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PubMed2006

Testis-specific expression and genomic multiplicity of the rat Rtdpoz genes that encode bipartite TRAF- and POZ/BTB-domain proteins.

Based on bioinformatics analysis, we previously hypothesized the existence of a bipartite TDPOZ protein family members of which carry the TRAF domain (TD) and POZ/BTB [Huang, C.-J., Chen, C.-Y., Chen, H.-H., Tsai, S.-F., Choo, K.-B., 2004. TDPOZ, a family of bipartite animal and plant proteins that contain the TRAF (TD) and POZ/BTB domains. Gene 324, 117-127.]. Conservation in animals and plants suggests important biological functions for the putative TDPOZ proteins. In this work, we report testis-specific expression of two new Tdpoz members, Rtdpoz-T1 and -T2, of the rat genome; the result clearly indicates that members of the hypothetical gene family are, indeed, expressed. T1 and T2 cDNA sequences were derived by rapid amplification of cDNA ends (RACE). The exons of the genes were determined by queries of the rat genome sequence draft and selectively confirmed in splicing assays. The results indicate that T1 and T2 share a common leader exon indicative of alternative splicing, and that the genes are uninterrupted by introns in their respective coding sequences. Database interrogations also reveal a combined 297 hits of Rtdpoz-like sequences on 7 chromosomes; however, the bulk of the hits (264) and 26 putative TDPOZ-encoding genes, including T1 and T2, are found in a approximately 2.5 Mb cluster in the Rn2_2148 supercontig on chromosome 2. Our data signify retrotransposition in the generation and expansion of the Rtdpoz repertoire in the rat genome. We also anticipate spatio-temporal-specific expression of many more TDPOZ members in the rat or other animals and plants.

Amino Acid Motifs↗

PubMed2026

miR-519d-3p inhibits gastric cancer progression by targeting the Beclin-1-dependent autophagy pathway.

Dysregulation of microRNA networks is a hallmark of gastric cancer pathogenesis, but the mechanisms driving early-stage disease remain poorly understood. This study utilized integrative bioinformatics analysis of the Gene Expression Omnibus dataset GSE158315 to identify tumor-suppressive microRNAs in early gastric cancer. We identified hsa-miR-519d-3p as a core downregulated microRNA in early-stage tissues. Functional assays in NUGC-3 and MKN-45 cell lines demonstrated that miR-519d-3p overexpression significantly suppressed cell migration and invasion, whereas its inhibition enhanced these malignant phenotypes. Dual-luciferase reporter assays confirmed that miR-519d-3p directly targets the 3' untranslated region of BECN1 (Beclin-1). Silencing Beclin-1 via siRNA mimicked the effects of miR-519d-3p overexpression, while rescue experiments showed that Beclin-1 knockdown reversed the pro-migratory and pro-invasive effects triggered by miR-519d-3p inhibition. Furthermore, monitoring of autophagic flux using mRFP-GFP-LC3 tandem reporters revealed that miR-519d-3p inhibition enhances autophagy in a Beclin-1-dependent manner. Clinical data analysis from The Cancer Genome Atlas further supported the upregulation of Beclin-1 in gastric cancer and its correlation with aggressive clinicopathological features. In conclusion, our findings establish the miR-519d-3p/Beclin-1 axis as a critical regulator of motility and autophagy in gastric cancer, representing a potential therapeutic target for early intervention.

Autophagy↗

PubMed2006

Identification and characterisation of the dopamine receptor II from the cat flea Ctenocephalides felis (CfDopRII).

