A block to efficient replication of simian immunodeficiency virus in C8166 cells can be overcome by duplication of the NF-κB binding site

Sequence analysis of the acutely lethal pbj14 strain of simian immunodeficiency virus (SIVpbj14) clone revealed among other differences from its less pathogenic counterparts a duplication of its binding site for nuclear factor kappa B (NF-B) in its long terminal repeats (LTR). We have investigated whether introducing a similar duplication into the pathogenic molecular clone SIVmac239 would alter its biological properties. We compared an SIV which possessed 2 NF-B sites to the wild type, a single NF-B site virus, with respect to its ability to replicate in vitro in established CD4+ T cell lines, primary peripheral blood mononuclear cells (PBMCs), and primary alveolar macrophages. The virus containing 2 NF-B sites exhibited no apparent difference from wild type in established cell lines 174×CEM, MT-2 and MT-4, or in primary PBMC or tissue macrophage cultures. However, the 2 B virus replicated well in the established cell line C8166, while the wild type, 1 B virus replicated very poorly in this cell type, suggesting that duplication of the NF-B site is capable of overcoming a block to efficient replication of SIVmac239 in C8166 cells. Interestingly, Em*, a macrophage tropic SIVmac that differs from SIVmac239 by 9 amino acids in the envelope region yet possesses only one NF-B binding site, also replicates well in C8166. The data suggest that the replication of wild type SIVmac239 is restricted in C8166 cells, but that this restriction can be overcome either by changes in the LTR or by changes in the envelope region.     

Characterization of phospholipase A2 activation by plasmin in cultured bovine endothelial cells

Treatment of cultured bovine carotid artery endothelial cells with 0.1 µM human plasmin has been reported to induce a receptor-mediated short burst of arachidonate release, which is a pertussis toxin-sensitive and extracellular calcium-dependent reaction. Plasmin-induced calcium influx in cells was significantly inhibited by pretreatment with pertussis toxin, indicating that the former was coupled with a pertussis toxin-sensitive guanosine 5-triphosphate (GTP)-binding protein. Plasmin significantly induced the formation of lysophosphatidylcholine but not lysophosphatidylethanolamine. A cellular phospholipase A₂ with an arachidonyl specificity at the sn-2 position of phosphatidylcholine, which required submicromolar calcium, was identified as a cytosolic phospholipase A₂ by immunoblot analysis. By a cell-free enzyme activity assay and immunoblot analysis, plasmin was found to induce a translocation of the cytosolic phospholipase A₂ from the cytosol to the membrane. Taken together, the results suggest that plasmin bound to its putative receptor and activated a GTP-binding protein coupled to calcium influx channel, followed by translocation and activation of cytosolic phospholipase A₂ in endothelial cells.     

Enzymes related to galactose utilization inPseudomonas cepacia

Pseudomonas cepacia produced a characteristic green sheen on EMB-galactose plates owing to production of galactonic acid by the constitutive membrane-associated glucose dehydrogenase of this bacterium. Mutants isolated as glucose dehydrogenase deficient (Gcd^–) also were deficient in membrane-associated galactose dehydrogenase. A strain that formed glucose dehydrogenase at 30°C but not at 40°C was also temperature sensitive with respect to formation of galactose dehydrogenase. The Gcd^– strains still utilized galactose. A second, NAD-specific, galactose dehydrogenase (not membrane associated) along with a transport system for galactose were induced during growth on galactose and constituted an alternative pathway of conversion of galactose to galactonate. Enzymes of the De Ley-Doudoroff pathway of conversion of galactonate to pyruvate and glyceraldehyde-3-phosphate were induced during growth on galactose. Unexpectedly, growth on galactose also elicited formation of enzymes of the Entner-Doudoroff (ED) route. Furthermore, mutants blocked in the ED pathway grew poorly on galactose. One interpretation of these findings is that glyceraldehyde-3-phosphate formed from galactose via the De Ley-Doudoroff route (by cleavage of 2-keto-3-deoxy-6-phosphogalaconate) is reconverted to hexose phosphate and metabolized via the ED pathway.     

Effect ofPseudomonas aeruginosa rhamnolipid on human neutrophil migration

The effect ofPseudomonas aeruginosa heat-stable hemolysin (rhamnolipid) on human neutrophil migration has been investigated. Rhamnolipid was prepared from culture filtrate and characterized by thin-layer chromatography. The lytic activity of rhamnolipid was quantitated by titration against neutrophils. Leukocyte migration response was measured using⁵¹Cr-labeled neutrophils with a double-filter technique in modified Boyden chambers. The results suggest rhamnolipid stimulated chemotaxis as well as chemokinesis. Moreover, rhamnolipid impaired a chemotactic response toN-formyl-methionyl-leucyl-phenylalanine. These effects may be important in host-parasite interactions.     

