Isolation of promoter for cytosolic phospholipase A2 (cPLA2)

Cytosolic phospholipase A2 (cPLA2) releases arachidonic acid from membrane phospholipids and is believed to be the rate-limiting enzyme in the arachidonic acid pathway. We report herein the isolation of a 3 kb fragment of rodent genomic DNA containing part of the first intron, the first exon and 5'-flanking sequence. The start site of transcription was mapped by 5'-rapid amplification of cDNA ends and corroborated by ribonuclease protection assay. The gene has a TATAless promoter with no classical Sp1 binding sites or initiator element. A microsatellite series of CA repeats was noted in the 5'-flanking region of both the rodent and human promoters. Deletion constructs have been analysed for luciferase activity and confirmed promoter activity.

AcT-2: A Novel Myotropic and Antimicrobial Type 2 Tryptophyllin from the Skin Secretion of the Central American Red-Eyed Leaf Frog, Agalychnis callidryas

Tryptophyllins are a diverse family of amphibian peptides originally found in extracts of phyllomedusine frog skin by chemical means. Their biological activities remain obscure. Here we describe the isolation and preliminary pharmacological characterization of a novel type 2 tryptophyllin, named AcT-2, from the skin secretion of the red-eyed leaf frog, Agalychnis callidryas. The peptide was initially identified during smooth muscle pharmacological screening of skin secretion HPLC fractions and the unique primary structure—GMRPPWF-NH2—was established by both Edman degradation and electrospray MS/MS fragmentation sequencing. A. cDNA encoding the biosynthetic precursor of AcT-2 was successfully cloned from a skin secretion-derived cDNA library by means of RACE PCR and this contained an open-reading frame consisting of 62 amino acid residues with a single AcT-2 encoding sequence located towards the C-terminus. A synthetic replicate of AcT-2 was found to relax arterial smooth muscle (EC50 = 5.1 nM) and to contract rat urinary bladder smooth muscle (EC50 = 9.3 μM). The peptide could also inhibit the growth of the microorganisms, Staphylococcus aureus, (MIC = 256 mg/L) Escherichia coli (MIC = 512 mg/L), and Candida albicans (128 mg/L). AcT-2 is thus the first amphibian skin tryptophyllin found to possess both myotropic and antimicrobial activities.

Ornithokinin (avian bradykinin) from the skin of the Chinese bamboo odorous frog, Odorrana versabilis

One of the most widespread and abundant families of pharmacologically active peptides in amphibian defensive skin secretions is the bradykinins and related peptides. Despite retaining certain primary structural attributes that assign them to this peptide family, bradykinins and related peptides are unique among amphibian skin peptides in that they exhibit a wide range of primary structural variations, post-translational modifications and/or N-terminal or C-terminal extensions. Initially it was believed that their high degree of primary structural heterogeneity was reflective of random gene mutations within species, but latterly, there is an increasing body of evidence that the spectrum of structural modifications found within this peptide family is reflective of the vertebrate predator spectrum of individual species. Here we report the discovery of ornithokinin (avian bradykinin – Thr6, Leu8-bradykinin) in the skin secretion of the Chinese bamboo odorous frog, Odorrana versabilis. Molecular cloning of its biosynthetic precursor-encoding cDNA from a skin secretion-derived cDNA library revealed a deduced open-reading frame of 86 amino acid residues, encoding a single copy of ornithokinin towards its C-terminus. The domain architecture of this ornithokinin precursor protein was consistent with that of a typical amphibian skin peptide and quite different to that of the ornithokininogen from chicken plasma. Ornithokinin was reported to induce hypotension in the chicken and to contract the chicken oviduct but to have no obvious effect on the rat uterus. However, in this study, synthetic ornithokinin was found to contract the rat ileum (EC50 = 539 nM) and to increase contraction frequency in the rat uterus (EC50 = 1.87 μM).