G protein-coupled receptors (GPCRs) represent a protein family with a wide range of functions. Approximately 30% of human drug targets are GPCRs, illustrating their pharmaceutical relevance. In contrast, the knowledge about invertebrate GPCRs is limited and is mainly restricted to model organisms like Drosophila melanogaster and Caenorhabditis elegans. Especially in ectoparasites like ticks and fleas, only few GPCRs are characterised. From the cat flea Ctenocephalides felis, a relevant parasite of cats and dogs, no GPCRs are known so far. Thus, we performed a bioinformatic analysis of available insect GPCR sequences from the honeybee Apis mellifera, the mosquito Anopheles gambiae, the fruit fly Drosophila melanogaster and genomic sequences from insect species. Aim of this analysis was the identification of highly conserved GPCRs in order to clone orthologs of these candidates from Ctenocephalides felis. It was found that the dopamine receptor family revealed highest conservation levels and thus was chosen for further characterisation. In this work, the identification, full-length cloning and functional expression of the first GPCR from Ctenocephalides felis, the dopamine receptor II (CfDopRII), are described.

Amino Acid Sequence↗

PubMed2006

Characterisation and expression of an Hsp70 gene from Parastrongyloides trichosuri.

Parastrongyloides trichosuri is a nematode parasite of Australian brushtail possums that has an alternative free-living life cycle which can be readily maintained indefinitely in a laboratory setting. The ability to maintain this parasite in a free-living cycle and induce it to parasitism at the free-living L1 stage makes this an excellent model for the study of genes associated with parasitism. A 70kD protein from infective larvae of P. trichosuri that appears to be immunogenic in infected possums has been identified as a heat shock protein (Hsp)70 homologue. The complete gene for Pt-Hsp70 was cloned and sequenced. The protein encoded by the Pt-Hsp70 gene is the likely orthologue of the Caenorhabditis elegans protein, Hsp70A, also known as hsp-1. Reverse transcriptase-PCR data indicate that Pt-Hsp70 (designated Pt-hsp-1) is expressed at readily detectable levels in all developmental stages of both the parasitic and free-living P. trichosuri life cycles and the promoter is mildly inducible by heat shock. Bioinformatic analysis of expressed sequence tag databases indicates that C. eleganshsp-1 homologues, together with C. eleganshsp-3 homologues, are the predominant members of the Hsp70 superfamily that are normally expressed in parasitic stages of the Strongyloididae family. Promoter fusions to a beta-galactosidase coding sequence were prepared and introduced into wild type C. elegans to produce transgenic nematodes. Reporter gene expression was clearly present within embryonic cells and within intestinal cells of larval and adult stages. Thus, the expression of the Pt-hsp-1 promoter within P. trichosuri and transgenic C. elegans appears similar to the known expression of C. elegans hsp-1. This promoter should be of value in efforts to develop genetic manipulation tools for P. trichosuri.

Amino Acid Sequence↗

PubMed2006

Not polymorphism but methylation of class II transactivator gene promoter IV associated with persistent HBV infection.

BACKGROUND: Class II transactivator (CIITA) is the major rate-limiting regulator for expression of class II major histocompability complex (MHC-II). Human CIITA gene expression is controlled by four distinct promoters (pIto pIV). OBJECTIVE: To evaluate the relationship among polymorphism and methylation status of CIITA gene promoters and persistent hepatitis B virus (HBV) infection. METHODS: We recruited 21 patients with hepatocellular carcinoma (HCC), 45 liver cirrhosis (LC), 65 chronic hepatitis B (CHB), 26 acute hepatitis B (AHB) and 95 healthy blood donors. Polymorphism of CIITA gene promoters was assayed by PCR-SSCP-sequencing. Bioinformatics analysis was employed to predict the existence of CpG islands. Methylation-specific PCR (MSP) was used to detect the methylation status of CIITA gene pIV. RESULTS: No sequence differences were observed at CIITA genes pI, III and IV among HCC, LC, CHB, AHB patients and healthy controls. No CpG islands were found in the pI, pII and pIII sequences, but there was a CpG island in pIV. The frequency of methylated POV was not significantly different within persistent HBV infection groups (patients with HCC, LC or CHB). Significance was found between the persistent infection group and acute HBV infection or healthy controls. CONCLUSIONS: CIITA gene promoter sequences are conserved. PIV is highly methylated and associated with host susceptibility to HBV persistent infection.

