Abrogation of the Fc gamma receptor IIA-mediated phagocytic signal by stem-loop Syk antisense oligonucleotides.

The role of Syk kinase in Fc gamma receptor (Fc gamma R) IIA-mediated phagocytosis was examined with two forms of antisense oligodeoxynucleotides (ODNs) designed to hybridize to human Syk mRNA. Monocytes were incubated with linear and stem-loop antisense ODNs targeted to Syk mRNA. When complexed with cationic liposomes, stem-loop Syk antisense ODN with phosphorothioate modification exhibited stability in fetal bovine and human serum. The stem-loop Syk antisense ODN at a concentration of 0.2 microM inhibited Fc gamma RIIA-mediated phagocytosis by 90% and completely eliminated Syk mRNA and protein in monocytes, whereas scrambled-control ODNs had no effect. The Syk antisense ODNs did not change beta-actin mRNA levels and Fc gamma RII cell-surface expression. In addition, stem-loop Syk antisense ODN inhibited Fc gamma RI and Fc gamma RIIIA-mediated phagocytosis. These data indicate the efficacy of stem-loop Syk antisense ODN for targeting and degrading Syk mRNA and protein and the importance of Syk kinase in Fc gamma receptor-mediated phagocytosis. Immunoblotting assay demonstrated that Fc gamma RII tyrosine phosphorylation after Fc gamma RII cross-linking did not change in the absence of Syk protein. These results indicate that Syk kinase is required for Fc gamma RIIA-mediated phagocytic signaling and that Fc gamma RII cross-linking leads to tyrosine phosphorylation of Fc gamma RII independent of Syk kinase.     

The amphotropic murine leukemia virus receptor gene encodes a 71-kilodalton protein that is induced by phosphate depletion.

The amphotropic murine leukemia virus (MuLV) can infect cells from a number of mammals, including humans, via its specific receptor. Basic knowledge of amphotropic MuLV receptor expression is likely to be useful in the development and improvement of gene therapy protocols based on amphotropic-pseudotyped vectors. To investigate the expression of the human receptor for the amphotropic MuLV (GLVR-2, newly termed Pit2), we determined its mRNA levels in several cell lines and found them to vary significantly. Induction of increased levels of mRNA after removal of phosphate from the media was observed in two osteosarcoma cell lines. The increase in GLVR-2 mRNA resulted in a concomitant rise in the levels of a 71-kDa protein specifically recognized by affinity-purified antibodies against GLVR-2. Using these antibodies, we were able to confirm the intracellular topology of the large hydrophilic domain between the proposed sixth and seventh transmembrane domains of the GLVR-2 protein. This assignment is in agreement with the fourth extracellular loop being outside the cell, consistent with the proposal that the fourth extracellular loop of GLVR-2 contains the envelope binding site.     

Separation of multiple forms of acidic glutathione S-transferase isozymes in a susceptible and a resistant strain of house fly,Musca domestica (L.)

The acidic glutathione S-transferases from a CSMA (susceptible) strain and a Cornell-R (resistant) strain of houseflies were purified and separated utilizing affinity chromatography followed by chromatofocusing. Nine fractions were isolated from each house fly strain. Fraction 1 had the highest 1-chloro-2,4-dinitrobenzene vs. 1,2-dichloro-4-nitrobenzene ratio (CDNB/DCNB ratio) in both strains and the ratio of all the other fractions tended to decrease as the isoelectrical points decreased except for fractions 4 and 9. Most fractions from the CSMA strain had higher CDNB conjugation activities than the fractions from the Cornell-R strain, but all the fractions from the CSMA strain had lower DCNB conjugation activities than fractions from the Cornell-R strain. Steady-state kinetics of all the fractions were examined. The Km values obtained from both strains ranged from 0.36 to 1.12 mM, while the Vmax value ranged from 3.0 to 32.6 μmol/min/mg. In the 100,000 g supernatant, the CDNB specific activities in the CSMA strain was about 1/3 of the activity in the Cornell-R strain but it was about 1.5-fold following affinity chromatography. The specific activity for DCNB measured in the CSMA strain was only 1/5 of the activities of the Cornell-R strain in the 100,000 g supernatant, but was about the same after affinity chromatography. The difference was due to the selectivity of the affinity column used in the current study. © 1995 Wiley-Liss, Inc.     

Occurrence of glycosphingolipids in chicken egg yolk.

Chicken egg yolk was found to contain a unique glycosphingolipid pattern not seen in other types of tissue or cell. These glycosphingolipids were isolated in pure form and their structures established by sequential enzymic hydrolysis and permethylation analysis. The major gangliosides in chicken egg yolk are N-acetylneuraminosylgalactosylceramide, N-acetylneuraminosyl-lactosylceramide and di-N-acetylneuraminosyl-lactosylceramide. The only neutral glycosphingolipid found in chicken egg yolk is galactosylceramide.     

