Macrophage precursors as natural killer cells against tumor cells and microorganisms

Macrophage precursors cells have been isolated from spleen and liver of mice and have characterized using F4/80 antibody, their proliferative response to CSF-1 and their maturation to macrophages. These nonadherent and nonphagocytic cells exert strong killing of Yac-1 tumor cells and of various microorganisms. Transplantation of these macrophage precursors into lethally irradiated allogenic hosts restores natural killer (NK) activity within 14 days. Macrophage precursors show enhanced NK activity when activated with interleukin 2. FACS analysis of F 4/80 presorted macrophage precursors reveals about 30% of the cells coexpressing NK 1.1. and F 4/80. These data support the assumption that at least a part of the NK cell compartment is derived from the myeloid lineage.     

A candidate gene family for the mouse t complex responder (Tcr) locus responsible for haploid effects on sperm function

The mouse t complex responder (Tcr) locus plays a central haploid-specific role in the transmission ratio distortion phenotype expressed during germ cell differentiation in t-carrying males. The accumulated data map Tcr to a region of less than 500 kb. Over 400 kb of this region has been cloned and consists entirely of sequences associated with a clustered family of large cross-hybridizing elements of 30 kb to 70 kb in size. We have characterized a gene family within this region that is expressed uniquely in male germ cells with a complex pattern of RNA processing. Antibodies produced against a product of the putative open reading frame recognize a testes-specific polypeptide. Genetic data support the hypothesis that this polypeptide(s) functions to effect the Tcr phenotype.     

Involvement of various organs in the initial plasma clearance of differently glycosylated rat liver secretory proteins

The initial plasma clearance and organ distribution of alpha 1-acid glycoprotein and alpha 2-macroglobulin carrying different types of oligosaccharide, side chains was studied in rats. The differently glycosylated proteins were synthesized by rat hepatocytes in culture in the presence of tunicamycin (unglycosylated form), swainsonine (hybrid type), or 1-deoxymannojirimycin (high-mannose type). Deglycosylated glycoproteins (Asn-GlcNAc) were obtained by endoglucosaminidase H treatment of high-mannose-type glycoproteins. Ten minutes after intravenous injection 3% of complex type, 26% of hybrid type, 84% of high-mannose type. 64% of unglycosylated and 80% of deglycosylated alpha 1-acid glycoprotein disappeared from the plasma. The respective values for alpha 2-macroglobulin were 26%, 42%, 59% and 67%. When the clearance of total hepatic secretory proteins was examined, major differences between glycosylated and unglycosylated (glyco)proteins were found, particularly in the case of low-molecular-mass polypeptides. Whereas complex-type alpha 1-acid glycoprotein and alpha 2-macroglobulin showed no accumulation in various organs, hybrid-type alpha 1-acid glycoprotein and alpha 2-macroglobulin were present in spleen and liver. High-mannose-type alpha 1-acid glycoprotein and alpha 2-macroglobulin also accumulated mainly in spleen and liver. Spleen had the highest specific activity; liver, due to its larger organ mass, represented the major organ for the uptake of high-mannose-type glycoproteins. Competition experiments with mannan and GlcNAc-bovine-serum-albumin showed a mannose/GlcNAc receptor-mediated removal. Whereas unglycosylated alpha 1-acid glycoprotein was taken up by the kidney, unglycosylated alpha 2-macroglobulin was found in the spleen. Deglycosylated glycoproteins (Asn-GlcNAc) were removed from the plasma via two different mechanisms: firstly, clearance by the kidney similar to the unglycosylated glycoproteins; secondly, clearance by a mannose/GlcNAc receptor-mediated uptake mainly into the spleen. We conclude that N-linked oligosaccharide side chains are important for the plasma survival of hepatic secretory glycoproteins and that unphysiologically glycosylated forms are cleared by different mechanisms.     

