Delineation of the IGF-II C domain elements involved in binding and activation of the IR-A, IR-B and IGF-IR.

OBJECTIVE: Human insulin-like growth factor-I and -II (IGF-I and -II) ligands share a high degree of sequence and structural homology. Despite their similarities, IGF-I and IGF-II exhibit differential receptor binding and activation characteristics. The C domains of IGF-I and IGF-II are the primary determinants of binding specificity to the insulin-like growth factor I receptor (IGF-IR), insulin receptor exon 11- (IR-A) and exon 11+ (IR-B) isoforms. DESIGN: Three IGF-II analogues were generated in order to delineate the C domain residues that confer the differential receptor binding affinity and activation properties of the IGFs. Chimeric IGF-II analogues IGF-IICI(N) and IGF-IICI(C) contained partial IGF-I C domain substitutions (IGF-I residues underlined) GYGSSSRRSR and SRVSRRAPQT, respectively. RESULTS: The IGF-IICI(N) analogue bound the IR-A and IGF-IR with high affinity but bound the IR-B with a relatively lower affinity than IGF-II, suggesting a negative interaction between the exon-11 encoded peptide in the IR-B and the C-domain. The ability of IGF-IICI(N) to activate receptors and elicit cell viability responses was generally proportional to its relative receptor binding affinity but appeared to act as a partial agonist equivalent to IGF-I when binding and activating the IGF-IR. In contrast, IGF-IICI(C) bound IGF-IR with high affinity but elicited lower receptor activation and cell viability responses. Analogue IGF-IICI(S) contained a truncated IGF-I C domain (GSSSRRAT) and generally displayed a relatively poor ability to bind, activate and elicit viability responses via each receptor. CONCLUSIONS: Together, the IGF analogues demonstrate that both flanks of the IGF-II C domain play important roles in the greater ability of IGF-II to bind and activate IR receptors than IGF-I.

Regulation of insulin secretion: role of mitochondrial signalling.

Pancreatic beta cells are specialised endocrine cells that continuously sense the levels of blood sugar and other fuels and, in response, secrete insulin to maintain normal fuel homeostasis. During postprandial periods an elevated level of plasma glucose rapidly stimulates insulin secretion to decrease hepatic glucose output and promote glucose uptake into other tissues, principally muscle and adipose tissues. Beta cell mitochondria play a key role in this process, not only by providing energy in the form of ATP to support insulin secretion, but also by synthesising metabolites (anaplerosis) that can act, both intra- and extramitochondrially, as factors that couple glucose sensing to insulin granule exocytosis. ATP on its own, and possibly modulated by these coupling factors, triggers closure of the ATP-sensitive potassium channel, resulting in membrane depolarisation that increases intracellular calcium to cause insulin secretion. The metabolic imbalance caused by chronic hyperglycaemia and hyperlipidaemia severely affects mitochondrial metabolism, leading to the development of impaired glucose-induced insulin secretion in type 2 diabetes. It appears that the anaplerotic enzyme pyruvate carboxylase participates directly or indirectly in several metabolic pathways which are important for glucose-induced insulin secretion, including: the pyruvate/malate cycle, the pyruvate/citrate cycle, the pyruvate/isocitrate cycle and glutamate-dehydrogenase-catalysed alpha-ketoglutarate production. These four pathways enable 'shuttling' or 'recycling' of these intermediate(s) into and out of mitochondrion, allowing continuous production of intracellular messenger(s). The purpose of this review is to present an account of recent progress in this area of central importance in the realm of diabetes and obesity research.

Holocarboxylase synthetase: correlation of protein localisation with biological function.

Holocarboxylase synthetase (HCS) governs the cellular fate of the essential micronutrient biotin (Vitamin H or B7). HCS is responsible for attaching biotin onto the biotin-dependent enzymes that reside in the cytoplasm and mitochondria. Evidence for an alternative role, viz the regulation of gene expression, has also been reported. Recent immunohistochemical studies reported HCS is primarily nuclear, inconsistent with the location of HCS activity. Improved understanding of biotin biology demands greater knowledge about HCS. Here, we investigated the localisation of HCS and its isoforms. Three variants were observed that differ at the N-terminus. All HCS isoforms were predominantly non-nuclear, consistent with the distribution of biotin protein ligase activity. Unlike the longer constructs, the Met(58) isoform was also detected in the nucleus--a novel observation suggesting shuttling activity between nucleus and cytoplasm. We resolved that the previous controversies in the literature are due to specificity and detection limitations that arise when using partially purified antibodies.