The pathways of assimilation of 13NH4+ by the cyanobacterium, Anabaena cylindrica.

The principal initial product of metabolism of 13N-labeled ammonium by Anabaena cylindrica grown with either NH4+ or N2 as nitrogen source is amide-labeled glutamine. The specific activity of glutamine synthetase is approximately half as great in NH4+-grown as in N2-grown filaments. After 1.5 min of exposure to 13NH4+, the ratio of 13N in glutamate to 13N in glutamine reaches a value of approximately 0.1 for N2- and 0.15 for NH4+-grown filaments, whereas after the same period of exposure to [13N]N2, that ratio has reached a value close to unity and is rising rapidly. During pulse-chase experiments, 13N is transferred from the amide group to glutamine into glutamate, and then apparently into the alpha-amino group of glutamine. Methionine sulfoximine, an inhibitor of glutamine synthetase, inhibits the formation of glutamine. In the presence of the inhibitor, direct formation of glutamate takes place, but accounts for only a few per cent of the normal rate of formation of that amino acid; and alanine is formed about as rapidly as glutamate. Azaserine reduces formation of [13N]glutamate approximately 100-fold, with relatively little effect on the formation of [13N]glutamine. Aminooxyacetate, an inhibitor of transaminase reactions blocks transfer of 13N to aspartate, citrulline, and arginine. We conclude, on the basis of these results and others in the literature, that the glutamine synthetase/glutamate synthase pathway mediates most of the initial metabolism of ammonium in A. cylindrica, and that glutamic acid dehydrogenase and alanine dehydrogenase have only a very minor role.     

Preferential assignment of allotype a1 globulin for the production of early IgM anti-para-azobenzenearsonate antibodies in heterozygous a1a3 rabbits.

Rabbits of allotype a1a3 were injected on days 0, 2, and 4 with mixtures containing equal amounts of pigeon erythrocytes (Prbc) coupled to para-azobenzenearsonate (AA) and to para-azobenzene-N-trimethylammonium (TMA). On day 6, the allotypes of antibody from plaque-forming cells (PFC) of the blood were determined by observing the inhibition of plaque formation by anti-allotype sera. Anti-AA PFC appeared to consist for the most part of cells making antibody of allotype a1 since 65% of them were inhibited by anti-a1 serum and only 8% by anti-a3. Anti-TMA PFC, on the other hand, appeared to consist mostly of cells making antibody of allotype a3, since less than 1% of them were inhibited by anti-a1 but 47% by anti-a3. Antibody allotype for spleen PFC was also determined on day 6 and was similar to that found for blood PFC. Anti-AA PFC were inhibited 74% by anti-a1 serum and 15% by anti-a3 whereas anti-TMA PFC were inhibited 19% by anti-a1 and 43% by anti-a3. Serum hemolysin specific for AA hapten from a1a3 animals was also strongly inhibited by anti-a1 serum but not by anti-a3 whereas the converse was true for hemolysin against TMA hapten. The a1a3 rabbits, in whcih the anti-AA was restricted to allotype a1, were mated to produced homozygous a3a3 animals. When the PFC and serum antibodies of these a3a3 offspring were examined by specific inhibition, the anti-AA activity was found to be of allotype a3 rather than being a-negative. The number of anti-AA PFC in the blood of a3a3 rabbits was lower than that in blood of a1a3 or a1a1 animals. In addition, the TMA hapten appeared to inhibit the response to the AA hapten. Thus a1a3 rabbits immunized with AA-Prbc alone had 14-fold more anti-AA PFC or 18-fold higher anti-AA hemolysin titer than a3a3 animals immunized with both AA-Prbc and TMA-Prbc. Our results are discussed in relation to various explanations which have been offered for an imbalance of allotypes in a given antibody.     

Heteroduplex analysis of the RNA of clone 3 Moloney murine sarcoma virus.

Heteroduplex analysis of the RNA isolated from purified virions of clone 3 Moloney murine sarcoma virus (M-MSV) hybridized to cDNA's from Moloney murine leukemia virus (M-MLV) and clone 124 M-MSV shows that the main physical component of clone 3 RNA is missing all or most of the 1.5-kilobase (kb) clone 124 M-MSV specific sequence denoted beta s (S. Hu et al. Cell 10:469--477, 1977). This sequence is either deleted in clone 3 RNA or substituted by a very short (0.3-kilobase) sequence. In other respects, clone 3 and clone 124 RNAs show the same heteroduplex structure relative to M-MLV. Since beta s is believed to contain the src gene(s) of clone 124 RNA, this result leaves as an unresolved question the nature of the src gene(s) of the clone 3 M-MSV RNA complex.