Remodeling of a rat hepatocyte plasma membrane glycoprotein. De- and reglycosylation of dipeptidyl peptidase IV

The present paper demonstrates the terminal de- and reglycosylation of a rat hepatocyte plasma membrane glycoprotein, dipeptidyl peptidase IV (DPP IV). Cultured hepatocytes were used in pulse-chase experiments with [3H]L-fucose and [14C]N-acetyl-D-mannosamine as markers for terminal carbohydrates, [3H]D-mannose as marker of a core-sugar, and [35S]L-methionine for labeling the protein backbone. Membrane DPP IV was immunoprecipitated with a polyclonal antibody which bound selectively at 4 degrees C to the cell-surface glycoprotein. The times of maximal labeling of hepatocyte plasma membrane DPP IV were 6-9 min for [3H]L-fucose, 20 min for [3H]D-mannose, and 25 min for [35S]L-methionine. When antibodies were bound to cell-surface DPP IV at 4 degrees C, the immune complex remained stable for more than 1 h after rewarming to 37 degrees C, despite ongoing metabolic and membrane transport processes. This was shown by pulse labeling with [35S]L-methionine at 37 degrees C, followed by cooling to 4 degrees C, and addition of antibody against plasma membrane DPP IV. During rewarming, the radioactivity in the complex remained constant. In a similar experiment with [3H]L-fucose, the radioactivity in the immune complex declined rapidly, indicating a defucosylation of the plasma membrane glycoprotein. Using the same experimental design with [3H]D-mannose, the radioactivity in the immune complex remained constant, showing that the core-sugar D-mannose is not cleaved from the membrane glycoprotein. Terminal reglycosylation (refucosylation and resialylation) was demonstrated as follows. Hepatocytes were maintained at 37 degrees C in a medium supplemented with tunicamycin in order to block the de novo synthesis of N-glycosidically bound carbohydrate chains. At 4 degrees C the antibody against DPP IV bound only to cell surface glycoprotein. During the rewarming period at 37 degrees C, radioactivity from [3H]L-fucose and [14C]N-acetyl-D-mannosamine became incorporated into the immune complex. This indicates a fucosylation and sialylation of the glycoprotein originally present at the cell surface. The mechanisms whereby terminal de- and reglycosylation of plasma membrane glycoproteins may occur during membrane recycling are discussed.     

Ganglioside biosynthesis in rat liver. Characterization of three sialyltransferases

Three sialyltransferase activities involved in ganglioside biosynthesis were studied in Golgi-enriched preparations of rat liver: the formation of GM3, GD3 and GD1a. The conditions for the quantitative assays of these enzymatic reactions were standardized and optimized, with Triton X-100 being used as detergent. The apparent Km values of each sialyltransferase for N-acetyl-2-(5'-cytidylyl)neuraminic acid (1.5 mM with GM3 synthase, 0.2 mM with GD3 synthase, and 0.5 mM with GD1a synthase) and the respective glycolipid substrates (0.08 mM for lactosylceramide, 0.1 mM for GM3, and 0.5 mM for GM1) were determined. Competition experiments showed that the three sialyltransferase activities are three individual catalytic entities. Moreover, evidence was found that product inhibition may play a role in the regulation of the activity of sialyltransferases.     

Cell proliferation-associated expression of a recently evolved isozyme of triosephosphate isomerase

An electrophoretically unique, thermolabile isozyme of triosephosphate isomerase (TPI; EC 5.3.1.1) accounts for 10–30% of the enzymatic activity in a range of mitotically active human cells and tissues. This type 2 form (subunit) of human TPI appears in two isozymes, an anodally migrating, relative to the constitutive TPI-1/1 homodimer, TPI-2/2 homodimer and the TPI-1/2 heterodimer with an intermediate mobility. Human cell types expressing the induced isozyme, which is the product of the same structural locus as the constitutive isozyme, include mitogen-stimulated lymphocytes, virally transformed B-lymphoblastoid cells, leukemia-derived T-lymphoblastoid cells, HeLa cells, both normal and transformed fibroblasts, and placental tissue. Extracts of nondividing or terminally differentiated human cells/tissues, such as erythrocytes, striated muscle, peripheral lymphocytes, and platelets, contain high levels of the constitutive TPI-1/1 isozyme but little or undetectable levels of the TPI-1/2 or TPI-2/2 isozyme. The cell division-associated TPI-1/2 and -2/2 isozymes are distinct in electrophoretic mobility from the deamidated forms of the constitutive isozyme. Extracts of dividing gorilla fibroblasts display an isozyme pattern identical to that of proliferating human cells, but various proliferating cells derived from the African green monkey, rabbit, and chicken express only the constitutive isozyme. Thus, expression of the cell division-associated isozyme of TPI is restricted to the hominoids, suggesting a recently evolved modification mechanism which is specifically activated in proliferating cells.Financial support was derived from Contract EY-77-C-02-2828 from the Department of Energy and Training Grant 5-T32-GM07544 from the National Institutes of Health.     

