The "Rhampholeon uluguruensis complex" (Squamata: Chamaeleonidae) and the taxonomic status of the pygmy chameleons in Tanzania.

The specific status of several pygmy chameleons endemic to mountain massifs in the Eastern Arc Mountains, Tanzania has long been controversial due to their lack of distinctive morphological characters. In this work we extend our previous sampling of Rieppeleon and Rhampholeon species, especially from the Rhampoleon moyeri/Rhampholeon uluguruensis complex, and add data from a new mitochondrial marker to address this problem. Our results show that there is geographical structure between populations of pygmy chameleons from different mountains. This structure is especially well defined for Rhampholeon (Rhinodigitum). Phylogenetic analyses confirm that both Rh. uluguruensis Tilbury and Emmrich, 1995 and Rh. moyeri Menegon, Salvidio and Tilbury, 2002 are distinct lineages, the former from the Uluguru Mountains and the latter from the Udzungwa Mountains. However, the paratype material used to erect Rh. moyeri belongs to a separate lineage from the holotype. Similarly, a number of additional lineages within the Rh. moyeri/Rh. uluguruensis complex recovered in the analysis may deserve specific status. At present, there is a lack of morphological characters that can be used to distinguish these lineages, suggesting that there are multiple cryptic taxa in this complex. 

A large 28S rDNA-based phylogeny confirms the limitations of established morphological characters for classification of proteocephalidean tapeworms (Platyhelminthes, Cestoda).

Proteocephalidean tapeworms form a diverse group of parasites currently known from 315 valid species. Most of the diversity of adult proteocephalideans can be found in freshwater fishes (predominantly catfishes), a large proportion infects reptiles, but only a few infect amphibians, and a single species has been found to parasitize possums. Although they have a cosmopolitan distribution, a large proportion of taxa are exclusively found in South America. We analyzed the largest proteocephalidean cestode molecular dataset to date comprising more than 100 species (30 new), including representatives from 54 genera (80%) and all subfamilies, thus significantly improving upon previous works to develop a molecular phylogeny for the group. The Old World origin of proteocephalideans is confirmed, with their more recent expansion in South America. The earliest diverging lineages are composed of Acanthotaeniinae and Gangesiinae but most of the presently recognized subfamilies (and genera) appear not to be monophyletic; a deep systematic reorganization of the order is thus needed and the present subfamilial system should be abandoned. The main characters on which the classical systematics of the group has been built, such as scolex morphology or relative position of genital organs in relation to the longitudinal musculature, are of limited value, as demonstrated by the very weak support for morphologically-defined subfamilies. However, new characters, such as the pattern of uterus development, relative ovary size, and egg structure have been identified, which may be useful in defining phylogenetically well-supported subgroups. A strongly supported lineage infecting various snakes from a wide geographical distribution was found. Although several improvements over previous works regarding phylogenetic resolution and taxon coverage were achieved in this study, the major polytomy in our tree, composed largely of siluriform parasites from the Neotropics, remained unresolved and possibly reflects a rapid radiation. The genus Spasskyellina Freze, 1965 is resurrected for three species of Monticellia bearing spinitriches on the margins of their suckers.

A serpin from the gut bacterium Bifidobacterium longum inhibits eukaryotic elastase-like serine proteases.

Serpins form a large class of protease inhibitors involved in regulation of a wide spectrum of physiological processes. Recently identified prokaryotic members of this protein family may provide a key to the evolutionary origins of the unique serpin fold and the associated inhibitory mechanism. We performed a biochemical characterization of a serpin from Bifidobacterium longum, an anaerobic Gram-positive bacterium that naturally colonizes human gastrointestinal tract. The B. longum serpin was shown to efficiently inhibit eukaryotic elastase-like proteases with a stoichiometry of inhibition close to 1. Porcine pancreatic elastase and human neutrophil elastase were inhibited with the second order association constants of 4.7 x 10(4) m(-1) s(-1) and 2.1 x 10(4) m(-1) s(-1), respectively. The B. longum serpin is expected to be active in the gastrointestinal tract, because incubation of the purified recombinant serpin with mouse feces produces a stable covalent serpin-protease adduct readily detectable by SDS-PAGE. Bifidobacteria may encounter both pancreatic elastase and neutrophil elastase in their natural habitat and protection against exogenous proteolysis may play an important role in the interaction between these commensal bacteria and their host.