DNA Methylation↗

PubMed2004

Expressed gene clusters associated with cellular sensitivity and resistance towards anti-viral and anti-proliferative actions of interferon.

Interferons (IFN) are multi-functional proteins that induce a large number of genes which mediate many biological processes including host defense, cell growth control, signaling, and metabolism. Bioinformatics analysis of the 3'-untranslated regions of IFN-stimulated genes (ISGs) showed that the AU-rich elements (ARE) exist in approximately 10% of the mRNA induced by IFN. The human epithelial cell lines, WISH and 293, and the human B cell lines, Daudi and RPMI 1788, were assessed for their response to type-I IFN. Due to their differential response to the anti-viral and anti-proliferative action of IFN-alpha, they were used as cellular models for genome wide ARE-gene expression. The anti-viral and anti-proliferative actions of IFN-alpha were substantially more potent against WISH and Daudi cells than 293 and RPMI 1788 cells, respectively. These results correlated with the Stat1-driven gene expression as assessed by monitoring the expression of Stat1-mediated IFN-inducible 6-16 mRNA. Interferons were able to induce a significant proportion of common and distinct ARE-genes, but the patterns of expression were different and dependent on the type of the cell, type of IFN, and status of the cellular sensitivity to IFN. Clustering algorithms generated two informative expressed gene clusters that were selectively associated with cellular sensitivity and resistance to the anti-viral and anti-proliferative action of IFN. Use of rationally designed microarray experiments in IFN biology yielded informative clusters that may provide candidate genes for diagnostic or for evaluation of therapeutic possibilities.

Antiviral Agents↗

PubMed2005

The axial channel of the 20S proteasome opens upon binding of the PA200 activator.

Proteasomes consist of a proteolytic core called the 20 S particle and ancillary factors that regulate its activity in various ways. PA200 has been identified as a large (200 kDa) nuclear protein that stimulates proteasomal hydrolysis of peptides. To characterize its interaction with the 20 S core, we have visualized PA200-20 S complexes by electron microscopy. Monomers of PA200 bind to one or both ends of the 20 S core. Reconstructed in three dimensions to 23 A resolution from cryo-electron micrographs of the singly bound complex, PA200 has an asymmetric dome-like structure with major and minor lobes. Taking into account previous bioinformatic analysis, it is likely to represent an irregular folding of an alpha-helical solenoid composed of HEAT-like repeats. PA200 makes contact with all alpha-subunits except alpha7, and this interaction induces an opening of the axial channel through the alpha-ring. Thus, the activation mechanism of PA200 is expressed via its allosteric effects on the 20 S core particle, perhaps facilitating release of digestion products or the entrance of substrates.

Allosteric Site↗

PubMed2006

Ig-like domains on bacteriophages: a tale of promiscuity and deceit.

The immunoglobulin (Ig) fold is one of the most important structures in biology, playing essential roles in the vertebrate immune response, cell adhesion, and many other processes. Through bioinformatic analysis, we have discovered that Ig-like domains are often found in the constituent proteins of tailed double-stranded (ds) DNA bacteriophage particles, and are likely displayed on the surface of these viruses. These phage Ig-like domains fall into three distinct sequence families, which are similar to the classic immunoglobulin domain (I-Set), the fibronectin type 3 repeat (FN3), and the bacterial Ig-like domain (Big2). The phage Ig-like domains are very promiscuous. They are attached to more than ten different functional classes of proteins, and found in all three morphogenetic classes of tailed dsDNA phages. In addition, they reside in phages that infect a diverse set of gram negative and gram positive bacteria. These domains are deceptive because many are added to larger proteins through programmed ribosomal frameshifting, so that they are not always detected by standard protein sequence searching procedures. In addition, the presence of unrecognized Ig-like domains in a variety of phage proteins with different functions has led to gene misannotation. Our results demonstrate that horizontal gene transfer involving Ig-like domain encoding DNA has occurred commonly between diverse classes of both lytic and temperate phages, which otherwise display very limited sequence similarities to one another. We suggest that phage may have been an important vector in the spread of Ig-like domains through diverse species of bacteria. While the function of the phage Ig-like domains is unknown, several lines of evidence suggest that they may play an accessory role in phage infection by weakly interacting with carbohydrates on the bacterial cell surface.