Biosynthesis and secretion of alpha 1 acute-phase globulin in primary cultures of rat hepatocytes

Experimental inflammation in rats led to a sevenfold increase in serum levels of alpha 1 acute-phase globulin. This increase is correlated with elevated levels of translatable mRNA for alpha 1 acute-phase globulin in the liver. Biosynthesis and secretion of alpha 1 acute-phase globulin were studied in rat hepatocyte primary cultures. An intracellular form of alpha 1 acute-phase globulin with an apparent relative molecular mass of 63 500 and a secreted form of 68 000 were found. The intracellular form of alpha 1 acute-phase globulin could be deglycosylated by endoglucosaminidase H treatment indicating that its oligosaccharide chains were of the high-mannose type. The secreted form of alpha 1 acute-phase globulin was not sensitive to endoglucosaminidase H, but was susceptible to the action of sialidase reflecting carbohydrate side-chains of the complex type. Pulse-chase experiments revealed a precursor-product relationship for the high-mannose and the complex type alpha 1 acute-phase globulin. In the hepatocyte medium newly synthesized alpha 1 acute-phase globulin was detected 30 min after the pulse. Unglycosylated alpha 1 acute-phase globulin was found in the cells as well as in the medium when the transfer of oligosaccharide chains onto the polypeptide chains was blocked by tunicamycin. Tunicamycin led to a marked delay in alpha 1 acute-phase globulin secretion.     

Characterization of a [3H]methyltrienolone (R1881) binding protein in rat liver cytosol

The binding of radiolabelled methyltrienolone 17 beta-hydroxy-17 alpha-methyl-estra-4,9,11-trien-3-one (R1881) to adult male rat liver cytosol has been characterized in the presence of Na-molybdate to stabilize steroid-hormone receptors, and triamcinolone acetonide to block progestin receptors. Using sucrose density gradient analysis, male liver cytosol contains a [3H] R1881 macromolecular complex which sediments in the 8-9S region. 8S binding of R1881 to male rat serum, female liver cytosol or cytosol from a tfm rat cannot be demonstrated. Further metabolism of [3H] R1881 following 20h incubation with male rat liver cytosol was excluded: In the 8S region 97% of [3H] R1881 was recovered by thin layer chromatography. Characteristics of this [3H] R1881-8S binding protein include high affinity (Kd = 2.3 +/- 41 nM) and low binding capacity (18.8 +/- 3.3 fmol/mg cytosol protein), precipitability in 0-33% ammonium sulfate, and translocation to isolated nuclei following in vivo R1881 treatment. Whereas, the cytosol R1881-receptor is competed for by dihydrotestosterone, testosterone, and estradiol, [3H] estradiol binding in the 8S region is not competitive with androgens but does compete with diethylstilbestrol. The nuclear androgen binding site has a Kd = 2.8 nM for [3H] R1881, and is androgen specific (testosterone greater than 5 alpha-dihydrotestosterone greater than estradiol greater than progesterone greater than cyproterone acetate greater than diethylstilbestrol greater than dexamethasone greater than triamcinolone). Since a number of liver proteins including the drug and steroid metabolizing enzymes are, in part, influenced by the sex-hormone milieu, the presence of a specific androgen receptor in male rat liver may provide valuable insight into the regulation of these proteins.