Molecular Cloning of a Novel Tryptophyllin Peptide from the Skin of the Orange-Legged Monkey Frog, Phyllomedusa hypochondrialis

Tryptophyllins are a group of small (4–14 amino acids), heterogenous peptides, mostly from the skins of hylid frogs from the genera, Phyllomedusa and Litoria. To date, more than forty TPHs have been discovered in species from these two genera. Here, we describe the identification of a novel tryptophyllin type 3 peptide, PhT-3, from the extracts of skin of the orange-legged monkey frog, Phyllomedusa hypochondrialis, and molecular cloning of its precursor-encoding cDNA from a cDNA library constructed from the same skin sample. Full primary structural characterization was achieved using a combination of direct Edman degradation, mass spectrometry and deduction from cloned skin-derived cDNA. The open-reading frame of the precursor cDNA was found to consist of 63 amino acid residues. The mature peptide arising from this precursor contains a post-translationally modified N-terminal pyroglutamate (pGlu) residue, formed from acid-mediated cyclization of an N-terminal Gln (Q) residue, and with the structure: pGlu-Asp-Lys-Pro-Phe-Trp-Pro-Pro-Pro-Ile-Tyr-Pro-Met. Pharmacological assessment of a synthetic replicate of this peptide on phenylephrine preconstricted rat tail artery segments, revealed a reduction in relaxation induced by bradykinin. PhT-3 was also found to mediate antiproliferative effects on human prostate cancer cell lines.

Feleucins: Novel Bombinin Precursor-Encoded Nonapeptide Amides from the Skin Secretion of Bombina variegata

The first amphibian skin antimicrobial peptide (AMP) to be identified was named bombinin, reflecting its origin from the skin of the European yellow-bellied toad (Bombina variegata). Bombinins and their related peptides, the bombinin Hs, were subsequently reported from other bombinid toads. Molecular cloning of bombinin-encoding cDNAs from skin found that bombinins and bombinin Hs were coencoded on the same precursor proteins. Here, we report the molecular cloning of two novel cDNAs from a skin secretion-derived cDNA library of B. variegata whose open-reading frames each encode a novel bombinin (GIGGALLNVGKVALKGLAKGLAEHFANamide) and a C-terminally located single copy of a novel nonapeptide (FLGLLGGLLamide or FLGLIGSLLamide). These novel nonapeptides were named feleucin-BV1 and feleucin-BV2, respectively. The novel bombinin exhibited 89% identity to homologues from the toads, B. microdeladigitora and B. maxima. The feleucins exhibited no identity with any amphibian AMP archived in databases. Synthetic feleucins exhibited a weak activity against Staphylococcus aureus (128–256 mg/L) but feleucin-BV1 exhibited a synergistic action with the novel bombinin. The present report clearly demonstrates that the skin secretions of bombinid toads continue to represent a source of peptides of novel structure that could provide templates for the design of therapeutics.

AaeAP1 and AaeAP2: Novel Antimicrobial Peptides from the Venom of the Scorpion, Androctonus aeneas: Structural Characterisation, Molecular Cloning of Biosynthetic Precursor-Encoding cDNAs and Engineering of Analogues with Enhanced Antimicrobial and Anticancer Activities

The main functions of the abundant polypeptide toxins present in scorpion venoms are the debilitation of arthropod prey or defence against predators. These effects are achieved mainly through the blocking of an array of ion channel types within the membranes of excitable cells. However, while these ion channel-blocking toxins are tightly-folded by multiple disulphide bridges between cysteine residues, there are additional groups of peptides in the venoms that are devoid of cysteine residues. These non-disulphide bridged peptides are the subject of much research interest, and among these are peptides that exhibit antimicrobial activity. Here, we describe two novel non-disulphide-bridged antimicrobial peptides that are present in the venom of the North African scorpion, Androctonus aeneas. The cDNAs encoding the biosynthetic precursors of both peptides were cloned from a venom-derived cDNA library using 3'- and 5'-RACE strategies. Both translated precursors contained open-reading frames of 74 amino acid residues, each encoding one copy of a putative novel nonadecapeptide, whose primary structures were FLFSLIPSVIAGLVSAIRN and FLFSLIPSAIAGLVSAIRN, respectively. Both peptides were C-terminally amidated. Synthetic versions of each natural peptide displayed broad-spectrum antimicrobial activities, but were devoid of antiproliferative activity against human cancer cell lines. However, synthetic analogues of each peptide, engineered for enhanced cationicity and amphipathicity, exhibited increases in antimicrobial potency and acquired antiproliferative activity against a range of human cancer cell lines. These data clearly illustrate the potential that natural peptide templates provide towards the design of synthetic analogues for therapeutic exploitation.