Amino Acid Sequence↗

PubMed2006

Identification of two phosphatidylinositol/phosphatidylcholine transfer protein genes that are predominately transcribed in the flowers of Arabidopsis thaliana.

The Sec14 protein (Sec14p) and its homologs are involved in the transfer of phosphatidylinositol/phosphatidylcholine phospholipids in eukaryotic cells. In the completely sequenced genome of Arabidopsis thaliana, multiple genes encoding putative Sec14p homologs have been predicted based on bioinformatic analysis. Here we report the identification of two yeast Sec14-like genes (designated as AtSFH3 and AtSFH12, respectively) that are predominately transcribed in Arabidopsis flowers. The deduced amino acid sequences of AtSfh3p and AtSfh12p exhibited high similarity to that of Sec14p. Ectopic expression of AtSfh3p or AtSfh12p corrected the high temperature sensitive phenotype caused by Sec14p functional deficiency in Saccharomyces cerevisiae, indicating that the two plant homologs are functional in the intracellular environment. AtSFH3 transcripts were detected in flowers, stems and immature siliques but not roots and leaves, with a relatively higher transcript level in the flowers. In contrast, AtSFH12 transcripts were only detectable in the flowers. Based on histochemical staining of beta-glucuronidase (GUS) activities in the transgenic Arabidopsis plants harboring promoter::GUS constructs, AtSFH3 transcription was first detected in the stigma papillae of the flowers at stage 11, and then in the pollen grains before and after fertilization. On the other hand, AtSFH12 transcription was only found in the mature and germinating pollen grains. The information from this study may provide useful clue for further analysis of the function of plant Sec14p homologs in the development of the male gametic cells and/or the fertilization process in higher plants.

Arabidopsis↗

PubMed2006

High performance computing in biology: multimillion atom simulations of nanoscale systems.

Computational methods have been used in biology for sequence analysis (bioinformatics), all-atom simulation (molecular dynamics and quantum calculations), and more recently for modeling biological networks (systems biology). Of these three techniques, all-atom simulation is currently the most computationally demanding, in terms of compute load, communication speed, and memory load. Breakthroughs in electrostatic force calculation and dynamic load balancing have enabled molecular dynamics simulations of large biomolecular complexes. Here, we report simulation results for the ribosome, using approximately 2.64 million atoms, the largest all-atom biomolecular simulation published to date. Several other nano-scale systems with different numbers of atoms were studied to measure the performance of the NAMD molecular dynamics simulation program on the Los Alamos National Laboratory Q Machine. We demonstrate that multimillion atom systems represent a 'sweet spot' for the NAMD code on large supercomputers. NAMD displays an unprecedented 85% parallel scaling efficiency for the ribosome system on 1024 CPUs. We also review recent targeted molecular dynamics simulations of the ribosome that prove useful for studying conformational changes of this large biomolecular complex in atomic detail.

Computational Biology↗

PubMed2026

Characterisation of Trichuris incognita n sp in Côte d'Ivoire: a morphological, genomic, and genome-wide association with drug sensitivity study.

