Islet hormones from the African bullfrog Pyxicephalus adspersus (Anura:Ranidae): structural characterization and phylogenetic implications.

The African bullfrog Pyxicephalus adspersus is generally classified along with frogs of the genus Rana in the subfamily Raninae of the family Ranidae but precise phylogenetic relationships between species are unclear. Pancreatic polypeptide (PP), insulin, and glucagon-like peptide (GLP-1) were isolated from an extract of P. adspersus pancreas and characterized structurally. A comparison of the amino acid sequence of Pyxicephalus PP (APSEPQHPGG(10)QATPEQLAQY(20)YSDLYQYITF(30)ITRPRF++ +. NH(2)) with those of the known amphibian PP molecules in a maximum parsimony analysis generates a single phylogenetic tree in which Pyxicephalus is the sister to the clade comprising the members of the genus Rana. The three orders of living amphibians form discrete clades with the representative of the Gymnophiona appearing as sister to the Caudata-Anura. In contrast, Pyxicephalus insulin (A chain, GIVEQCCHSA(10)CSLYDLENYC(20)N; B-chain, LANQHLCGSH(10)LVEALYMVCG(20)ERGFFYYPKS(30)) and and GLP-1 (HAEGTFTSDM(10)TSYLEEKAAK(20)EFVDWLIKGR(30)PK) resemble more closely the corresponding peptides from the cane toad Bufo marinus than the peptides from any species of Rana. Cladistic analysis based upon the amino acid sequences of insulin produced a polyphyletic assemblage with the Gymnophiona nesting within an unresolved clade containing the non-ranid frogs. The data support the assertion that the amino acid sequence of PP, but not those of the other islet hormones, is of value as a molecular marker for inferring phylogenetic relationships between early tetrapod species.

Peptides with antimicrobial activity from four different families isolated from the skins of the North American frogs Rana luteiventris, Rana berlandieri and Rana pipiens.

The skins of frogs of the genus Rana synthesize a complex array of antimicrobial peptides that may be grouped into eight families on the basis of structural similarity. A total of 24 peptides with differential growth-inhibitory activity towards the Gram-positive bacterium Staphylococcus aureus, the Gram-negative bacterium Escherichia coli and the yeast Candida albicans were isolated from extracts of the skins of three closely related North American frogs, Rana luteiventris (spotted frog), Rana berlandieri (Rio Grande leopard frog) and Rana pipiens (Northern leopard frog). Structural characterization of the antimicrobial peptides demonstrated that they belonged to four of the known families: the brevinin-1 family, first identified in skin of the Asian frog Rana porosa brevipoda; the esculentin-2 family, first identified in the European frog Rana esculenta; the ranatuerin-2 family, first identified in the North American bullfrog Rana catesbeiana; and the temporin family, first identified in the European frog Rana temporaria. Peptides belonging to the brevinin-2, ranalexin, esculentin-1 and ranatuerin-1 families were not identified in the extracts. Despite the close phylogenetic relationship between the various species of Ranid frogs, the distribution and amino-acid sequences of the antimicrobial peptides produced by each species are highly variable and species-specific, suggesting that they may be valuable in taxonomic classification and molecular phylogenetic analysis.

Peptides with antimicrobial activity of the brevinin-1 family isolated from skin secretions of the southern leopard frog, Rana sphenocephala.

Three peptides with growth-inhibitory activity towards the gram-negative bacterium Eschericia coli were isolated from electrically stimulated secretions from the skin of the southern leopard frog, Rana sphenocephala. Structural characterization demonstrated that the peptides [brevinin-1Sa, minimum inhibitory concentration (MIC) = 55 microM; brevinin-1Sb, MIC = 17 microM; brevinin-1Sc, MIC = 14 microM] represent new members of the brevinin-1 family of antimicrobial peptides, previously isolated from several other species of frogs of the genus Rana. Their high concentration in skin secretions and extreme variability in amino acid sequence suggest that the brevinin family of peptides may be of value as molecular markers for the identification and taxonomic classification of Ranid frogs.

Amino acid sequence diversity of pancreatic polypeptide among the amphibia.

It has been suggested that the amino acid sequence of pancreatic polypeptide (PP) may provide a useful molecular marker with which to study evolutionary relationships between tetrapods but few PP sequences from amphibia are available to test this hypothesis. PPs have been purified from the pancreata of five species belonging to the different orders of amphibians. Their amino acid sequences were established as: APSEPEHPGD10 NASPDELAKY20 YSDLWQYITF30 VGRPRY for the lesser siren, Siren intermedia (Caudata); GPTEPIHPGK10 DATPEELTKY20 YSDLYDYITL30 VGRSRW for the caecilian, Typhlonectes natans (Gymnophiona); and TPSEPQHPGD10 QASPEQLAQY20 YSDLWQYITF30 VTRPRF for the cane toad, Bufo marinus (Anura). The structure of Rana sylvatica PP is the same as that of Rana catesbeiana PP whereas PP from the green frog Rana ridibunda contains one substitution (His6 --> Gln). The data provide further support for the conclusion that the amino acid sequence of PP has been poorly conserved during evolution with only 17 residues invariant among the eight species of amphibia yet studied and only 8 residues (Pro5, Pro8, Gly9, Ala12, Leu24, Tyr27, Arg33, and Arg35) invariant among all tetrapods. A maximum parsimony analysis based upon the amino acid sequence of PP and using the sequence of frog PYY as outgroup to polarize the in-group taxa generates a consensus phylogenetic tree in which the Amniota and Amphibia form two distinct clades. However, such a tree does not permit valid conclusions to be drawn regarding branching order within the Amphibia.

Primary structure of insulin from the african lungfish, Protopterus annectens.

Among the extant Sarcopterygii, the interrelationship between the Dipnoi (lungfishes), Actinistia (coelacanths), and Tetrapoda (tetrapods) is controversial. Insulin has been purified from an extract of the pancreas of the African lungfish Protopterus annectens and its primary structure established as A-chain, Gly-Ile-Val-Glu-Gln-Cys-Cys-His-Lys-Pro10-Cys-Ser-Leu- Tyr -Glu-Leu-Glu-Asn-Tyr-Cys20-Asn-Val-Pro; and B-chain, Ala-Val-Leu-Asn-Gln-His-Leu-Cys-Gly-Ser10-His-Leu-Val- Glu- Ala-Leu-Tyr-Leu-Val-Cys20-Ala-Asp-Asn-Gly-Phe- Phe-Tyr-Lys-Pro-Ser30-Gly. Lungfish insulin contains unusual structural features, such as the dipeptide extension to the C-terminus of the A-chain and the substitution Arg --> Asn at position B-23 in the putative receptor binding region of insulin, which may be expected to influence appreciably its biological potency relative to mammalian insulins. Lungfish insulin also contains amino acid substitutions such as Gly --> Ala at position B-21, Glu --> Asp at position B-22, and a Lys --> Ser residue at position B-30, previously found in insulins from amphibia. This observation is consistent with paleontological data suggesting that lungfish and amphibia share a close phylogenetic relationship.

Rana pipiens complex: hemoglobin phenotypes of sympatric and allopatric populations in Arizona.

Electrophoretic comparison of hemoglobin samples from numerous populations of Rana pipiens in Arizona reveals three distinct phenotypes that closely correlate with morphological differences. Hemoglobin samples from sympatric locations contain parental phenotypes with only the occasional occurrence of a hybrid. These data support the contention that the Rana pipiens complex consists of several species.