Silencing of the insulin receptor isoform A favors formation of type 1 insulin-like growth factor receptor (IGF-IR) homodimers and enhances ligand-induced IGF-IR activation and viability of human colon carcinoma cells.

Insulin receptor (IR) overexpression is common in cancers, with expression of the A isoform (IR-A, exon 11-) predominating over the B isoform. The IR-A signals a proliferative, antiapoptotic response to IGF-II, which itself can be secreted by tumors to establish an autocrine proliferative loop. Therefore, IGF-II signaling via the IR-A could mediate resistance to type 1 IGF receptor (IGF-IR) inhibitory drugs that are currently in development. This study addressed the role of the IR-A, using a small interfering RNA-based approach in SW480 human colon adenocarcinoma cells that coexpress the IGF-IR. Clonogenic survival was inhibited by depletion of the IGF-IR but not the IR-A, and dual receptor depletion had no greater effect than IGF-IR knockdown alone, suggesting that the IR-A could not compensate for IGF-IR loss. IGF-IR knockdown also resulted in a decrease in viability, whereas IR-A depletion resulted in increased viability. Consistent with this, upon IR-A depletion, we found a concomitant enhancement of IGF-IR activation by IGF-I and IGF-II, reduced formation of IGF-IR:IR-A hybrid receptors and increased IGF-IR homodimer formation. Together, these results suggest that IGF bioactivity is mediated more effectively by the IGF-IR than by the IR-A or receptor hybrids and that signaling via the IGF-IR is dominant to the IR-A in colon cancer cells that express both receptors.

Artifactual detection of biotin on histones by streptavidin.

Biotinylation is a recent addition to the list of reported posttranslational modifications made to histones. Holocarboxylase synthetase (HCS) and biotinidase have been implicated as biotinylating enzymes. However, the details of the mechanism and the regulation of biotin transfer on and off histones remains unclear. Here we report that in a cell culture system low biotin availability reduces biotinylation of carboxylases, yet apparent biotinylation of histones is unaffected. This is despite biotin depletion having detrimental effects on cell viability and proliferation. Further analysis of the widely used method for detecting biotin on histones, streptavidin blotting, revealed that streptavidin interacts with histones independently of biotin binding. Preincubation of streptavidin with free biotin reduced binding to biotinylated carboxylases but did not block binding to histones. To investigate biotinylation of histones using an alternative detection method independent of streptavidin, incorporation of 14C biotin into biotinylated proteins was analyzed. Radiolabeled biotin was readily detectable on carboxylases but not on histones, implying very low levels of biotin in the nucleus attached to histone proteins (< 0.03% biotinylation). In conclusion, we would caution against the use of streptavidin for investigating histone biotinylation.

Anaplerotic roles of pyruvate carboxylase in mammalian tissues.

Pyruvate carboxylase (PC) catalyzes the ATP-dependent carboxylation of pyruvate to oxaloacetate. PC serves an anaplerotic role for the tricarboxylic acid cycle, when intermediates are removed for different biosynthetic purposes. In liver and kidney, PC provides oxaloacetate for gluconeogenesis. In adipocytes PC is involved in de novo fatty acid synthesis and glyceroneogenesis, and is regulated by the peroxisome proliferator-activated receptor-gamma, suggesting that PC is involved in the metabolic switch controlling fuel partitioning toward lipogenesis. In islets, PC is necessary for glucose-induced insulin secretion by providing oxaloacetate to form malate that participates in the 'pyruvate/malate cycle' to shuttle 3C or 4C between mitochondria and cytoplasm. Hyperglycemia and hyperlipidemia impair this cycle and affect glucose-stimulated insulin release. In astrocytes, PC is important for de novo synthesis of glutamate, an important excitatory neurotransmitter supplied to neurons. Transcriptional studies of the PC gene pinpoint some transcription factors that determine tissue-specific expression.

Evaluation of gilthead sea bream, Sparus aurata, sperm quality after cryopreservation in 5 ml macrotubes.

