Inhibition of apoptosis by calcium ionophores in IL-3-dependent bone marrow cells is dependent upon production of IL-4+.

Calcium ionophores inhibit apoptosis in the IL-3-dependent cell line BAF3 and maintain the cells in a viable noncycling state. In this report, an identical effect of ionophore was also demonstrated on the multipotent IL-3-dependent progenitor cell line FDCP-MIX and on the primary IL-3-dependent cell population that could be cultured from murine bone marrow. Inhibition of apoptosis required extracellular calcium and could be blocked by cyclosporin A. Nuclei from IL-3-dependent cells were found to lack a calcium-activatable nuclease that degrades chromatin in the linker region between nucleosomes, unlike the nuclei of lymphoid cells. The mechanism of action of calcium ionophore could be divided into two distinct steps. First, ionophore induced the production of a survival factor that stimulated DNA synthesis and was identified as IL-4. Second, ionophore inhibited the cell cycle of the various IL-3-dependent cells. IL-4 production could be inhibited by cyclosporin A and required extracellular calcium, whereas cell cycle arrest did not. This implied that factor production was the step that was necessary for inhibition of apoptosis and maintenance of cell viability. This was confirmed by the use of an anti-IL-4R antibody, which blocked the inhibition of apoptosis induced by calcium ionophores.     

Murine Ia-associated invariant chain's processing to complex oligosaccharide forms and its dissociation from the I-Ak complex

The processing of murine invariant chain (Ii) to a cell surface form bearing complex N-linked oligosaccharides has been demonstrated in the B cell lymphoma, AKTB-1b. In addition, the rate of processing of pulse-labeled Ii has been determined relative to its rate of dissociation from the alpha/beta complex of I-Ak. Ii, alpha-, and beta-chains were immunoprecipitated with anti-I-Ak or anti-Ii monoclonal antibodies. The heretofore uncharacterized complex oligosaccharide form of Ii (Ii-c) was identified in gel-purified immunoprecipitates by peptide mapping with reverse-phase HPLC. Ii-c is resistant to deglycosylation by Endo H, which is specific for high-mannose N-linkages, but can be digested with Endo F, a glycosidase capable of cleaving both complex and high-mannose N-linked oligosaccharides. Immunoprecipitation of surface iodinated cells indicates that Ii-c is expressed on the plasma membrane. Pulse-chase metabolic labeling data show that the processing of Ii to Ii-c occurs with a t1/2 of about 120 min. In contrast, the processing of both alpha- and beta-chains of I-Ak to complex forms occurs with a t1/2 of 15 to 20 min. Our data show that Ii-hm begins to dissociate rapidly from the I-Ak complex after 100 to 120 min of chase. Only a small amount (less than 5% on a per mole basis) of Ii-c was found associated with the I-Ak complexes after 300 min of continuous metabolic labeling. These results are consistent with Ii serving as a carrier for Ia antigens as they are transported to the cell surface. In addition, they suggest that the processing of Ii to Ii-c, or a late processing event of the alpha- and beta-chains, such as their sialylation, may be a possible mechanism for inducing the dissociation of Ii from the I-Ak complex.     

Iron-sulfur centers in the photosynthetic reaction center complex fromChlorobium vibrioforme. Differences from and similarities to the iron-sulfur centers in Photosystem I

The photosynthetic reaction center complex from the green sulfur bacteriumChlorobium vibrioforme has been isolated under anaerobic conditions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals polypeptides with apparent molecular masses of 80, 40, 30, 18, 15, and 9 kDa. The 80- and 18-kDa polypeptides are identified as the reaction center polypeptide and the secondary donor cytochromec ₅₅₁ encoded by thepscA andpscC genes, respectively. N-terminal amino acid sequences identify the 40-kDa polypeptide as the bacteriochlorophylla-protein of the baseplate (the Fenna-Matthews-Olson protein) and the 30-kDa polypeptide as the putative 2[4Fe-4S] protein encoded bypscB. Electron paramagnetic resonance (EPR) analysis shows the presence of an iron-sulfur cluster which is irreversibly photoreduced at 9K. Photoaccumulation at higher temperature shows the presence of an additional photoreduced cluster. The EPR spectra of the two iron-sulfur clusters resemble those of F_A and F_B of Photosystem I, but also show significantly differentg-values, lineshapes, and temperature and power dependencies. We suggest that the two centers are designated Center I (with calculatedg-values of 2.085, 1.898, 1.841), and Center II (with calculatedg-values of 2.083, 1.941, 1.878). The data suggest that Centers I and II are bound to thepscB polypeptide.     