Characterization of native outer membrane vesicles from lpxL mutant strains of Neisseria meningitidis for use in parenteral vaccination.

Native outer membrane vesicles (NOMV) of Neisseria meningitidis consist of intact outer membrane and contain outer membrane proteins (OMP) and lipooligosaccharides (LOS) in their natural conformation and membrane environment. NOMV have been safely used intranasally in P1 studies with encouraging results, but they are too toxic for parenteral vaccination. We now report the preparation and characterization of lpxL mutants that express LOS with reduced toxicity, and the evaluation of the potential of NOMV from these strains for use as a parenteral vaccine. A series of deletion mutants were prepared with knockouts of one or more of the lpxL1, lpxL2, or synX genes. The deltalpxL2 mutants had a reduced growth rate, reduced level of LOS expression, and increased sensitivity to surfactants. In addition, deltasynX deltalpxL2 double mutants had reduced viability in stationary phase. The deltalpxL1 deltalpxL2 double mutant behaved essentially the same as the deltalpxL2 single mutant. LOS from both lpxL mutant strains exhibited altered migration on polyacrylamide gels. The LOS of deltalpxL2 mutants of L3,7 strains were fully sialylated. NOMV prepared from lpxL2 mutants was about 200-fold less active than wild-type NOMV in rabbit pyrogen tests and in tumor necrosis factor alpha release assays. Bactericidal titers induced in animals by deltalpxL2 mutant NOMV were lower than those induced by deltalpxL1 or wild-type NOMV. However, immunogenicity could be largely restored by use of an adjuvant. These results provide evidence that NOMV from deltalpxL2 mutant strains will be safe and immunogenic in humans when given parenterally.

Deletion analysis identifies key functional domains of the cytadherence-associated protein HMW2 of Mycoplasma pneumoniae.

Mycoplasma pneumoniae attachment to host cells requires biogenesis of a functional attachment organelle, including proper localization of the adhesion protein P1 to this structure. Mutations in the hmw2 gene result in the inability to cytadhere, failure to localize P1 to the attachment organelle, altered cell morphology and accelerated turnover of the cytadherence-associated proteins HMW1, HMW3 and P65. The hmw2 gene encodes HMW2 (190 kDa) and P28 (28 kDa), the latter apparently the product of internal translation initiation near the 3' end of the hmw2 coding region. Transformation of hmw2 mutant I-2 with recombinant wild-type hmw2 restores a wild-type phenotype. In the current study, a severely truncated hmw2 gene with an in frame internal deletion of 80% of the HMW2 coding region that leaves the P28-encoding region intact restored cytadherence to mutant I-2. Transformants produced the expected 38 kDa HMW2 derivative (HMW2Deltamid) at levels comparable to that of HMW2 in wild-type cells; like HMW2, HMW2Deltamid exhibited marked Triton X-100 insolubility. HMW3, P65 and P28 were fully restored, but not HMW1. These transformants were morphologically similar to wild-type M. pneumoniae but failed to localize P1 to the attachment organelle. Finally, a C-terminally truncated HMW2 derivative was partly Triton X-100 soluble and incapable of restoring HMW1, HMW3 and P65 to wild-type levels. These data are consistent with a model in which the C-terminal domain of HMW2 imparts normal localization to the protein, and this localization itself is required for productive interactions with downstream cytadherence-associated proteins. Furthermore, these results emphasize the association of HMW1 with P1 clustering.