Reversion of the gal3 mutation of Escherichia coli: Partial deletion of the insertion sequence

Summary The gal3 mutation of E. coli is an insertion of a DNA sequence, 1,100 base pairs in length, into the operator-promoter region of the galactose operon. This mutation reverts spontaneously to gal⁺ by excision of the insertion to produce stable, inducible revertants, or by tandem duplications of the gal operon to produce unstable, constitutive revertants. The nature of a third class of revertants, which are stable and constitutive, is the subject of the present study.The stable, constitutive class of revertants included approximately 30% of all gal⁺ revertants obtained from a gal3() strain. Although the constitutive reversions could be transduced by , the efficiency was found to be extremely poor and the rare transductants which did appear seemed to originate from abnormal transducing particles. It was concluded that these reversions were not normally packaged by .In order to facilitate the packaging of these reversions, the chlD-pgl region was deleted from the parent gal3() strain. Unexpectedly, the gal3 mutation in the majority of these deletions reverted to produce stable, constitutive reversions exclusively. The explanation proposed was that the chlD-pgl deletions had also removed part of the gal operator-promoter up to the gal3 insertion, so that simple excisions of the insertion yielded stable, constitutive revertants by connecting the gal structural genes to a different promoter. These revertants were not considered to be true representatives of the stable, constitutive class. The specificity of deletion end-points at the insertion was found only in the gal3() strain, and not in gal ⁺, gal ⁺(), or gal3 strains. Moreover, the frequency of spontaneous chlD-pgl deletions increased 10- to 15-fold in presence of the gal3 insertion.A gal phage bearing a true stable, constitutive reversion (gal ^c 200) was isolated from the revertant strain by subsequent deletion of the chlD-pgl segment (31). Electron micrographs of gal ⁺ and gal ^c 200 31(chlD pgl) DNA heteroduplexes were interpreted to indicate that the stable, constitutive reversion had arisen by a deletion of 3/4 of the gal3 insertion sequence.The main conclusions are: (i) the stable, constitutive reversions of gal3 can arise by partial deletions of the insertion sequence, apparently by elimination of the nucleotide sequence which causes polarity; (ii) the chlD-pgl deletions may exhibit preferential termination at the right extremity of the gal3 insertion in presence of prophage ; and (iii) the gal3 insertion appears to inhibit the production of gal particles by providing a nucleotide sequence which is recognized and degraded by a specific endonuclease. It is suggested that inhibition of transducing particle formation by gal3 and the preferred termination of deletions at gal3 might represent related phenomena.     

Natural and Unanticipated Modifiers of RNAi Activity in Caenorhabditis elegans

Organisms used as model genomics systems are maintained as isogenic strains, yet evidence of sequence differences between independently maintained wild-type stocks has been substantiated by whole-genome resequencing data and strain-specific phenotypes. Sequence differences may arise from replication errors, transposon mobilization, meiotic gene conversion, or environmental or chemical assault on the genome. Low frequency alleles or mutations with modest effects on phenotypes can contribute to natural variation, and it has proven possible for such sequences to become fixed by adapted evolutionary enrichment and identified by resequencing. Our objective was to identify and analyze single locus genetic defects leading to RNAi resistance in isogenic strains of Caenorhabditis elegans. In so doing, we uncovered a mutation that arose de novo in an existing strain, which initially frustrated our phenotypic analysis. We also report experimental, environmental, and genetic conditions that can complicate phenotypic analysis of RNAi pathway defects. These observations highlight the potential for unanticipated mutations, coupled with genetic and environmental phenomena, to enhance or suppress the effects of known mutations and cause variation between wild-type strains.     

Purification of rabies virus glycoprotein produced in Drosophila melanogaster S2 cells: An efficient immunoaffinity method

Most rabies vaccines are based on inactivated virus, which production process demands a high level of biosafety structures. In the past decades, recombinant rabies virus glycoprotein (RVGP) produced in several expression systems has been extensively studied to be used as an alternative vaccine. The immunogenic characteristics of this protein depend on its correct conformation, which is present only after the correct post-translational modifications, typically performed by animal cells. The main challenge of using this protein as a vaccine candidate is to keep its trimeric conformation after the purification process. We describe here a new immunoaffinity chromatography method using a monoclonal antibody for RVGP Site II for purification of recombinant rabies virus glycoprotein expressed on the membrane of Drosophila melanogaster S2 cells. RVGP recovery achieved at least 93%, and characterization analysis showed that the main antigenic proprieties were preserved after purification.     

In vitro induction of neoantigen-specific T cells in myelodysplastic syndrome,a disease with low mutational burden

Therapies that utilize immune checkpoint inhibition work by leveraging mutation-derived neoantigens and have shown greater clinical efficacy in tumors with higher mutational burden. Whether tumors with a low mutational burden are susceptible to neoantigen-targeted therapy has not been fully addressed. To examine the feasibility of neoantigen-specific adoptive T-cell therapy, the authors studied the T-cell response against somatic variants in five patients with myelodysplastic syndrome (MDS), a malignancy with a very low tumor mutational burden. DNA and RNA from tumor (CD34⁺) and normal (CD3⁺) cells isolated from the patients’ blood were sequenced to predict patient-specific MDS neopeptides. Neopeptides representing the somatic variants were used to induce and expand autologous T cells ex vivo, and these were systematically tested in killing assays to determine the proportion of neopeptides yielding neoantigen-specific T cells. The authors identified a total of 32 somatic variants (four to eight per patient) and found that 21 (66%) induced a peptide-specific T-cell response and 19 (59%) induced a T-cell response capable of killing autologous tumor cells. Of the 32 somatic variants, 11 (34%) induced a CD4⁺ response and 11 (34%) induced a CD8⁺ response that killed the tumor. These results indicate that in vitro induction of neoantigen-specific T cells is feasible for tumors with very low mutational burden and that this approach warrants investigation as a therapeutic option for such patients.