Cryopreservation produces several types of damage in spermatozoa, leading to fertility impairment. The reduction arises both from a lower viability post-thaw and from sublethal dysfunctions in some of the surviving cells. In the present study, we have analysed the effect of cryopreservation in 5 ml macrotubes on the quality of post-thawed gilthead sea bream sperm. Several standard sperm quality parameters were determined: pH and osmolarity of seminal plasma, sperm concentration, and motility. An exhaustive determination of sperm quality before and after cryopreservation was investigated. Several parameters related with spermatozoal status were determined: ATP content, plasma membrane integrity and functionality, mitochondrial functionality, and sperm fertility. Our results demonstrated that gilthead sea bream spermatozoa suffer several types of damage after freezing/thawing. The percentage of viable cells slightly decreased after cryopreservation, however plasma membrane was affected by cryopreservation, since cells could not resist the hyperosmotic shock. Mitochondrial status was affected by cryopreservation since there was a decrease in the parameters of sperm motility, ATP content (3.17 nmol ATP/10(5) spermatozoa to 1.7 nmol ATP/10(5) spermatozoa in 1:20 frozen samples) and an increase of the percentage of cells with mitochondrial depolarized membranes (11% for fresh and 27% for 1:20 frozen samples). Fertility rate was similar either using fresh or frozen/thawed sperm (77 and 75% hatched larvae, respectively).

Interaction of insulin-like growth factor (IGF)-I and -II with IGF binding protein-2: mapping the binding surfaces by nuclear magnetic resonance.

The interaction of IGF binding protein-2 (IGFBP-2) with IGF-I and -II has been investigated in solution using nuclear magnetic resonance (NMR) spectroscopy. Chemical shift perturbations in 15N- and 2H/15N-labelled IGF-I or -II upon binding to unlabelled thioredoxin-tagged bovine IGFBP-2 (Trx(1-279)IGFBP-2) have been monitored to identify residues involved directly in the binding interaction as well as any affected by conformational changes associated with the interaction. A key step in obtaining high-quality spectra of the complexes was the use of transverse relaxation optimised spectroscopy (TROSY) methods with partially deuterated ligands. Indeed, because the effects of conformational averaging and aggregation are eliminated in IGF-I and -II bound to IGFBP-2, the spectra of the complexes are actually superior to those of the free ligands. Comparison of our results with the crystal structure of the complex between IGF-I and an N-terminal fragment of IGFBP-5 allowed identification of those residues perturbed by the C-domain of IGFBP-2. Other perturbations, such as those of Gly 19 and Asp 20 of IGF-I (and the corresponding residues in IGF-II) - which are located in a reverse turn linking N-domain and C-domain interactive surfaces - are due to local conformational changes in the IGF-I and -II. Our results confirm that the C-domain of IGFBP-2 plays a key role in binding regions of IGF-I and -II that are also involved in binding to the type-1 IGF receptor and thereby blocking ligand binding to this receptor.

Dihydrothymine lesion in X-irradiated DNA: characterization at the molecular level and detection in cells.

PURPOSE: To study the formation of the dihydrothymine lesion produced in DNA by ionizing radiation in an anaerobic environment. MATERIALS AND METHODS: The dihydrothymine lesion, along with other lesions, was isolated from an X-irradiated aqueous solution of the dinucleoside monophosphate d(TpA) and analysed by correlated two-dimensional nuclear magnetic resonance spectroscopy. The dihydrothymine lesion was obtained by enzymatic digestion of irradiated DNA in the form of modified dinucleoside monophosphates, d(T(d)A), where T(d) stands for dihydrothymidine. Liquid chromatography-tandem mass spectrometry was used to detect the lesion in the DNA of X-irradiated mouse fibroblast cells. RESULTS: The modified dinucleoside monophosphate, d(T(d)pA), fragments by two pathways so that altogether the lesion could be detected using two different sets of tandem mass spectrometry (precursor ion mass/daughter ion mass) values. CONCLUSION: The dihydrothymine lesion is a significant lesion in cells exposed to ionizing radiation in an anaerobic environment.

Effect of different cryoprotectants and vitrificant solutions on the hatching rate of turbot embryos (Scophthalmus maximus).