Attenuation of urokinase activity during experimental ischaemia protects the cerebral barrier from damage through regulation of matrix metalloproteinase-2 and NAD(P)H oxidase

Ischaemic injury impairs the integrity of the blood-brain barrier (BBB). In this study, we investigated the molecular causes of this defect with regard to the putative correlations among NAD(P)H oxidase, plasminogen-plasmin system components, and matrix metalloproteinases. Hence, the activities of NAD(P)H oxidase, matrix metalloproteinase-2, urokinase-type plasminogen activator (uPA), and tissue-type plasminogen activator (tPA), and superoxide anion levels, were assessed in human brain microvascular endothelial cells (HBMECs) exposed to oxygen-glucose deprivation (OGD) alone or OGD followed by reperfusion (OGD + R). The integrity of an in vitro model of BBB comprising HBMECs and astrocytes was studied by measuring transendothelial electrical resistance and the paracellular flux of albumin. OGD with or without reperfusion (OGD ± R) radically perturbed barrier function while concurrently enhancing uPA, tPA and NAD(P)H oxidase activities and superoxide anion release in HBMECs. Pharmacological inactivation of NAD(P)H oxidase attenuated OGD ± R-mediated BBB damage through modulation of matrix metalloproteinase-2 and tPA, but not uPA activity. Overactivation of NAD(P)H oxidase in HBMECs via cDNA electroporation of its p22-phox subunit confirmed the involvement of tPA in oxidase-mediated BBB disruption. Interestingly, blockade of uPA or uPA receptor preserved normal BBB function by neutralizing both NAD(P)H oxidase and matrix metalloproteinase-2 activities. Hence, selective targeting of uPA after ischaemic strokes may protect cerebral barrier integrity and function by concomitantly attenuating basement membrane degradation and oxidative stress.

A Review on Bradykinin-Related Peptides Isolated from Amphibian Skin Secretion

Amphibian skin secretion has great potential for drug discovery and contributes hundreds of bioactive peptides including bradykinin-related peptides (BRPs). More than 50 BRPs have been reported in the last two decades arising from the skin secretion of amphibian species. They belong to the families Ascaphidae (1 species), Bombinatoridae (3 species), Hylidae (9 speices) and Ranidae (25 species). This paper presents the diversity of structural characteristics of BRPs with N-terminal, C-terminal extension and amino acid substitution. The further comparison of cDNA-encoded prepropeptides between the different species and families demonstrated that there are various forms of kininogen precursors to release BRPs and they constitute important evidence in amphibian evolution. The pharmacological activities of isolated BRPs exhibited unclear structure–function relationships, and therefore the scope for drug discovery and development is limited. However, their diversity shows new insights into biotechnological applications and, as a result, comprehensive and systematic studies of the physiological and pharmacological activities of BRPs from amphibian skin secretion are needed in the future.

Transcriptome analysis of a parasitic clade V nematode:comparative analysis of potential molecular anthelmintic targets in Cylicostephanus goldi

Clade V nematodes comprise several parasitic species that include the cyathostomins, primary helminth pathogens of horses. Next generation transcriptome datasets are available for eight parasitic clade V nematodes, although no equine parasites are included in this group. Here, we report next generation transcriptome sequencing analysis for the common cyathostomin species, Cylicostephanus goldi. A cDNA library was generated from RNA extracted from 17 C. goldi male and female adult parasites. Following sequencing using a 454 GS FLX pyrosequencer, a total of 475,215 sequencing reads were generated, which were assembled into 26,910 contigs. Using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases, 27% of the transcriptome was annotated. Further in-depth analysis was carried out by comparing the C. goldi dataset with the next generation transcriptomes and genomes of other clade V nematodes, with the Oesophagostomum dentatum transcriptome and the Haemonchus contortus genome showing the highest levels of sequence identity with the cyathostomin dataset (45%). The C. goldi transcriptome was mined for genes associated with anthelmintic mode of action and/or resistance. Sequences encoding proteins previously associated with the three major anthelmintic classes used in horses were identified, with the exception of the P-glycoprotein group. Targeted resequencing of the glutamate gated chloride channel α4 subunit (glc-3), one of the primary targets of the macrocyclic lactone anthelmintics, was performed for several cyathostomin species. We believe this study reports the first transcriptome dataset for an equine helminth parasite, providing the opportunity for in-depth analysis of these important parasites at the molecular level. Sequences encoding enzymes involved in key processes and genes associated with levamisole/pyrantel and macrocyclic lactone resistance, in particular the glutamate gated chloride channels, were identified. This novel data will inform cyathostomin biology and anthelmintic resistance studies in future.