BACKGROUND: Trichuriasis is a neglected tropical disease that affects up to 500 million individuals and can cause considerable morbidity. For decades, trichuriasis was thought to be caused by one species of whipworm, Trichuris trichiura. The aim of this study was to investigate the origin of differences in response rates to the best available anthelmintic treatment for trichuriasis-a combination of albendazole and ivermectin-in Côte d'Ivoire by analysing the parasite population. METHODS: In this morphological, genomic, and genome-wide association study (GWAS) with drug sensitivity we used long-read and short-read sequencing approaches and assembled a high-quality reference genome of Trichuris incognita n sp isolated in a primary interventional study conducted in the Lagunes district of Côte d'Ivoire. Children aged 6-12 years were screened between July 14, 2022, and July 31, 2022; children positive for T trichiura on duplicate Kato-Katz smears and with infection intensity of 200 eggs per gram or more were eligible and treated first with albendazole (400 mg) and ivermectin (200 μg/kg) then with oxantel pamoate (20 mg/kg). We constructed a species tree of the Trichuris genus using 12 434 orthologous groups. We sequenced individual worms, which were used to confirm the phylogenetic placement and investigate patterns of adaptation through comparative genomic analyses. Finally, we conducted a GWAS to compare albendazole-ivermectin sensitive worms to drug non-sensitive worms. FINDINGS: 670 children were screened, of whom 243 were enrolled and from whom 271 worms were isolated after the first treatment and 827 worms after the second treatment. Sufficient DNA was recovered from 747 worms of which 721 were suitable for further bioinformatic analysis; of these, 179 were albendazole-ivermectin sensitive worms and 542 were drug non-sensitive worms. We present and characterise a new, human-infecting Trichuris species named T incognita n sp, which is morphologically indistinguishable from T trichiura, but forms a distinct phylogenetic clade, closer to Trichuris suis than to the canonical human-infective T trichiura. Comparative genomic analysis of genes suspected to confer resistance to either albendazole or ivermectin in helminths revealed a high number of β-tubulin orthologs, present in the whole population of T incognita n sp, compared with the canonical T trichiura species, but these genes were not associated with a resistant phenotype. The GWAS did not provide conclusive evidence of adaptation to drug pressure within the same species. INTERPRETATION: Our results demonstrate that trichuriasis can be caused by multiple whipworm species, and that differences in response rates might result from species responding differently to drug treatment, rather than from the intraspecies establishment of resistance. This discovery, coupled with the high tolerability of T incognita n sp to albendazole-ivermectin, marks a substantial shift in how we understand and approach whipworm infections. FUNDING: European Research Council.

Trichuris↗

PubMed2004

Triple genetic variation in the HNF-4alpha gene is associated with early-onset type 2 diabetes mellitus in a philippino family.

Maturity-onset diabetes of the young-type 1 (MODY1) is a form of monogenic type 2 diabetes mellitus (T2DM) with long-term complications due to mutations in the HNF-4alpha gene. The HNF-4alpha gene is involved in hepatic differentiation and expression of genes regulating glucose transport, glycolysis, and lipid metabolism. The abnormal glucose-stimulated insulin secretion in MODY1 subjects may be due to reduced glucose transport and glycolysis. To date, 14 mutations in the HNF-4alpha gene have been identified as a cause of either MODY1 or late-onset type 2 diabetes. So far, no screening has been performed in subjects from the Philippines. We recruited a Philippino family with autosomal dominant early-onset type 2 diabetes and screened the proband for mutations in the genes for HNF-1alpha, GCK, HNF-4alpha, IPF-1, HNF-6, and NGN3. We identified a new missense mutation in exon 5 (V199I) of the HNF-4alpha gene and 2 new single-nucleotide substitutions in intron 4, IVS4-nt4 (G --> A) and IVS4-nt20 (C --> T), all cosegregating with diabetes in the 3 affected available siblings. These variations were not present in 100 normal healthy subjects. Bioinformatic analysis suggests that these variations in the whole, and overall the IVS4-nt4 variation located at splicing site, may affect the splicing potential of intron 4. We have biochemically and clinically characterized the Philippine-1 family. We suggest that the V199I missense mutation located in the ligand binding/dimerization domain of HNF-4alpha contributes to type 2 diabetes in the Philippine-1 family. The intron variations may contribute susceptibility to diabetes.

Adult↗

PubMed2006

Retinoic acid regulation of eye and testis-specific transcripts within a complex locus.