Vitrification could provide a promising tool for the cryopreservation of fish embryos. However, in order to achieve a vitrifiable medium, a high concentration of permeable cryoprotectants must be employed, and the incorporation of high molecular weight compounds should also be considered. The toxicity of these permeable and non-permeable agents has to be assessed, particularly when high concentrations are required. In the present study, permeable and non-permeable cryoprotectant toxicity was determined in turbot embryos at two development stages (F stage-tail bud and G stage-tail bud free). Embryos treated with pronase (2mg/ml, 10 min at 22 degrees C) were incubated in dimethyl sulfoxide (Me2SO), methanol (Meth.) or ethylene glycol (EG) in concentrations ranging from 0.5 to 6M for periods of 10 or 30 min, and in 5, 10, and 15% polyvinylpyrrolidone (PVP), 10, 15, and 20% sucrose or 0.1, 1, and 2% X-1000 for 2 min. The embryos were then washed well and incubated in seawater until hatching. The toxicity of permeable cryoprotectants increased with concentration and exposure time. There were no significant differences between permeable cryoprotectants. However, embryos tolerated higher concentrations of Me2SO than other cryoprotectants. Exposure to permeable cryoprotectants did not affect the hatching rate except at G stage with X-1000 treatment and 20% sucrose. Taking into account the cryoprotectant toxicity and the vitrification ability of cryoprotectant mixtures, three vitrification solutions (V1, V2, and V3), and one protocol for stepwise incorporation were designed. The tested solutions contained 5M Me2SO+2M Meth+1M EG plus 5% PVP, 10% sucrose or 2% X-1000. The hatching rate of embryos that had been exposed to the the vitrification solutions was analyzed and no significant differences were noticed compared with the controls. Our results demonstrate that turbot embryos can be subject to this cryoprotectant protocol without deleterious effect on the hatching rate.

The insulin receptor isoform exon 11- (IR-A) in cancer and other diseases: a review.

The insulin receptor plays a vital role in mediating the actions of insulin. These include metabolic and mitogenic effects. This review will focus on the role of the insulin receptor isoforms in normal development and the pathogenesis of certain cancers and type 2 diabetes. There are two insulin receptor isoforms arising from the alternative splicing of exon 11 resulting in either the exon 11+ (IR-B) isoform (including 12 amino acids encoded by exon 11) or the exon 11- (IR-A) isoform. The isoforms have different affinities for insulin, IGF-II and IGF-I with the exon 11- isoform binding both insulin and IGF-II with high affinities. Interestingly, differential expression of the insulin receptor isoforms has been demonstrated in disease. Several cancer cell types that also overexpress IGF-II preferentially express the exon 11- isoform. Activation of the exon 11- insulin receptor by IGF-II and insulin results in mitogenic effects and a potentiation of the cancer phenotype. Also hyperinsulinemia has been associated with increased risk of cancer. Differential expression of the insulin receptor isoforms has also been demonstrated in type 2 diabetes although there is some discrepancy in the literature as to which isoform is expressed.

The C-terminal domain of biotin protein ligase from E. coli is required for catalytic activity.

Biotin protein ligase of Escherichia coli, the BirA protein, catalyses the covalent attachment of the biotin prosthetic group to a specific lysine of the biotin carboxyl carrier protein (BCCP) subunit of acetyl-CoA carboxylase. BirA also functions to repress the biotin biosynthetic operon and synthesizes its own corepressor, biotinyl-5'-AMP, the catalytic intermediate in the biotinylation reaction. We have previously identified two charge substitution mutants in BCCP, E119K, and E147K that are poorly biotinylated by BirA. Here we used site-directed mutagenesis to investigate residues in BirA that may interact with E119 or E147 in BCCP. None of the complementary charge substitution mutations at selected residues in BirA restored activity to wild-type levels when assayed with our BCCP mutant substrates. However, a BirA variant, in which K277 of the C-terminal domain was substituted with Glu, had significantly higher activity with E119K BCCP than did wild-type BirA. No function has been identified previously for the BirA C-terminal domain, which is distinct from the central domain thought to contain the ATP binding site and is known to contain the biotin binding site. Kinetic analysis of several purified mutant enzymes indicated that a single amino acid substitution within the C-terminal domain (R317E) and located some distance from the presumptive ATP binding site resulted in a 25-fold decrease in the affinity for ATP. Our data indicate that the C-terminal domain of BirA is essential for the catalytic activity of the enzyme and contributes to the interaction with ATP and the protein substrate, the BCCP biotin domain.

Bioactive dahlein peptides from the skin secretions of the Australian aquatic frog Litoria dahlii: sequence determination by electrospray mass spectrometry.

Eleven dahlein peptides are present in the skin secretion of the Australian aquatic frog Litoria dahlii. All peptides have been sequenced using a combination of electrospray mass spectrometry (ES-MS) and Lys-C digestion/MS, with each sequence confirmed by automated Edman sequencing. The 13-residue dahlein 1 peptides (e.g. dahlein 1.1 GLFDIIKNIVSTL-NH(2)) exhibit weak wide-spectrum antimicrobial activity but no significant activity in the anticancer testing program of the National Cancer Institute (Washington). There are no potent antimicrobial peptides present in the glandular secretion, but the dahleins 5 strongly inhibit the formation of NO by neuronal nitric oxide synthase (e.g. dahlein 5.1 GLLGSIGNAIGAFIANKLKP-OH).