Recombinant Subgroup B Human Respiratory Syncytial Virus Expressing Enhanced Green Fluorescent Protein Efficiently Replicates in Primary Human Cells and Is Virulent in Cotton Rats

Human respiratory syncytial virus (HRSV) is the most important viral cause of severe respiratory tract disease in infants. Two subgroups (A and B) have been identified, which cocirculate during, or alternate between, yearly epidemics and cause indistinguishable disease. Existing in vitro and in vivo models of HRSV focus almost exclusively on subgroup A viruses. Here, a recombinant (r) subgroup B virus (rHRSV(B05)) was generated based on a consensus genome sequence obtained directly from an unpassaged clinical specimen from a hospitalized infant. An additional transcription unit containing the gene encoding enhanced green fluorescent protein (EGFP) was introduced between the phosphoprotein and matrix genes (position 5) of the genome to generate rHRSV(B05)EGFP(5). The recombinant viruses replicated efficiently in both HEp-2 cells and in well-differentiated normal human bronchial cells grown at air-liquid interface. Intranasal infection of cotton rats (Sigmodon hispidus) resulted in high numbers of EGFP(+) cells in epithelia of the nasal septum and conchae. When administered in a relatively large inoculum volume, the virus also replicated efficiently in bronchiolar epithelial cells and spread extensively in both the upper and lower respiratory tracts. Virus replication was not observed in ciliated epithelial cells of the trachea. This is the first virulent rHRSV strain with the genetic composition of a currently circulating wild-type virus. In vivo tracking of infected cells by means of EGFP fluorescence in the absence of cytopathic changes increases the sensitivity of virus detection in HRSV pathogenesis studies.

IMPORTANCE

Virology as a discipline has depended on monitoring cytopathic effects following virus culture in vitro. However, wild-type viruses isolated from patients often do not cause significant changes to infected cells, necessitating blind passage. This can lead to genetic and phenotypic changes and the generation of high-titer, laboratory-adapted viruses with diminished virulence in animal models of disease. To address this, we determined the genome sequence of an unpassaged human respiratory syncytial virus from a sample obtained directly from an infected infant, assembled a molecular clone, and recovered a wild-type recombinant virus. Addition of a gene encoding enhanced green fluorescent protein allowed this wild-type virus to be tracked in primary human cells and living animals in the absence of significant cytopathic effects. Imaging of fluorescent cells proved to be a highly valuable tool for monitoring the spread of virus and may help improve assays for evaluating novel intervention strategies.

pLR-HL: a novel amphibian Bowman-Birk-type trypsin inhibitor from the skin secretion of the broad-folded frog, Hylarana latouchii

In this study, we report a novel heptadecapeptide (LIGGCWTKSIPPKPCLV) of the pLR/ranacyclin family, named pLR-HL, whose structure was deduced from its biosynthetic precursor-encoding cDNA cloned from the skin secretion-derived cDNA library of the broad-folded frog, Hylarana latouchii, by employing a "shotgun" cloning technique. It contains a disulphide loop between Cys5 and Cys15 which is consistent with Bowman-Birk-type protease inhibitors. The primary structure of pLR-HL deduced from the cDNA sequence was confirmed by fractionating the skin secretion using reverse phase HPLC and subsequent analysis using MALDI-TOF mass spectrometry and LC/MS/MS fragmentation sequencing. On the basis of the establishment of unequivocal amino acid sequence, a synthetic replicate was synthesised by solid-phase Fmoc chemistry, and it displayed a moderately potent trypsin inhibition with a Ki of 143 nM. The substitution of Lys-8 by Phe (Phe8 -pLR-HL) resulted in abolition of trypsin inhibition but generation of modest inhibition on chymotrypsin with a Ki of 2.141 μM. Additionally, both the disulphide loops of pLR-HL and Phe8 -pLR-HL were synthesised and tested. Both of the catalytic loops retained similar inhibitory potencies towards trypsin or chymotrypsin in comparison with the original intact molecules. Thus, the replacement of reactive site residues could alter the specificity of these protease inhibitors, while the canonical reactive loop alone can independently constitute biologically-active moiety.

