Living in an Extremely Polluted Environment: Clues from the Genome of Melanin-Producing Aeromonas salmonicida subsp. pectinolytica 34melT

Aeromonas salmonicida subsp. pectinolytica 34mel^T can be considered an extremophile due to the characteristics of the heavily polluted river from which it was isolated. While four subspecies of A. salmonicida are known fish pathogens, 34mel^T belongs to the only subspecies isolated solely from the environment. Genome analysis revealed a high metabolic versatility, the capability to cope with diverse stress agents, and the lack of several virulence factors found in pathogenic Aeromonas. The most relevant phenotypic characteristics of 34mel^T are pectin degradation, a distinctive trait of A. salmonicida subsp. pectinolytica, and melanin production. Genes coding for three pectate lyases were detected in a cluster, unique to this microorganism, that contains all genes needed for pectin degradation. Melanin synthesis in 34mel^T is hypothesized to occur through the homogentisate pathway, as no tyrosinases or laccases were detected and the homogentisate 1,2-dioxygenase gene is inactivated by a transposon insertion, leading to the accumulation of the melanin precursor homogentisate. Comparative genome analysis of other melanogenic Aeromonas strains revealed that this gene was inactivated by transposon insertions or point mutations, indicating that melanin biosynthesis in Aeromonas occurs through the homogentisate pathway. Horizontal gene transfer could have contributed to the adaptation of 34mel^T to a highly polluted environment, as 13 genomic islands were identified in its genome, some of them containing genes coding for fitness-related traits. Heavy metal resistance genes were also found, along with others associated with oxidative and nitrosative stresses. These characteristics, together with melanin production and the ability to use different substrates, may explain the ability of this microorganism to live in an extremely polluted environment.     

Capping of HLA antigens in human lymphocytes as followed by immunogold label-fracture

We used immunogold label-fracture to follow the migration of HLA I class and HLA II class antigens during capping as induced by specific monoclonal antibodies. Capping is achieved through a process of clustering and "consolidation" of clusters into larger patches and, finally, a single cap. All receptors appear to cluster from the very start, with no "stray" molecules joining already formed patches. Characterization of exoplasmic and protoplasmic fracture-faces of capping cells fails to reveal any corresponding accumulation of intramembrane particles and/or subtler rugosities. Our results are consistent with the concepts that view the migration of capping molecules as contemporaneous with the efflux of noncapping integral membrane proteins.     

The effects of serial passage of a nucleopolyhedrosis virus through an alternate host system

The multiple-embedded velvetbean caterpillar nucleopolyhedrosis virus (VBC-NPV) ofAnticarsia gemmatalis Hübner was shown to be infectious of a variety of noctuid hosts. The mortality data demonstrated the importance of defining the dosage used in host range analysis. Serial passage of the VBC-NPV through the soybean looper,Pseudoplusia includens, was done to select for host range variants of this NPV. After several passages of the VBC-NPV through this insect the virulence of the progeny virus remained unchanged indicating heterogeneity in the host and not the virus population. However, between the 3rd and 5th serial passage throughPseudoplusia a latent NPV identical to a single-embedded NPV previously associated fromA. gemmatalis was activated. The biological and biochemical characteristics of this isolate demonstrated it to be distinct from the original VBC-NPV. A single passage of this activated virus throughA. gemmatalis resulted in the production of viral progeny having characteristics of the original VBC-NPV. Serial passage of this virus preparation throughP. includens resulted in virus progeny having biological properties associated with both the original VBC-NPV and the activated NPV isolate.     

Association of Cellular Prion Protein with Gangliosides in Plasma Membrane Microdomains of Neural and Lymphocytic Cells

