Superantigen-staphylococal-enterotoxin-A-dependent and antibody-targeted lysis of GD2-positive neuroblastoma cells

 Superantigens such as the staphylococcal enterotoxin A (SEA) are among the most potent T cell activators known. They bind to major histocompatibility complex (MHC) class II molecules and interact with T cells depending on their T cell receptor (TCR) Vβ expression. Superantigens also induce a variety of cytokines and trigger a direct cytotoxic effect against MHC-class-II-positive target cells. In order to extend superantigen-dependent cell-mediated cytotoxicity (SDCC) to MHC-class-II-negative neuroblastoma cells, SEA was linked to the anti-ganglioside GD₂ human/mouse chimeric monoclonal antibody (mAb) ch14.18. Ganglioside GD₂ is expressed on most tumours of neuroectodermal origin but is expressed to a lesser extent on normal tissues. The linkage of ch14.18 to SEA was achieved either with a protein-A–SEA fusion protein or by chemical coupling. Both constructs induced T-cell-mediated cytotoxicity towards GD₂-positive neuroblastoma cells in an effector-to-target(E:T)-ratio-and dose-dependent manner in vitro. To reduce the MHC class II affinity of SEA, a point mutation was introduced in the SEA gene (SEAm9) that resulted in 1000-fold less T cell killing of MHC-class-II-expressing cells as compared to native SEA. However, a protein-A–SEAm9 fusion protein mediated cytotoxicity similar to that of protein-A–SEA on ch14.18-coated, MHC-class-II-negative neuroblastoma cells. Taken together, these findings suggest that superantigen-dependent and monoclonal-antibody-targeted lysis may be a potent novel approach for neuroblastoma therapy. Received: 15 March 1995 / Accepted: 22 May 1995     

MMK2, a novel alfalfa MAP kinase, specifically complements the yeast MPK1 function

Mitogen-activated protein (MAP) kinases are serine/threonine protein kinases that are activated in response to a variety of stimuli. Here we report the isolation of an alfalfa cDNA encoding a functional MAP kinase, termedMMK2. The predicted amino acid sequence ofMMK2 shares 65% identity with a previously identified alfalfa MAP kinase, termedMMK1. Both alfalfa cDNA clones encode functional kinases when expressed in bacteria, undergoing autophosphorylation and activation to phosphorylate myelin basic protein in vitro. However, only MMK2 was able to phosphorylate a 39 kDa protein from the detergent-resistant cytoskeleton of carrot cells. The distinctiveness ofMMK2 was further shown by complementation analysis of three different MAP kinase-dependent yeast pathways; this revealed a highly specific replacement of the yeastMPK1 (SLT2) kinase byMMK2, which was found to be dependent on activation by the upstream regulators of the pathway. These results establish the existence of MAP kinases with different characteristics in higher plants, suggesting the possibility that they could mediate different cellular responses.     

Evolutionary Relationships Among the Eukaryotic Crown Taxa Taking into Account Site-to-Site Rate Variation in 18S rRNA

In this study we constructed a bootstrapped distance tree of 500 small subunit ribosomal RNA sequences from organisms belonging to the so-called crown of eukaryote evolution. Taking into account the substitution rate of the individual nucleotides of the rRNA sequence alignment, our results suggest that (1) animals, true fungi, and choanoflagellates share a common origin: The branch joining these taxa is highly supported by bootstrap analysis (bootstrap support [BS] > 90%), (2) stramenopiles and alveolates are sister groups (BS = 75%), (3) within the alveolates, dinoflagellates and apicomplexans share a common ancestor BS > 95%), while in turn they both share a common origin with the ciliates (BS > 80%), and (4) within the stramenopiles, heterokont algae, hyphochytriomycetes, and oomycetes form a monophyletic grouping well supported by bootstrap analysis (BS > 85%), preceded by the well-supported successive divergence of labyrinthulomycetes and bicosoecids. On the other hand, many evolutionary relationships between crown taxa are still obscure on the basis of 18S rRNA. The branching order between the animal-fungal-choanoflagellates clade and the chlorobionts, the alveolates and stramenopiles, red algae, and several smaller groups of organisms remains largely unresolved. When among-site rate variation is not considered, the inferred tree topologies are inferior to those where the substitution rate spectrum for the 18S rRNA is taken into account. This is primarily indicated by the erroneous branching of fast-evolving sequences. Moreover, when different substitution rates among sites are not considered, the animals no longer appear as a monophyletic grouping in most distance trees. Received: 11 June 1997 / Accepted: 21 July 1997     

General secretion pathway (eps) genes required for toxin secretion and outer membrane biogenesis in Vibrio cholerae.

