Are all lymphoid blasts in the cell cycle?

Labelling index after one or repeated intravenous injections of 3H-thymidine was measured for various subpopulations of lymphatic cells in different canine lymphoid compartments and correlated with cell morphology. High doses of tritiated thymidine were injected and exposure times of up to 211 days were used. The labelling indices of lymphoid blasts were comparable in all tissues investigated. Labelling index varied from 100% in immunoblasts to 4% in small-sized lymphocytes. Approximately 80% of immunoblasts were labelled 1 h after 3H-thymidine application and 100% labelling was obtained after 12 h repetitive 3H-thymidine labelling. In contrast with mediumsized and large lymphocytes, immunoblasts seem to be rapidly proliferating cells in the dog with almost no G_o cells. Supported by the Deutsche Forschungsgemeinschaft DFG Grant SFB 112     

Analysis of the human regulators of complement activation (RCA) gene cluster with yeast artificial chromosomes (YACs).

The human regulators of complement activation gene cluster (RCA cluster) have been partially characterized with yeast artificial chromosomes (YACs). While the data confirm many points previously elucidated, the finer resolution of YAC mapping has allowed the discovery and/or localization of partial gene duplications, the determination of gene orientations, and the measurement of gaps between known genes. Here nine overlapping YACs that encompass a genomic region of 800 kb, encoding four RCA genes and three gene-like elements, are described. The encoded genes and two of the gene-like elements share the same orientation and are ordered (5' to 3') DAF, CR2, CR1, MCP-like, CR1-like, and MCP. A C4bp-like region lies upstream from DAF and is likely to correspond to one recently observed by F. Pardo-Manuel, J. Rey-Campos, A. Hillarp, B. Dahlback, and S. Rodriguez de Cordoba (1990, Proc. Natl. Acad. Sci. USA 87: 4529-4533). MCP-like, a new genetic element, was discovered and found to be homologous to the 5' portion of the MCP gene. Two large gaps of 85 kb (between CR2 and DAF) and 110 kb (between DAF and the C4bp-like element) could carry additional RCA genes. The arrangement of CR1, MCP-like, CR1-like, and MCP, in that order, strongly suggests that this region was generated by a single duplication of neighboring CR1/CR1-like and MCP/MCP-like forerunners. The RCA YACs will now serve as convenient DNA sources for the subcloning and further characterization of this region.     

Expression of functional c-kit receptors rescues the genetic defect of W mutant mast cells.

Loss-of-function mutations in the gene for the c-kit tyrosine kinase receptor are strongly implicated in the developmental abnormalities of W mutant mice. To dissect further the relationship between kit and the W phenotype, retroviruses carrying the normal murine c-kit gene were constructed. In infected cells, the level of c-kit expression from these vectors varied markedly with different promoter elements, the 5' viral LTR proving to be the most effective. When introduced into cells which normally do not express c-kit, ectopic kit receptors transduced a ligand (Steel factor)-dependent proliferative signal in IL-3-dependent DA-1 myeloid cells and induced transformation in fibroblasts. Primary mutant mast cells were used to examine the effects of reconstituting functional kit expression in cells affected by W mutations. Exogenous c-kit expression rescued the defective proliferative response to Steel factor of cells from both W/Wv and W/W mutant mice. Moreover, functional kit expression also restored the capacity of W/Wv mast cells to survive and differentiate in vivo. These results imply that defective c-kit receptor function is sufficient to generate the W mutant phenotype.     

Purification of a form of protease nexin 1 that binds heparin with a low affinity

A form of protease nexin 1 (PN-1) that binds heparin with a low affinity (L-PN-1) was purified and studies since altered interactions with glycosaminoglycans could affect its inhibition of certain serine proteases. Purification of L-PN-1 and PN-1 was achieved by fractionating serum-free conditioned culture medium from human fibroblasts over dextran sulfate-Sepharose followed by immunoaffinity fractionation over a PN-1 monoclonal antibody-Sepharose column. The first step separated L-PN-1 from PN-1, and the second step resulted in apparently homogeneous L-PN-1 and PN-1. Comparisons of the two proteins showed that they could not be distinguished by the following properties: (a) molecular weight; (b) proteases complexed; (c) molecular weights of protease-L-PN-1 and protease-PN-1 complexes; (d) CNBr peptide maps; and (e) immunological cross-reactivity. Studies on activities that depend on the heparin binding domain revealed that heparin equally accelerated the rate of formation of 125I-thrombin-L-PN-1 and 125I-thrombin-PN-1 complexes even when the ratio of heparin to L-PN-1 or PN-1 was varied from 0.01 to 100. A functional difference, however, between L-PN-1 and PN-1 was observed in studies on the ability of the fibroblast surface to accelerate their reactions. Fixed fibroblasts accelerated the formation of 125I-thrombin-L-PN-1 complexes 2-fold, whereas they accelerated the formation of 125I-thrombin-PN-1 complexes 5-fold. The availability of purified L-PN-1 will permit studies on its functional relationship to PN-1.     

