Phylogenetic characterization of the blue filamentous bacterial community from an Icelandic geothermal spring.

Molecular phylogenetic analysis of a blue filamentous community from an alkaline thermal spring (79-83 degrees C) in Iceland revealed that the blue filaments were affiliated with the Aquificales. The dominant sequence type, pIce1, was most closely related to a sequence (SRI-48) found in a white filamentous community from a separate Icelandic thermal spring and the pink filaments (EM17) from Yellowstone National Park. Fluorescent in situ hybridization with clone-specific oligonucleotide probes showed that the sample analyzed was essentially a monoculture of a single phylotype.

Identification of a new subset of cells exhibiting dendritic phenotypes in patients affected by breast proliferative disorders.

We report that a subset of circulating cells reacting with a monoclonal antibody raised against a protein marker is significantly increased in the peripheral blood of women carrying benign or malignant breast diseases, particularly in patients under 55 years of age with ductal mammary carcinomas. These cells were statistically (confidence level of 99%) less represented in a control population including healthy women or women carrying carcinomas of origin other than breast. Double staining analysis showed that they harbor markers of dendritic cells and exhibit endo- cytic activity, as determined by their ability to internalize FITC-dextran particles. Their dendritic morphology was further demonstrated by electron microscopy of sorted antibody-positive cells. However, expression of surface molecules, such as CD34 and CD14, usually not present in differentiated populations of dendritic cells was also observed. Adherent cells of patients with breast ductal carcinoma including mostly cells of this new subset were efficient stimulators of mixed lymphocyte reaction, attaining maximal stimulatory activity attained after TNFalpha treatment. In conclusion, we have shown that a subset of cells characterized by a phenotype suggestive of a yet undescribed stage of maturation of the dendritic cell lineage is accumulated in the blood of patients affected by breast proliferative disorders.

A mutation in the DE loop of the VP1 protein that prevents polyomavirus transcription and replication.

Natural mutants of the DE loop of the Polyomavirus (Py) major coat protein VP1 have been previously shown to display an altered host specificity (L. Ricci, R. Maione, C. Passananti, A. Felsani, and P. Amati, 1992, J. Virol. 66, 7153-7158). To better understand the role of this outfacing loop of the VP1 protein in Py infectivity, we constructed and characterized a Py mutant (Py M17) harboring a deletion of 7 AA within the tip of the DE loop. The mutant virions obtained after DNA transfection were unable to replicate and initiate early transcription in fibroblast cells. Complementation experiments performed to rescue the deficient M17 replication by means of wt functions revealed the cis-dominance of the mutation. In situ cell fractionation experiments demonstrated that the Py mutant, like the Py wt, enters the cells, reaches the nucleus and that both the viral DNA and VP1 protein are found tightly bound to the nuclear matrix. These data suggest that the VP1 protein, associated to the viral DNA, conditions early viral gene expression and that the DE loop of the protein must be involved in this process.

Nodule invasion and symbiosome differentiation during Rhizobium etli-Phaseolus vulgaris symbiosis.

By means of a detailed ultrastructural analysis of nodules induced by Rhizobium etli on the roots of Phaseolus vulgaris, we observe that the development of host-invaded cells is not synchronous. An accumulation of mitochondria was found in freshly invaded host cells, containing only a few symbiosomes (SBs) that are released from highly branched intracellular ramification of the infection threads. Moreover, besides the fusion between the SB membrane with host secretory vesicles, we observe also a great number of fusions between the outer leaflets of adjoining SB membranes, thus resulting in structures that resemble the tight junction network (zona occludens with a five-layered structure) of epithelian cells. This process was found to be induced strongly and earlier both in the invaded host cells of ineffective nodules (elicited by Fix- mutant strains of R. etli) and in the older (senescence) invaded cells of effective nodules, whereas bacteroid division is seldom if ever observed. Our observations strongly suggest that multiple-occupancy SBs also arise by fusion of single-occupancy SBs and the physiological consequence of this process is discussed.

The Rhizobium etli trpB gene is essential for an effective symbiotic interaction with Phaseolus vulgaris.

A mutant strain (CTNUX4) of Rhizobium etli carrying Tn5 unable to grow with ammonium as the sole nitrogen source was isolated and characterized. Sequence analysis showed that Tn5 is inserted into a trpB (tryptophan synthase)-homologous gene. When tested on the roots of Phaseolus vulgaris, strain CTNUX4 was able to induce only small, slightly pink, ineffective (Fix-) nodules. However, under free-living conditions, strain CTNUX4 was unable to produce flavonoid-inducible lipo-chitin oligosaccharides (Nod factors) unless tryptophan was added to the growth medium. These data and histological observations indicate that the lack of tryptophan biosynthesis affects the symbiotic behavior of R. etli.

Immuno-cross-reactivity of CUT-1 and cuticlin epitopes between ascaris lumbricoides, Caenorhabditis elegans, and Heterorhabditis.

Cuticlin is the insoluble residue of nematode cuticle. It has been proved that cuticlin and CUT-1-like epitopes are conserved between the free-living Caenorhabditis elegans and the entomopathogenic nematode Heterorhabditis sp. The cloning of a cut-1 homologous gene from the animal intestinal parasite Ascaris lumbricoides has allowed us to extend the study of immuno-cross-reactivity at the ultrastructural level to this important species. Antibodies against recombinant CUT-1 protein and against cuticlin from Ascaris as well as from C. elegans were used for immuno-labeling ultrathin sections of high-pressure cryoprocessed worms. All the antisera used showed the same specific pattern of localization on sections of C. elegans of Heterorhabditis dauer larvae, and of Ascaris larvae in mature eggs. It was also shown that sera raised against the cuticlin residue contain anti-CUT-1 antibodies. CUT-1-like proteins are thus possibly important components in the immune response of hosts to invading nematodes. The results presented support the use of C. elegans as a model for the study of vertebrate parasitic nematodes.

Immuno-gold-labelling of CUT-1, CUT-2 and cuticlin epitopes in Caenorhabditis elegans and Heterorhabditis sp. processed by high pressure freezing and freeze-substitution.

CUT-1 and CUT-2 are two distinct protein components of cuticlin, the insoluble residue of the cuticles of nematodes. In previous experiments of gold-immuno-labelling on sections of chemically fixed Caenorhabditis elegans, CUT-1 and CUT-2 epitopes were specifically lost. Cryo-immobilization of C. elegans under high pressure followed by freeze-substitution, however, resulted in a good preservation of these antigenic sites and of the ultrastructure of the worms. The entomopathogenic nematode Heterorhabditis sp. processed by the same cryopreparation protocol has shown a strong reactivity with anti-sera raised against CUT-1, CUT-2 and against the whole cuticlin residue of C. elegans. The localization of these epitopes was conserved across the two species.

Ultrastructural immuno-localization of CUT-1 and CUT-2 antigenic sites in the cuticles of the nematode Caenorhabditis elegans.

CUT-1 and CUT-2 are two distinct proteins found in cuticlin, the insoluble residue of the cuticles of the nematode Caenorhabditis elegans. They are the products of genes which have been previously characterized molecularly. These proteins have been expressed as recombinant in Escherichia coli and specific antisera have been raised against them. The experiments reported here regard their ultrastructural immuno-gold localization either on purified cuticles or on whole worms of various stages of development of Caenorhabditis elegans. A location in the cortical layer of the isolated cuticles is common to all stages, whereas there is a dauer specific location in the fibrous ribbon underneath the alae. These localizations are compared with immuno-labelling obtained using a serum raised against the whole cuticlin residue. CUT-1 and CUT-2 epitopes are easily and specifically lost during conventional chemical fixation.