The general secretion pathway (GSP) of Vibrio cholerae is required for secretion of proteins including chitinase, enterotoxin, and protease through the outer membrane. In this study, we report the cloning and sequencing of a DNA fragment from V. cholerae, containing 12 open reading frames, epsC to -N, which are similar to GSP genes of Aeromonas, Erwinia, Klebsiella, Pseudomonas, and Xanthomonas spp. In addition to the two previously described genes, epsE and epsM (M. Sandkvist, V. Morales, and M. Bagdasarian, Gene 123: 81-86, 1993; L. J. Overbye, M. Sandkvist, and M. Bagdasarian, Gene 132:101-106, 1993), it is shown here that epsC, epsF, epsG, and epsL also encode proteins essential for GSP function. Mutations in the eps genes result in aberrant outer membrane protein profiles, which indicates that the GSP, or at least some of its components, is required not only for secretion of soluble proteins but also for proper outer membrane assembly. Several of the Eps proteins have been identified by use of the T7 polymerase-promoter system in Escherichia coli. One of them, a pilin-like protein, EpsG, was analyzed also in V. cholerae and found to migrate as two bands on polyacrylamide gels, suggesting that in this organism it might be processed or otherwise modified by a prepilin peptidase. We believe that TcpJ prepilin peptidase, which processes the subunit of the toxin-coregulated pilus, TcpA, is not involved in this event. This is supported by the observations that apparent processing of EpsG occurs in a tcpJ mutant of V. cholerae and that, when coexpressed in E. coli, TcpJ cannot process EpsG although the PilD peptidase from Neisseria gonorrhoeae can.     

Competition between Different Variegating Rearrangements for Limited Heterochromatic Factors in Drosophila Melanogaster

Position effect variegation (PEV) results from the juxtaposition of a euchromatic gene to heterochromatin. In its new position the gene is inactivated in some cells and not in others. This mosaic expression is consistent with variability in the spread of heterochromatin from cell to cell. As many components of heterochromatin are likely to be produced in limited amounts, the spread of heterochromatin into a normally euchromatic region should be accompanied by a concomitant loss or redistribution of the protein components from other heterochromatic regions. We have shown that this is the case by simultaneously monitoring variegation of a euchromatic and a heterochromatic gene associated with a single chromosome rearrangement. Secondly, if several heterochromatic regions of the genome share limited components of heterochromatin, then some variegating rearrangements should compete for these components. We have examined this hypothesis by testing flies with combinations of two or more different variegating rearrangements. Of the nine combinations of pairs of variegating rearrangements we studied, seven showed nonreciprocal interactions. These results imply that many components of heterochromatin are both shared and present in limited amounts and that they can transfer between chromosomal sites. Consequently, even nonvariegation portions of the genome will be disrupted by re-allocation of heterochromatic proteins associated with PEV. These results have implications for models of PEV.     

The lethal effects of biguanides on cysts and trophozoites of Acanthamoeba castellanii

The effects of a range of biocides on trophozoite and encysted forms of Acanthamoeba castellanii were investigated. Viable acanthamoebae were enumerated by a plaque assay technique. The cyst form of Acanthamoeba castellanii was more resistant to all biocides tested than the trophozoite form. Of the biocides tested, chlorhexidine diacetate (CHA) and polyhexamethylene biguanide (PHMB) were the most effective. Their lethal effects were time- and concentration-dependent. CHA was very effective when formulated in 0.1% EDTA combined with Tris buffer pH 7.8 whereas PHMB activity was reduced by 0.1% EDTA. Three per cent dimethylsulphoxide (DMSO) enhanced the activity of CHA but not of PHMB.     

Overexpression of HOXA10 in murine hematopoietic cells perturbs both myeloid and lymphoid differentiation and leads to acute myeloid leukemia.

Multiple members of the A, B, and C clusters of Hox genes are expressed in hematopoietic cells. Several of these Hox genes have been found to display distinctive expression patterns, with genes located at the 3' side of the clusters being expressed at their highest levels in the most primitive subpopulation of human CD34+ bone marrow cells and genes located at the 5' end having a broader range of expression, with downregulation at later stages of hematopoietic differentiation. To explore if these patterns reflect different functional activities, we have retrovirally engineered the overexpression of a 5'-located gene, HOXA10, in murine bone marrow cells and demonstrate effects strikingly different from those induced by overexpression of a 3'-located gene, HOXB4. In contrast to HOXB4, which causes selective expansion of primitive hematopoietic cells without altering their differentiation, overexpression of HOXA10 profoundly perturbed myeloid and B-lymphoid differentiation. The bone marrow of mice reconstituted with HOXA10-transduced bone marrow cells contained in high frequency a unique progenitor cell with megakaryocytic colony-forming ability and was virtually devoid of unilineage macrophage and pre-B-lymphoid progenitor cells derived from the transduced cells. Moreover, and again in contrast to HOXB4, a significant proportion of HOXA10 mice developed a transplantable acute myeloid leukemia with a latency of 19 to 50 weeks. These results thus add to recognition of Hox genes as important regulators of hematopoiesis and provide important new evidence of Hox gene-specific functions that may correlate with their normal expression pattern.     