Structural and promoter regions of the murine pyruvate carboxylase gene.

We have cloned and sequenced the gene encoding mouse pyruvate carboxylase (mPC) [EC 6.4.1.1]. The coding region contains 19 exons, one 5'-untranslated region exon, and 19 introns in 22 kb of genomic DNA. This gene's exon/intron organization is highly conserved with respect to rat and human PC genes. The mPC gene promoter lacks canonical TATA and CCAAT boxes, in common with a number of housekeeping genes. Transient expressions in COS-1 of a luciferase reporter gene under the control of 5'-nested deletions of the 5'-flanking sequence of the mPC gene have identified the 166-bp minimal sequence required for basal transcription. Alternative splicing at the 5'-untranslated region exon of the mouse PC gene results in the production of two alternate transcripts bearing different 5'-noncoding regions. Both transcripts are highly expressed in kidney and liver and moderately expressed in heart and testis and expressed at a low level in spleen.

Linkers for improved cleavage of fusion proteins with an engineered alpha-lytic protease.

Addition of an N-terminal fusion partner can greatly aid the expression and purification of a recombinant protein in Escherichia coli. We investigated two genetically engineered proteases designed to remove the fusion partner after the protein of interest has been expressed. Recombinant human insulin-like growth factor-II (hIGF-II) has been produced from E. coli-derived fusion proteins using a novel enzymatic cleavage system that uses a mutant of alpha-lytic protease. Initially, two potential fusion protein linkers were designed, Pro-Ala-Pro-His (PAPH) and Pro-Ala-Pro-Met (PAPM), and were tested as substrates in the form of synthetic dodecapeptides. Using mass spectrometry and reverse-phase HPLC, the position of cleavage was confirmed and the kinetics of synthetic peptide cleavage were examined. Use of the linkers in hIGF-II fusion proteins produced in E. coli was then evaluated. The fusion proteins constructed consist of the first 11 amino acids of porcine growth hormone linked N-terminally to hIGF-II by six amino acids that include the dipeptide Val-Asn followed by a variable tetrapeptide protease cleavage motif. Mass spectrometry and N-terminal sequencing confirmed that proteolytic cleavage of the fusion proteins had occurred at the predicted sites. Using the fusion proteins as substrates, the cleavage of the rationally designed motifs by the alpha-lytic protease mutant was compared. The fusion protein containing the motif PAPM had a k(cat)/K(M) ratio indicating a 1.6-fold preference over the PAPH fusion protein for cleavage by this enzyme. Furthermore, when hIGF-II fusion proteins containing the designed cleavable linkers were processed with the engineered alpha-lytic protease, they gave greatly improved yields of native hIGF-II compared to an analogous fusion protein cleaved by H64A subtilisin. Comparison of the peptide and protein cleavage studies shows that the efficient proteolysis of the cleavage motifs is an inherent property of the designed sequences and is not determined by secondary or tertiary structure in the fusion proteins.

BIAcore analysis of bovine insulin-like growth factor (IGF)-binding protein-2 identifies major IGF binding site determinants in both the amino- and carboxyl-terminal domains.

In the absence of a complete tertiary structure to define the molecular basis of the high affinity binding interaction between insulin-like growth factors (IGFs) and IGF-binding proteins (IGFBPs), we have investigated binding of IGFs by discrete amino-terminal domains (amino acid residues 1-93, 1-104, 1-132, and 1-185) and carboxyl-terminal domains (amino acid residues 96-279, 136-279, and 182-284) of bovine IGFBP-2 (bIGFBP-2). Both halves of bIGFBP-2 bound IGF-I and IGF-II in BIAcore studies, albeit with different affinities ((1-132)IGFBP-2, K(D) = 36.3 and 51.8 nm; (136-279)IGFBP-2HIS, K(D) = 23.8 and 16.3 nm, respectively). The amino-terminal half appears to contain components responsible for fast association. In contrast, IGF binding by the carboxyl-terminal fragment results in a more stable complex as reflected by its K(D). Furthermore, des(1-3)IGF-I and des(1-6)IGF-II exhibited reduced binding affinity to (1-279)IGFBP-2HIS, (1-132)IGFBP-2, and (136-279)IGFBP-2HIS biosensor surfaces compared with wild-type IGF. A charge reversal at positions 3 and 6 of IGF-I and IGF-II, respectively, affects binding interactions with the amino-terminal fragment and full-length bIGFBP-2 but not the carboxyl-terminal fragment.

