriction-enzyme polymorphisms (RFLPs) of coagulant F.IX, TaqI and XmnI, have been examined in five ethnic groups: white Americans, black Americans, East Indians, Chinese, and Malays. There is a distinct "cline" in the frequencies of both polymorphisms, from white Americans to Malays. The rarer type 2 alleles of both polymorphisms, in which middle recognition sites are present--and which in our sample reach their highest frequencies in white Americans--are marginally higher in four groups of Europeans previously reported by others. The frequencies of the rarer alleles are significantly higher in Europeans than in black Americans and East Indians, and these alleles are essentially absent in Chinese and Malays. The frequency of heterozygosity diminishes in the same order, being zero in Malays for both polymorphisms. The polymorphisms are in strong linkage disequilibrium, and in all groups the type 1 allele for TaqI is disproportionately accompanied by the type 1 allele for XmnI. The paucity of type 2 alleles and the low rate of heterozygosity in four non-European groups suggest that the polymorphisms will be of little diagnostic value south of Gibraltar and east of Suez. This prediction is confirmed by the observed haplotype frequencies in the black American and the Oriental groups.     

L-641,953 (R-8-fluoro-dibenzo[b, f]thiepin-3-carboxylic acid-5-oxide): a novel thromboxane-prostaglandin endoperoxide antagonist

The effects of L-641,953 (R-8-fluoro-dibenzo[b, f]thiepin-3-carboxylic acid-5-oxide) have been studied on pulmonary and other smooth muscle preparations in vitro and in vivo. When studied in vitro on guinea-pig tracheal chains, L-641,933 produced significant shifts in the dose-response curves to the prostaglandin endoperoxide analogues, U-44069 (pA2 7.06) and U-46619 (pA2 7.14), and prostaglandin (PG) F2 alpha (pA2 6.33) had minimal activity against contractions induced by histamine (pA2 4.38), 5-hydroxytryptamine (pA2 4.63), and acetylcholine (pA2 4.56) and slightly enhanced relaxation induced by PGE2. When tested on the guinea-pig gall bladder strip in vitro, L-641,953 antagonized contractions induced by U-44069 (pA2 7.03) but was less active against those induced by PGF2 alpha (pA2 6.03), PGE1 (pA2 5.62), and histamine (pA2 4.84). When tested in vitro on the guinea-pig pulmonary artery, L-651-953 significantly antagonized contractions induced by U-44069 (pA2 7.04), U-46619 (pA2 7.14), and PGF2 alpha (pA2 7.16) but was less effective against contractions induced by histamine (pA2 4.19). Schild analysis indicated that L-641,953 was fully competitive against contractions of either the guinea-pig tracheal chain induced by U-46619 or the guinea-pig pulmonary artery induced by U-44069 and U-46619. When tested on human platelets in vitro L-641,953 inhibited aggregation induced by U-44069 (IC50 1.3 X 10(-6) M) but not ADP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Different routes for integral protein insertion into Ricinus communis protein-body and glyoxysome membranes

Total polyadenylated RNA from ripening or germinating Ricinus communis L. endosperm was translated in rabbit reticulocyte lysate in the absence or presence of canine pancreatic microsomes. The products were immunoprecipitated using antibodies raised againts Triton X-114-extracted integral membrane proteins of protein bodies or glyoxysomes. While the proteins of proteinbody membranes were found to insert co-translationally into added microsomes, this was not observed in the case of glyoxysomal proteins. This observation was confirmed using antibodies raised against a purified glyoxysome membrane protein, alkaline lipase. These results indicate that different routes exist for the insertion of membrane proteins into the two organelles. In both cases membrane-protein insertion does not appear to be accompanied by proteolytic processing.Abbreviations anti-PB antiserum to integral protein-body membrane proteins - anti-G antiserum to integral glyoxysomal membrane proteins - anti-L antiserum to alkaline lipase - ER endoplasmic reticulum - M_r relative molecular mass - mRNA poly(A)-rich messenger RNA - PAGE polyacrylamide gel electrophoresis - poly(A) polyadenylic acid - SDS sodium dodecyl sulphate