Effect of a Cloned DNA Fragment from Streptomyces chrysomallusNo. 2 on the Metabolism of Certain Streptomyces Strains

The effects on a cloned DNA fragment carrying an actinomycin resistance determinant on physiological processes in strains of streptomycetes with various potencies in producing this antibiotic, their inactive mutants, and the model strain ofStreptomyces lividans66 were studied. This fragment was shown to modulate bacterial resistance to actinomycin and biosynthesis of antibiotics.     

Asp537, Asp812 are essential and Lys631, His811 are catalytically significant in bacteriophage T7 RNA polymerase activity.

To define catalytically essential residues of bacteriophage T7 RNA polymerase, we have generated five mutants of the polymerase, D537N, K631M, Y639F, H811Q and D812N, by site-directed mutagenesis and purified them to homogeneity. The choice of specific amino acids for mutagenesis was based upon photoaffinity-labeling studies with 8-azido-ATP and homology comparisons with the Klenow fragment and other DNA/RNA polymerases. Secondary structural analysis by circular dichroism indicates that the protein folding is intact in these mutants. The mutants D537N and D812N are totally inactive. The mutant K631M has 1% activity, confined to short oligonucleotide synthesis. The mutant H811Q has 25% activity for synthesis of both short and long oligonucleotides. The mutant Y639F retains full enzymatic activity although individual kinetic parameters are somewhat different. Kinetic parameters, (kcat)app and (Km)app for the nucleotides, reveal that the mutation of Lys to Met has a much more drastic effect on (kcat)app than on (Km)app, indicating the involvement of K631 primarily in phosphodiester bond formation. The mutation of His to Gln has effects on both (kcat)app and (Km)app; namely, three- to fivefold reduction in (kcat)app and two- to threefold increase in (Km)app, implying that His811 may be involved in both nucleotide binding and phosphodiester bond formation. The ability of the mutant T7 RNA polymerases to bind template has not been greatly impaired. We have shown that amino acids D537 and D812 are essential, that amino acids K631 and H811 play significant roles in catalysis, and that the active site of T7 RNA polymerase is composed of different regions of the polypeptide chain. Possible roles for these catalytically significant residues in the polymerase mechanism are discussed.     

Construction of a human cytochrome c gene and its functional expression in Saccharomyces cerevisiae

The nucleotide sequences of a partial cDNA and three pseudogenes of human cytochrome c were determined. The complete nucleotide sequences which encode human cytochrome c were constructed on the basis of one of the pseudogenes by in vitro mutagenesis. The constructed human cytochrome c was functionally expressed in Saccharomyces cerevisiae. The recombinant human cytochrome c was purified and characterized.     

Characterization of phosphotyrosyl-protein phosphatase activity associated with calcineurin

Calcineurin purified from bovine brain is shown to possess phosphotyrosyl -protein phosphatase activity towards proteins phosphorylated by the epidermal growth factor receptor/kinase. The phosphatase activity is augmented by Ca2+/calmodulin or divalent cation (Ni2+ greater than Mn2+ greater than Mg2+ greater than Co2+). In the simultaneous presence of all three effectors, the enzymatic activity is synergistically increased. Ca2+/calmodulin activates the Mg2+-supported activity by decreasing the Km value for phosphotyrosyl -casein from 2.2 to 0.6 microM, and increasing the Vmax from 0.4 to 4.6 nmol/min/mg. These results represent the first demonstration that calcineurin can dephosphorylate phosphotyrosyl -proteins and suggest a novel mechanism of activation of this enzyme.     

Analysis of factors restricting the use of binomial statistics for studying transmitter release in neuromuscular junctions

Binomial parameters of transmitter secretion were calculated on the basis of analysis of synaptic potentials in the frog sartorius muscle. Negative values of the parameter p were found in some synapses. This happened most often in low Ca²⁺ concentrations and with low amplitude of miniature end-plate potentials. The results were interpreted in terms of a model assuming spatial heterogeneity of probability of transmitter quantum release at different release points. Simulation of transmitter secretion by computer showed that the appearance of negative values of the parameter p and incorrect estimates of n experimentally are connected with the form of distribution of probability of transmitter quantum release in the synapse and with the amplitude of miniature potentials.S. V. Kurashov Kazan' Medical Institute, Ministry of Health of the RSFSR. Translated from Neirofiziologiya, Vol. 16, No. 2, pp. 182–189, March–April, 1984.