Polyphyly of Asian Tree Toads,Genus Pedostibes Günther, 1876 (Anura: Bufonidae), and the Description of a New Genus from Southeast Asia

The Asian Tree Toad genus Pedostibes, as currently understood, exhibits a conspicuously disjunct distribution, posing several immediate questions relating to the biogeography and taxonomy of this poorly known group. The type species, P. tuberculosus and P. kempi, are known only from India, whereas P. hosii, P. rugosus, and P. everetti are restricted to Southeast Asia. Several studies have shown that these allopatric groups are polyphyletic, with the Indian Pedostibes embedded within a primarily South Asian clade of toads, containing the genera Adenomus, Xanthophryne, and Duttaphrynus. Southeast Asian Pedostibes on the other hand, are nested within a Southeast Asian clade, which is the sister lineage to the Southeast Asian river toad genus Phrynoidis. We demonstrate that Indian and Southeast Asian Pedostibes are not only allopatric and polyphyletic, but also exhibit significant differences in morphology and reproductive mode, indicating that the Southeast Asian species’ are not congeneric with the true Pedostibes of India. As a taxonomic solution, we describe a new genus, Rentapia gen. nov. to accommodate the Southeast Asian species.     

Rapid determination of substrate specificity of Clostridium histolyticum beta-collagenase using an immobilized peptide library.

The molecular basis of the substrate specificity of Clostridium histolyticum beta-collagenase was investigated using a combinatorial method. An immobilized positional peptide library, which contains 24,000 sequences, was constructed with a 7-hydroxycoumarin-4-propanoyl (Cop) fluorescent group attached at the N terminus of each sequence. This immobilized peptide library was incubated with C. histolyticum beta-collagenase, releasing fluorogenic fragments in the solution phase. The relative substrate specificity (k(cat)/K(m)) for each member of the library was determined by measuring fluorescence intensity in the solution phase. Edman sequencing was used to assign structure to subsites of active substrate mixtures. Collectively, the substrate preference for subsites (P(3)-P(4)') of C. histolyticum beta-collagenase was determined. The last position on the C-terminal side in which the identity of the amino acids affects the activity of the enzyme is P(4)', and an aromatic side chain is preferred in this position. The optimal P(1)'-P(3)' extended substrate sequence is P(1)'-Gly/Ala, P(2)'-Pro/Xaa, and P(3)'-Lys/Arg/Pro/Thr/Ser. The Cop group in either the P(2) or P(3) position is required for a high substrate activity with C. histolyticum beta-collagenase. S(2) and S(3) sites of the protease play a dominant role in fixing the substrate specificity. The immobilized peptide library proved to be a powerful approach for assessing the substrate specificity of C. histolyticum beta-collagenase, so it may be applied to the study of other proteases of interest.     

Protistan microbial observatory in the Cariaco Basin,Caribbean. I. Pyrosequencing vs Sanger insights into species richness

Microbial diversity and distribution are topics of intensive research. In two companion papers in this issue, we describe the results of the Cariaco Microbial Observatory (Caribbean Sea, Venezuela). The Basin contains the largest body of marine anoxic water, and presents an opportunity to study protistan communities across biogeochemical gradients. In the first paper, we survey 18S ribosomal RNA (rRNA) gene sequence diversity using both Sanger- and pyrosequencing-based approaches, employing multiple PCR primers, and state-of-the-art statistical analyses to estimate microbial richness missed by the survey. Sampling the Basin at three stations, in two seasons, and at four depths with distinct biogeochemical regimes, we obtained the largest, and arguably the least biased collection of over 6000 nearly full-length protistan rRNA gene sequences from a given oceanographic regime to date, and over 80 000 pyrosequencing tags. These represent all major and many minor protistan taxa, at frequencies globally similar between the two sequence collections. This large data set provided, via the recently developed parametric modeling, the first statistically sound prediction of the total size of protistan richness in a large and varied environment, such as the Cariaco Basin: over 36 000 species, defined as almost full-length 18S rRNA gene sequence clusters sharing over 99% sequence homology. This richness is a small fraction of the grand total of known protists (over 100 000–500 000 species), suggesting a degree of protistan endemism.     

Calcium binding mode of gamma-carboxyglutamic acids in conantokins.

Conantokin-T (con-T) and conantokin-G (con-G) are two highly homologous peptide toxins found in Conus venom. The former is a 21-residue peptide with four gamma-carboxyglutamic acid (Gla) residues (at positions 3, 4, 10 and 14), while the latter is a 17-residue peptide with five gamma-carboxyglutamic acid residues (at positions 3, 4, 7, 10 and 14). Despite the apparent similarity in number and relative positions of the gamma-carboxyglutamic acid residues, (113)Cd-NMR studies indicated a distinct metal binding behavior for con-G and con-T. There appears to be four binding sites in con-G in contrast to one metal binding site in con-T. To elucidate the mode of calcium binding by the gamma-carboxyglutamic acid residues in these conantokins, we designed various analogous peptides with their gamma-carboxyglutamic acid replaced by other amino acid residues. (113)Cd-NMR experiments on conantokin analogues reveal that the major difference in the number of metal binding sites between con-G and con-T is due to the residue at position 7. We also performed molecular simulations to calculate the relative binding free energies of several potential binding sites. Based on our theoretical and experimental results, we propose a 'four-site' binding model for conantokin-G and a 'single-site' binding model for conantokin-T.     

Human endothelin converting enzyme-2 (ECE2): characterization of mRNA species and chromosomal localization.

Generation of the functionally pleiotropic members of the endothelin vasoactive peptide family is critically catalyzed by unique type II metalloproteases, termed endothelin converting enzymes (ECE). Isolation of human ECE-2 (EC 3.4.24.71) cDNAs revealed deduced open reading frames of 787 and 765 amino acids with approximately 60% identity with human ECE-1. Characterization of mRNA variants revealed mRNA structural diversity at the 5'-terminus. Two mRNA species exist containing distinct first and second exons. Furthermore, in one of these species, an in-frame deletion of the intracytoplasmic domain removed 29 amino acids. Because of the previously reported human genetic diseases ascribed to germline mutations of member genes of the endothelin family, ECE2 was localized in human chromosomes with fluorescence in situ hybridization and radiation hybrid mapping to 3q28-q29 and SHGC-20171/D3S1571, respectively.