In this report we demonstrated that cellular prion protein is strictly associated with gangliosides in microdomains of neural and lymphocytic cells. We preliminarily investigated the protein distribution on the plasma membrane of human neuroblastoma cells, revealing the presence of large clusters. In order to evaluate its possible role in tyrosine signaling pathway triggered by GEM, we analyzed PrP^c presence in microdomains and its association with gangliosides, using cholera toxin as a marker of GEM in neuroblastoma cells and anti-GM3 MoAb for identification of GEM in lymphoblastoid cells. In neuroblastoma cells scanning confocal microscopical analysis revealed a consistent colocalization between PrP^c and GM1 despite an uneven distribution of both on the cell surface, indicating the existence of PrP^c-enriched microdomains. In lymphoblastoid T cells PrP^c molecules were mainly, but not exclusively, colocalized with GM3. In addition, PrP^c was present in the Triton-insoluble fractions, corresponding to GEM of cell plasma membrane. Additional evidence for a specific PrP^c-GM3 interaction in these cells was derived from the results of TLC analysis, showing that prion protein was associated with GM3 in PrP^c immunoprecipitates. The physical association of PrP^c with ganglioside GM3 within microdomains of lymphocytic cells strongly suggests a role for PrP^c-GM3 complex as a structural component of the multimolecular signaling complex involved in T cell activation and other dynamic lymphocytic plasma membrane functions.     

WNT1 and WNT3a promote expansion of melanocytes through distinct modes of action

Summary WNT1 and WNT3a have been described as having redundant roles in promoting the development of neural crest-derived melanocytes (NC-Ms). We used cell lineage restricted retroviral infections to examine the effects of WNT signaling on defined cell types in neural crest cultures. RCAS retroviral infections were targeted to melanoblasts (NC-M precursor cells) derived from transgenic mice that express the virus receptor, TVA, under the control of a melanoblast promoter (DCT). As expected, over 90% of DCT-TVA+ cells expressed early melanoblast markers MITF and KIT. However, by following the fate of infected cells in standard culture conditions, we find that only 5% of descendents were NC-Ms. The majority of the descendents were not NC-Ms, but expressed smooth muscle cell markers, demonstrating that mammalian melanoblasts are not committed to the NC-M lineage. RCAS infection of DCT-TVA+ cells demonstrated that overexpression of canonical WNT signaling genes (betaCAT, WNT3a or WNT1) can increase NC-M numbers in an endothelin dependent manner. However, WNT1 and WNT3a have different modes of action with respect to melanoblast fate. Intrinsic over-expression of betaCAT or WNT3a can increase NC-M numbers by biasing the fate of DCT-TVA+ cells to NC-Ms. In contrast, the DCT-TVA+ melanoblasts cannot respond to WNT1 signaling and do not alter their fate towards NC-M. Instead, WNT1 only increases NC-M numbers through paracrine signaling on melanoblast precursors to increase the numbers of neural crest cells that become NC-Ms.     

Co-purification and localization of Munc18-1 (p67) and Cdk5 with neuronal cytoskeletal proteins

Munc18-1 (p67, nSec1, rbSec1), a neuron-specific 67kDa protein was independently identified as a syntaxin-binding protein, and as a component that co-purifies with, and regulates the kinase activity of cyclin dependent kinase (Cdk5). Gene knockout studies have demonstrated a role for Munc18-1 in synaptic vesicle docking and neurotransmitter release. Mice lacking Munc18-1 gene were synaptically silent, but the gene deletion did not prevent normal brain assembly, including the formation of layered structures, fiber pathways and morphologically defined synapses. Previous study has shown that Munc18-1 facilitates Cdk5 mediated phosphorylation of KSPXK domains of the neuronal cytoskeletal elements, suggesting that Munc18-1 may function in the regulation of cytoskeletal dynamics. Present study demonstrates the co-purification and co-localization of Munc18 with cytoskeletal elements and forms first step towards understanding the role for Munc18-1 in cytoskeletal dynamics. Conversely, the cytoskeletal proteins and Cdk5 co-purifies with Munc18-1 in a Munc18-1 immuno-affinity chromatography, suggesting a strong protein-protein interaction. Findings from immunofluorescence studies in PC12 cells have shown co-localization of Munc18-1 and Cdk5 with neurofilaments and microtubules. Further, immunohistochemical and immuno-electron microscopic studies of rat olfactory bulb also demonstrated co-localization of Munc18-1 and Cdk5 with cytoskeletal elements. Thus, the biochemical evidence of strong interaction between Munc18-1 with cytoskeletal proteins and morphological evidence of their (Munc18 and cytoskeletal elements) identical sub-cellular localization is suggestive of the possible role for Munc18-1 in cytoskeletal dynamics.