We previously used a yeast-based enhancer trap to identify a strong, retinoic acid response element (RARE). We have now characterized testis and eye transcripts that are adjacent to this regulatory element. Bioinformatics analysis of expressed sequence tag (EST) clones and RNase protection, reverse transcription-PCR, and Northern blot assays indicate that these two RNAs are transcribed from the same locus on opposite template strands. This positions the RARE upstream of the testis transcript and downstream of the eye transcript. Additionally, these two RNAs are embedded within the third intron of the 329kbp gene that encodes the Zinc Finger and BTB domain containing 7C protein (Zbtb7C). We present evidence indicating that the testis transcript is expressed primarily in spermatocytes and/or early round spermatids. Furthermore, our analyses of transcript levels in eyes and testes isolated from vitamin A deficient mice or from mice with defects in retinoid storage or signaling indicate that retinoids are required for expression in vivo.

Journal Article↗

PubMed2004

Genomic organization and gene expression in a chromosomal region of Leishmania major.

Little is known about the relation between the genome organization and gene expression in Leishmania. Bioinformatic analysis can be used to predict genes and find homologies with known proteins. A model was proposed, in which genes are organized into large clusters and transcribed from only one strand, in the form of large polycistronic primary transcripts. To verify the validity of this model, we studied gene expression at the transcriptional, post-transcriptional and translational levels in a unique locus of 34kb located on chr27 and represented by cosmid L979. Sequence analysis revealed 115 ORFs on either DNA strand. Using computer programs developed for Leishmania genes, only nine of these ORFs, localized on the same strand, were predicted to code for proteins, some of which show homologies with known proteins. Additionally, one pseudogene, was identified. We verified the biological relevance of these predictions. mRNAs from nine predicted genes and proteins from seven were detected. Nuclear run-on analyses confirmed that the top strand is transcribed by RNA polymerase II and suggested that there is no polymerase entry site. Low levels of transcription were detected in regions of the bottom strand and stable transcripts were identified for four ORFs on this strand not predicted to be protein-coding. In conclusion, the transcriptional organization of the Leishmania genome is complex, raising the possibility that computer predictions may not be comprehensive.

Animals↗

PubMed2004

Response of REV3 promoter to N-methyl-N'-nitro-N-nitrosoguanidine.

Previously, we have shown that low concentration of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) led to the upregulation of REV3 gene at transcriptional level in cultured human amnion FL cells. In this study, using bioinformatic analysis the putative binding sites for different transcription factors were found to exist in REV3 gene promoter region. A 2570-bp fragment of the 5' flanking region of REV3 gene was amplified by PCR from PAC clone RP3-415N12 and inserted into the pGL3-Basic reporter vector. Dual-luciferase reporter assay demonstrated that the reconstructed plasmid did respond to MNNG exposure in transfected FL cells. Several variants of the reporter plasmids with different deletions of the REV3 promoter region were also constructed and their promoter strength was analyzed. It was found that the MNNG response element might locate at the REV3 gene promoter region -404 to -102 between two Sma1 sites. The shortest responsive fragment containing the putative binding sites for transcription factors CREBP, AP-2, NF-kappaB, and SP1 was also identified.

Base Sequence↗

PubMed2026

Targeting SUV4-20H2-mediated H4K20 methylation restrains growth and migration in pediatric high-grade astrocytomas.