Native structure and physical properties of bovine brain kinesin and identification of the ATP-binding subunit polypeptide

Kinesin was extensively purified from bovine brain cytosol by a microtubule-binding step in the presence of 5'-adenylyl imidodiphosphate (AMP-PNP), followed by gel filtration chromatography and sucrose gradient ultracentrifugation. The products consistently contained 124,000 (124K) and 64,000 (64K) dalton polypeptides. These two polypeptides appear to represent heavy and light chains of kinesin, respectively, because they copurified on sucrose gradients to a constant and equimolar stoichiometry and bound stably to microtubules in the presence of AMP-PNP but not ATP. The mobilities of 124K and 64K in sodium dodecyl sulfate-polyacrylamide gels under reducing conditions were the same as under nonreducing conditions. A diffusion coefficient of (2.24 +/- 0.21) X 10(-7) cm2 s-1 and a sedimentation coefficient of (9.56 +/- 0.34) X 10(-13) s were determined for native kinesin by gel filtration and sucrose gradient ultracentrifugation, respectively. These values were used to calculate a native molecular weight of about 379,000 and suggest that kinesin has an axial ratio of approximately 20. Extensively purified kinesin exhibited microtubule-activated ATPase activity, and only the 124K subunit incorporated ATP in photoaffinity labeling experiments using [32P]ATP. Collectively, these data favor the interpretation that bovine brain kinesin is a highly elongated, microtubule-activated ATPase comprising two subunits each of 124,000 and 64,000 daltons, that the subunits are not linked to one another by disulfide bonds, and that the heavy chains are the ATP-binding subunits.     

Distinct synthetic and structural characteristics of proteoglycans produced by cultured artery smooth muscle cells of atherosclerosis-susceptible pigeons

Proteoglycan (PG) metabolism by aortic smooth muscle cell cultures derived from atherosclerosis-susceptible White Carneau (WC) and -resistant Show Racer (SR) pigeons was compared using [35S]sodium sulfate and [3H]serine or [3H]glucosamine as labeling precursors. Chondroitin sulfate (CS) PG and dermatan sulfate (DS) PG were the major PG secreted into the medium by both cell types. Total PG production, whether measured by incorporation of radiolabel into either core protein or glycosaminoglycan chains, was consistently lower in WC compared to SR cultures at several time points. This difference was due in part to lower (30-37%) PG synthesis in WC cells, but degradation of newly synthesized PG was an important contributor. A pulse-chase study indicated that of the total radiolabeled PG present at time O, only 47% was present at 24 h in WC cultures compared to 88% in SR cultures. The large CS-PG appeared to be the primary target for degradation in WC cells, and this selective processing resulted in a higher DS-PG:CS-PG ratio in these cultures. Structural studies indicated similar core protein and glycosaminoglycan chain sizes within a PG type for both cell types. PG monomer composition differed, however, by a higher sulfation of WC CS-PG compared to SR CS-PG and by a disaccharide sulfation position favoring 6-sulfation in WC PG and 4-sulfation in SR PG.     

Binding of 2'(3')-O-(2,4,6-trinitrophenyl)-adenosine 5'-diphosphate opens the pathway for protons through the chloroplast ATPase complex

The effect of 2'(3')-O-(2,4,6-trinitrophenyl)-adenosine 5'-diphosphate (TNP-ADP) on photophosphorylation and on the proton conductivity of the thylakoid membrane has been investigated. The results show that TNP-ADP is a potent competitive inhibitor of photophosphorylation (Ki = 1-2 microM). Moreover, in the absence of ADP and Pi, TNP-ADP accelerates basal electron transport of chloroplasts. Addition of ADP, which promotes release of the analogue from CF1, completely reverses this effect of TNP-ADP; likewise Pi alone reverses stimulation of electron transport by TNP-ADP. Dicyclohexylcarbodiimide treatment, which is known to close CF0 to H+, completely abolishes the effect of TNP-ADP. The measurements of the alkalization of the medium and the acidification of the thylakoid lumen following single turnover flashes showed that binding of TNP-ADP to CF1 increased membrane permeability for H+. Further results suggest that binding of TNP-ADP to the catalytic site of CF1 opens the CF0-CF1 complex for H+. Since ADP, as well as Pi alone, reverses the effect, it is concluded that TNP-ADP induces a conformation of the CF0-CF1 complex similar to the one triggered by simultaneous binding of ADP plus Pi. This may be achieved by interaction of the TNP residue with the Pi binding site. Thus it seems that the status of the catalytic site(s) in CF1 can be transmitted to the CF0 part to control proton flux through the ATPase complex in an economically reasonable way.     

Function of the CD4 and CD8 molecules on human cytotoxic T lymphocytes: regulation of T cell triggering

The function of the T cell differentiation antigens CD4 (Leu-3/T4) and CD8 (Leu-2/T8) on human cytotoxic T lymphocytes (CTL) is presently seen only in conjugate formation between CTL and target cell via class II or class I MHC antigens rather than in the later killing steps. In this study, human CD4+ and CD8+ CTL clones were used to investigate the effects of monoclonal antibodies against these differentiation antigens on nonspecific triggering of cytotoxicity. Cytotoxicity was induced either by antibodies against the CD3 (T3) antigen or by the lectins Con A and PHA. Anti-CD4 or anti-CD8 antibodies specifically inhibited all types of cytotoxicity of CD4+ or CD8+ CTL, respectively, regardless of the specificity of the CTL for class I or class II HLA antigens and regardless of whether target cells expressed class I or class II antigens. These results are incompatible with an exclusive role of the CD4 and CD8 molecules in MHC class recognition and are discussed with respect to a function as negative signal receptors for these molecules on CTL.