PH-sauvagine from the skin secretion of Phyllomedusa hypochondrialis: a novel CRF-like peptide with smooth muscle contraction activity

Amphibian skin, and particularly that of south/Central American phyllomedusine frogs, is supposed to be "a huge factory and store house of a variety of active peptides". The 40 amino acid amphibian CRF-like peptide, sauvagine, is a prototype member of a unique family of these Phyllomedusa skin peptides. In this study, we describe for the first time the structure of a mature novel peptide from the skin secretion of the South American orange-legged leaf frog, Phyllomedusa hypochondrialis, which belongs to the amphibian CRF/sauvagine family. Partial amino acid sequence from the N-terminal was obtained by automated Edman degradation with the following structure: pGlu-GPPISIDLNMELLRNMIEI-. The biosynthetic precursor of this novel sauvagine peptide, consisted of 85 amino acid residues and was deduced from cDNA library constructed from the same skin secretion. Compared with the standard sauvagine from the frog, Phyllomedusa sauvagei, this novel peptide was found to exert similar contraction effects on isolated guinea-pig colon and rat urinary bladder smooth muscle preparations.

Uncovering functionally relevant signaling pathways using microarray-based expression profiling

The introduction of microarray technology to the scientific and medical communities has fundamentally altered the way in which we now address basic biomedical questions. Microarrays technology facilitates a more complete and inclusive experimental approach where alterations in the transcript level of entire genomes can be simultaneously assayed in response to a variety of stimuli. Conceptually different approaches to the development of microarray technology have resulted in the generation of two different array formats: oligonucleotide arrays and cDNA arrays. The application of microarray and related technologies to identify specific targets of defined genes that have clearly been implicated in cancer progression requires a specific experimental approach. The objective of tiffs approach is to define changes in transcriptional profile that occur in response to modulating the expression level of the gene to be studied. The resulting altered expression profile can then be viewed as a blueprint by which that gene effects its cellular function. We have used oligonucleotide array-based expression profiling in collaboration with Affymetrix to identify downstream transcriptional targets of the BRCA1 tumor-suppressor gene as a means of defining its function. BRCA1 has been implicated in at least three functional pathways, namely, mediating the cellular response to DNA damage, as a cell cycle checkpoint protein and in the regulation of transcription. The physiological significance of these properties and their implications for the function of BRCA1 as a tumor-suppressor gene remain to be defined.

Mycosis fungoides shows concurrent deregulation of multiple genes involved in the TNF signaling pathway:an expression profile study

Mycosis fungoides (MF) is the most frequent type of cutaneous T-cell lymphoma, whose diagnosis and study is hampered by its morphologic similarity to inflammatory dermatoses (ID) and the low proportion of tumoral cells, which often account for only 5% to 10% of the total tissue cells. cDNA microarray studies using the CNIO OncoChip of 29 MF and 11 ID cases revealed a signature of 27 genes implicated in the tumorigenesis of MF, including tumor necrosis factor receptor (TNFR)-dependent apoptosis regulators, STAT4, CD40L, and other oncogenes and apoptosis inhibitors. Subsequently a 6-gene prediction model was constructed that is capable of distinguishing MF and ID cases with unprecedented accuracy. This model correctly predicted the class of 97% of cases in a blind test validation using 24 MF patients with low clinical stages. Unsupervised hierarchic clustering has revealed 2 major subclasses of MF, one of which tends to include more aggressive-type MF cases including tumoral MF forms. Furthermore, signatures associated with abnormal immunophenotype (11 genes) and tumor stage disease (5 genes) were identified.

A role for neurotensin in bicalutamide resistant prostate cancer cells

BACKGROUND: Anti-androgens are administered as a principal treatment for prostate cancer. Aggressive hormone refractory disease is characterized in some cases by the development of a neuroendocrine phenotype. However little attention has been paid to resistance pathways selected for by long-term treatment with non-steroidal anti-androgens.

METHODS: Using a resistant sub-line, LNCaP-Bic, we performed a comparative gene expression profiling using cDNA microarrays and target validation by qRT-PCR. Targets were then explored using cell proliferation, cell cycle analysis and in vitro invasion assays using siRNA technology.

RESULTS: Neurotensin/Neuromedin N (NTS) was upregulated in the LNCaP-Bic line at both the transcript and protein level. The resistant line was found to have an increased proliferation rate, more rapid cell cycle progression and increased invasiveness through Matrigel. Each phenotypic difference could be reduced using siRNA knockdown of NT.

CONCLUSION: Increased expression of NT in bicalutamide resistant prostate cancer cells induces cell proliferation and invasion suggesting that this peptide may contribute to the development of bicalutamide resistant prostate cancer.