Oligonucleotide N3'-->P5' phosphoramidates as antisense agents.

Uniformly modified oligonucleotide N3'-->P5' phosphoramidates, where every 3'-oxygen is replaced by a 3'-amino group, were synthesized. These compounds have very high affinity to single-stranded RNAs and thus have potential utility as antisense agents. As was shown in this study, the oligonucleotide phosphoramidates are resistant to digestion with snake venom phosphodiesterase, to nuclease activity in a HeLa cell nuclear extract, or to nuclease activity in 50% human plasma, where no significant hydrolysis was observed after 8 h. These compounds were used in various in vitro cellular systems as antisense compounds addressed to different targeted regions of c-myb, c-myc and bcr-abl mRNAs. C-myb antisense phosphoramidates at 5 microM caused sequence and dose-dependent inhibition of HL-60 cell proliferation and a 75% reduction in c-myb protein and RNA levels, as determined by Western blot and RT-PCR analysis. Analogous results were observed for anti-c-myc phosphoramidates, where a complete cytostatic effect for HL-60 cells was observed at 1 microM concentration for fully complementary, but not for mismatched compounds, which were indistinguishable from untreated controls. This was correlated with a 93% reduction in c-myc protein level. Moreover, colony formation by the primary CML cells was also inhibited 75-95% and up to 99% by anti-c-myc and anti-bcr-abl phosphoramidate oligonucleotides, respectively, in a sequence- and dose-dependent manner within a 0.5 nM-5 microM dose range. At these concentrations the colony-forming ability of normal bone marrow cells was not affected. The presented in vitro data indicate that oligonucleotide N3'-->P5' phosphoramidates could be used as specific and efficient antisense agents.     

Satellite telemetry of the winter migration of Adélie penguins (Pygoscelis adeliae)

Adélie penguins (Pygoscelis adeliae), after breeding in Antarctica during the austral summer, undergo a winter migration before returning to the breeding grounds 8 months later. It is the major source of adult mortality, with about a quarter of them not returning. Here we describe the first attempt to track the winter migration of Adélie penguins using satellite telemetry. Transmitters were attached to two penguins on 16 February 1991 after their post-breeding moult at Cape Bird, Ross Island, Antarctica. Transmissions were received from one penguin (bird #1) for 4.4 months, during which time it travelled 2792.6 km from the rookery (nearly 1500 km straight-line distance). Transmissions were received from the other penguin (bird #2) for 2.5 months during which time it followed a path remarkably similar to that of bird #1. The penguins travelled northwards up the coast of Victoria Land, keeping within 100 km of the coast, rounding Cape Adare soon after 29 March and were midway between the Balleny Islands and the Antarctic coast on 3 May. Thereafter, the record from bird #1 shows that it travelled further westwards until, when opposite the Mastusevich Glacier Tongue of the Mastusevich Glacier, it turned due north and moved away from the coast. By 29 June, when transmissions ended, its progression had slowed and it was northwest of the Balleny Islands near a zone where pack ice covered 75% of the surface of the sea. Two novel points that arise from this study are: (1) that Adélie penguins from Cape Bird undergo winter migrations of not less than 5000 km, and (2) that they may be travelling to common overwinter feeding grounds.     

The mmsA locus of Streptococcus pneumoniae encodes a RecG-like protein involved in DNA repair and in three-strand recombination

We describe the characterization of a mutant strain of Streptococcus pneumoniae previously isolated on the basis of its sensitivity to Methyl Methane Sulphonate (MMS). The mutant strain also exhibited increased sensitivity to UV light and to X-rays, together with a reduced capacity for recombination and Hex-mediated generalized mismatch repair. We show that the original mutant contains two unlinked mutations in the mmsA and in the pms genes. The mmsA wild-type region was cloned and the nucleotide sequence of the mmsA gene was determined. mmsA encodes a polypeptide of 671 amino acids related to a large family of DNA–RNA helicases, with the highest similarity to Escheri-chia coli RecG, a protein involved in the branch migration of Holliday junctions. A plasmid carrying the intact mmsA coding region was shown to restore UV resistance to E. coli recG mutant strains. An mmsA -null mutant constructed by insertion of a chloramphenicol-resistance gene exhibited a 25-fold reduction in recombination during transformation. We suggest that MmsA recognizes and branch migrates three-strand transformation intermediates to extend donor–recipient heteroduplex regions. The mmsA -null mutant exhibited the other phenotypes of the original mutant, apart from mismatch-repair deficiency and, in addition, an alteration in colony-forming ability was noticed. In the pms mutant background, all phenotypes caused by the mmsA mutation were attenuated. Therefore, the pms mutation, although it affected mismatch repair and, to some extent, DNA repair and recombination, acted as a suppressor of the mmsA mutation.