Pediatric astrocytomas are characterized by increased molecular and clinical heterogeneity with epigenetic alterations contributing to aggressiveness and therapy resistance. The repressive histone mark H4K20 trimethylation (H4K20me3) and the methyltransferase SUV4-20H2 (KMT5C) are critical regulators of chromatin integrity and genome stability, with limited investigation in pediatric astrocytomas. KMT5C mRNA levels were evaluated in a publicly available pediatric gliomas database using bioinformatic analysis. Investigation of SUV4-20H2 and H4K20me3 expression was performed in a cohort of 43 pediatric astrocytoma tissues by immunohistochemistry. Their functional role and mechanism of action was investigated in pediatric glioma cell lines by using the substrate-competitive inhibitor of SUV4-20, A-196. Cell viability, apoptosis and migration were assessed using XTT, cleaved PARP, and wound healing assays, respectively. Effects of treatment on H4K20 methylation, DNA damage, mitotic stress [Polo-like kinase (PLK1) expression], and invasion markers (N-cadherin, β-catenin expression) were examined by western immunoblotting. KMT5C mRNA was significantly enriched in pediatric high-grade astrocytomas compared to low-grade tumors. A significant elevation of SUV4-20H2 and H4K20me3 expression was detected in astrocytoma tissues indicating epigenetic dysregulation contributing to malignancy. Treatment with A-196 reduced cell proliferation of pediatric glioma cell lines and induced apoptosis in a dose-dependent manner. It further impaired cell migration, accompanied by reduced N-cadherin and β-catenin expression. Mechanistically, inhibition of SUV4-20 depleted H4K20me3, inducing chromatin destabilization, replication-associated DNA damage and was associated with increased PLK1 expression, consistent with activation of a mitotic stress response. Our findings indicate that SUV4-20H2-mediated H4K20 activity in pediatric high-grade astrocytomas maintains their growth and migratory potential by regulating chromatin integrity and may serve as potential therapeutic target.

H4K20me2/3↗

PubMed2005

Amphibian skin peptides and their corresponding cDNAs from single lyophilized secretion samples: identification of novel brevinins from three species of Chinese frogs.

Brevinins are peptides of 24 amino acid residues, originally isolated from the skin of the Oriental frog, Rana brevipoda porsa, by nature of their microbicidal activity against a wide range of Gram-positive and Gram-negative bacteria and against strains of pathogenic fungi. cDNA libraries were constructed from lyophilized skin secretion of three, unstudied species of Chinese frog, Odorrana schmackeri, Odorrana versabilis and Pelophylax plancyi fukienensis, using our recently developed technique. In this report, we describe the "shotgun" cloning of novel brevinins by means of 3'-RACE, using a "universal" degenerate primer directed towards a highly conserved nucleic acid sequence domain within the 5'-untranslated region of previously characterized frog skin peptide cDNAs. Novel brevinins, deduced from cloned cDNA open-reading frames, were subsequently identified as mature peptides in the same samples of respective species skin secretions. Bioinformatic analysis of both prepro-brevinin nucleic acid sequences and translated open-reading frame amino acid sequences revealed a highly conserved signal peptide domain and a hypervariable anti-microbial peptide-encoding domain. The experimental approach described here can thus rapidly provide robust structural data on skin anti-microbial peptides without harming the donor amphibians.

Amino Acid Sequence↗

PubMed2005

Histamine-releasing and antimicrobial peptides from the skin secretions of the dusky gopher frog, Rana sevosa.

Seven novel peptides were isolated from the skin secretions of the North American dusky gopher frog, Rana sevosa, on the basis of antimicrobial activity and histamine release from rat peritoneal mast cells. The peptides were purified to homogeneity using HPLC and characterized by electrospray ion-trap mass spectrometry, MALDI-TOF mass spectrometry and Edman sequencing. Bioinformatic analysis of primary structures revealed that the novel peptides could be assigned to four established families of ranid frog antimicrobial peptides, namely esculentin-1, esculentin-2, brevinin-1 and ranatuerin-2. The peptides were named in accordance with accepted terminology as ranatuerin 2SEa, etc., reflecting the peptide family name, the species of origin (SE for sevosa) and the isotype (a). Of major interest was the fact that brevinin 1SE displayed significant structural similarity to ponericin W5, an antibacterial venom peptide from the ant, Pachyconyla goeldii. This is a further example of amphibian skin defensive peptides showing striking structural similarities to peptides from insects. These data may shed some light on the functional biological relevance of defensive peptides that possess both antimicrobial and histamine-releasing activities.

Animals↗