Structural aspects of myosin subfragment 1 as studied by subtilisin digestion of trypsin-split S-1 (27 kDa-50 kDa-20 kDa)

Limited subtilisin digestion of myosin subfragment 1 (S-1) was carried out, varying the enzyme: substrate weight ratio from 1:200 to 1:10, and changes in structure, and in the MgATPase activities of S-1 and acto-S-1 after proteolysis, were followed. When the starting material--tryptically-cleaved S-1 (27 kDa-50 kDa-20 kDa) ("split S-1")--was subjected to further subtilisin digestion, it was found that with increasing enzyme concentration, the 50 kDa fragment degraded into an 18 kDa fragment via a 33 kDa peptide (50----33----18 kDa), which was not cross-linked with F-actin. On the other hand, the 27 and 20 kDa fragments were rather stable at lower subtilisin concentrations and started to degrade only at higher subtilisin concentrations. These degradations lowered the MgATPase activities of S-1 and acto-S-1. The losses of MgATPase activities of S-1 and of acto-S-1 were mainly due to the degradations of the 27 and 20 kDa fragments, respectively. Addition of EDTA did not affect the subtilisin cleavage pattern of split S-1 but the breakdown of the 50 kDa fragment was extremely depressed, suggesting that some conformational change of the 50 kDa fragment is induced by the binding of divalent cation. The binding of MgADP to split S-1 accelerated the degradation of the 27 kDa fragment and produced a new cut in the 27 kDa fragment (27----20 kDa), resulting in a further loss of the S-1 MgATPase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selection of mouse macrophage-like sublines that differ in leukemogenic potential and characterization

The murine macrophage-like cell line (Mm-1), which is nonleukemogenic to syngeneic SL mice, was originally derived from spontaneously differentiated cells of a clonal line of mouse myeloid leukemia cells (M1). In the present experiment, variant cell lines with a high (Mm-A), moderate (Mm-P), and little or no (Mm-S1 and Mm-S2) leukemogenic potential were obtained from the Mm-1 cells. The mean survival times of syngeneic SL mice inoculated i.p. with 5 X 10(6) Mm-A and Mm-P cells were 17 and 33 days, respectively, whereas almost all the mice inoculated with Mm-S1 or Mm-S2 cells survived for more than 90 days. These variant cell lines did not lose their macrophage-like characteristics in vitro. These variant cell lines phagocytized latex beads and sensitized sheep erythrocytes, produced lysozyme, and adhered to culture dishes. The four variant cell lines showed no significant difference in proliferation rates in vitro in liquid medium containing 10% calf serum, but Mm-A cells could grow both in soft agar medium in the absence of ascitic fluid containing colony-stimulating factor (CSF) and in liquid medium containing 1% serum, whereas Mm-P cells could grow in the liquid medium but not in soft agar medium without ascitic fluid, and Mm-S1 and Mm-S2 cells could not grow in either medium. The ratio of the nuclear area to the cell area (NCR) of Mm-A cells was a high (51%) but those of Mm-S1 and Mm-S2 cells were low (40-41%), and that of Mm-P cells was intermediate (44%). The leukemogenicity of Mm-1 cell lines was roughly correlated with their NCR. The possibility that interactions between Mm-1 variant cells and host immune cells might be involved in the mechanisms of their different leukemogenicities was not supported by results on the in vitro susceptibilities of Mm-1 variant cells to the cytostatic actions by normal macrophages and spleen cells and on leukemogenicities of the Mm-1 variant cells in athymic nude mice. A possible method of control of the leukemogenicity of Mm-1 variant cells is discussed.     

Characterization of a factor inducing differentiation of mouse myeloid leukemic cells purified from conditioned medium of mouse Ehrlich ascites tumor cells

A factor inducing differentiation of mouse myeloid leukemic cells (MI) into macrophages was purified to apparent homogeneity from 168 1 of CM of Ehrlich ascites tumor cells. The purified factor was half-maximally active at 2 X 10(-11) M. The factor was analyzed by radioiodination, SDS-polyacrylamide gel electrophoresis and autoradiography. Its Mr was 40 000-50 000. On reduction, the factor lost activity, but showed no subunit structure. Treatment of the factor with endo-beta-N-acetylglucosaminidase F, but not endo-beta-N-acetylglucosaminidase H, gave rise to a molecule of Mr 20 000-28 000. The activity of the factor from Ehrlich cells was completely neutralized by antiserum to the factor of Mr 50 000-70 000 from mouse fibroblast L929 cells.     

Angiogenic activity of syndecan-binding laminin peptide AG73 (RKRLQVQLSIRT)

The AG73 peptide (RKRLQVQLSIRT, mouse laminin alpha 1 chain 2719-2730) promotes cell adhesion and tumor metastasis, and interacts with transmembrane syndecan proteoglycans. Here, we demonstrate AG73 peptide angiogenic activity using in vitro, ex vivo, and in vivo models. AG73 induced murine endothelial cell (SVEC4-10) tube formation on Cultrex Basement Membrane Extract (Cultrex BME) and stimulated sprouting of aortic rings. None of the homologous sequences from the laminin alpha2, alpha3, alpha4, or alpha5 chains was as active as AG73 in promoting sprouting formation. AG73 also mediated angiogenesis in the chick chorioallantonic membrane (CAM) assay. Using subcutaneously injected Cultrex BME supplemented with AG73, we observed a large angiogenic response. Furthermore, AG73-conjugated to a chitosan membrane promoted a strong angiogenic response in the CAM assay. These results indicate that the AG73 peptide is a potent syndecan-binding angiogenesis stimulator and may be useful for therapeutic application to treat ischemic injuries.     

Electrical Properties of the Pacemaker Neurons in the Heart Ganglion of a Stomatopod, Squilla oratoria

In the Squilla heart ganglion, the pacemaker is located in the rostral group of cells. After spontaneous firing ceased, the electrophysiological properties of these cells were examined with intracellular electrodes. Cells respond to electrical stimuli with all-or-none action potentials. Direct stimulation by strong currents decreases the size of action potentials. Comparison with action potentials caused by axonal stimulation and analysis of time relations indicate that with stronger currents the soma membrane is directly stimulated whereas with weaker currents the impulse first arises in the axon and then invades the soma. Spikes evoked in a neuron spread into all other neurons. Adjacent cells are interconnected by electrotonic connections. Histologically axons are tied with the side-junction. B spikes of adjacent cells are blocked simultaneously by hyperpolarization or by repetitive stimulation. Experiments show that under such circumstances the B spike is not directly elicited from the A spike but is evoked by invasion of an impulse or electrotonic potential from adjacent cells. On rostral stimulation a small prepotential precedes the main spike. It is interpreted as an action potential from dendrites.     

Analysis of proteolytic processing during specific antigen presentation

In this report we have studied the effect of protease inhibitors on B-cell-antigen processing. As a source of antigen-presenting B cells we have utilized transformants transfected with a vector carrying immunoglobulin (Ig) genes specific for the hapten trinitrophenyl (TNP). B-cell-specific (TNP-proteins) and nonspecific antigen-presentation activities were blocked to the same extent upon addition of inhibitors for protease and endosomal function. Interestingly, the effect of leupeptin, a thiol protease inhibitor, varied depending on the antigen and helper T cells utilized. These results suggest that specific groups of proteases may be required for antigen processing so that discrete antigenic epitopes in association with major histocompatibility complex molecules can be recognized by interacting T cells.