Mutational analysis of protein substrate presentation in the post-translational attachment of biotin to biotin domains.

Biotinylation in vivo is an extremely selective post-translational event where the enzyme biotin protein ligase (BPL) catalyzes the covalent attachment of biotin to one specific and conserved lysine residue of biotin-dependent enzymes. The biotin-accepting lysine, present in a conserved Met-Lys-Met motif, resides in a structured domain that functions as the BPL substrate. We have employed phage display coupled with a genetic selection to identify determinants of the biotin domain (yPC-104) of yeast pyruvate carboxylase 1 (residues 1075-1178) required for interaction with BPL. Mutants isolated using this strategy were analyzed by in vivo biotinylation assays performed at both 30 degrees C and 37 degrees C. The temperature-sensitive substrates were reasoned to have structural mutations, leading to compromised conformations at the higher temperature. This interpretation was supplemented by molecular modeling of yPC-104, since these mutants mapped to residues involved in defining the structure of the biotin domain. In contrast, substitution of the Met residue N-terminal to the target lysine with either Val or Thr produced mutations that were temperature-insensitive in the in vivo assay. Furthermore, these two mutant proteins and wild-type yPC-104 showed identical susceptibility to trypsin, consistent with these substitutions having no structural effect. Kinetic analysis of enzymatic biotinylation using purified Met --> Thr/Val mutant proteins with both yeast and Escherichia coli BPLs revealed that these substitutions had a strong effect upon K(m) values but not k(cat). The Met --> Thr mutant was a poor substrate for both BPLs, whereas the Met --> Val substitution was a poor substrate for bacterial BPL but had only a 2-fold lower affinity for yeast BPL than the wild-type peptide. Our data suggest that substitution of Thr or Val for the Met N-terminal of the biotinyl-Lys results in mutants specifically compromised in their interaction with BPL.

Contribution of residues A54 and L55 of the human insulin-like growth factor-II (IGF-II) A domain to Type 2 IGF receptor binding specificity.

The underlying specificity of the interaction between insulin-like growth factor-II (IGF-II) and mammalian Type 2 insulin-like growth factor/cation-independent mannose 6 phosphate receptor (IGF2R) is not understood. We have mutated residues A54 and L55 of IGF-II in the second A domain helix to arginine (found in the corresponding positions of IGF-I) and measured IGF2R binding. There is a 4- and 3.3-fold difference in dissociation constants for A54R IGF-II and L55R IGF-II, respectively, and a 6.6-fold difference for A54R L55R IGF-II compared with IGF-II as measured by BlAcore analysis using purified rat IGF2R. This is also confirmed using cross-linking and soluble rat placental membrane receptor binding assays. Binding to the type I IGF receptor (IGF1R) and IGF binding protein-2 (IGFBP-2) is not altered. We can, therefore, conclude that residues at positions 54 and 55 in IGF-II are important for and equally contribute to IGF2R binding.

The antibiotic and anticancer active aurein peptides from the Australian Bell Frogs Litoria aurea and Litoria raniformis. Part 2. Sequence determination using electrospray mass spectrometry.

Sixteen aurein peptides are present in the host defence secretion from the granular dorsal glands of the Green and Golden Bell Frog Litoria aureus and seventeen from those of the related Southern Bell Frog Litoria raniformis. All peptides have been sequenced using a combination of electrospray mass spectrometry and Lys-C digestion, with each sequence confirmed by automated Edman sequencing. The peptides are named in five groups, viz. aureins 1-5. Ten of these peptides are common to both species of frog. Thirteen of the aurein peptides show wide-spectrum antibiotic and anticancer activity. Amongst the more active peptides are aurein 1.2 (GLFDIIKKIAESF-NH2), the smallest peptide from an anuran reported to have both antibiotic and anticancer activity; aurein 2.2 (GLLDIVKKVIGAFGSL-NH2) and aurein 3.1 (GLFDIVKKIAGHIAGSI-NH2). The aurein 4 and 5 peptides, e.g. aurein 4.1 (GLIQTIKEKLKELAGGLVTGIQS-OH) and aurein 5.1 (GLLDIVTGLLGNLIVDVLKPKTPAS-OH), show neither antibacterial nor anticancer activity.