Mammalian Sequences Research Articles

124 Functional analysis of porcine OCT4 transcriptional regulatory region-based reporter system

Gene OCT4 plays pivotal roles in maintaining pluripotency of early mammalian embryonic development and embryonic stem cells. It is essential to establish a reporter system based on the OCT4 promoter region for the study of pluripotency. However, there is still a lack of sufficient information about the porcine OCT4 upstream reporter system. To improve our understanding of the porcine OCT4 regulatory region, first, we conducted an investigation to find conserved regions in the porcine OCT4 promoter upstream region by sequence-based comparative analysis using various mammalian genome sequences. A similarity of nucleotide sequences of the 5′ upstream region was low among mammalian species. However, the OCT4 promoter and 4 regulatory regions including distal and proximal enhancer elements have a high similarity. Next, a functional analysis of the porcine OCT4 promoter region was conducted. Luciferase reporter assay indicated that the porcine OCT4 distal enhancer and proximal enhancer are highly activated in mouse embryonic stem cells and embryonic carcinoma cells, respectively (n=3). Comparison analysis of naïve (Tbx3, Nr0b1, Rex1, Esrrb, Nanog, Klf2) or primed (Gata6, Mixl1, Fgf5, Otx2) state marker gene expression in a dual-reporter assay using pOCT4-DE-eGFP and pOCT4-PE-DsRed2 showed that expression of naïve and primed markers were up-regulated in cells with high green fluorescent protein and red fluorescent protein expression, respectively (n=3). Porcine OCT4-upstream region-based reporter constructs showed exclusive expression patterns depending on the state of pluripotency. This work could provide basic information for the porcine OCT4 upstream region and the various porcine OCT4-fluorescence reporter constructs, which can be applied to study species-specific pluripotency in early embryo development and for the establishment of embryonic stem cells in pigs. This work was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through the Development of High Value-Added Food Technology Program, funded by the Ministry of Agriculture, Food and Rural Affairs (MAFRA, 118042-03-1-HD020).

Targeted Protection of Mitochondria of Mesophyll Cells in Transgenic CYP11A1 CDNA Expressing Tobacco Plant Leaves After NaCl-Induced Stress Damage

Abstract Recently we have showed that the expression of the mammalian CYP11A1 cDNA in plants confers their resistance to abiotic and biotic stresses. To determine the role of heterologous expression of cytochrome P450scc cDNA in resistance to ROS (radical oxygen species) dependent abiotic stresses, the structural changes of mitochondria and peroxisomes were studied under 150 mM NaCl-induced 14-day salinity treatment on juvenile tobacco plants in in vitro culture. Ultrastructural analysis of mesophyll cells of transgenic tobacco leaves constitutively expressing CYP11A1 cDNA was performed. Under NaCl stress, a change in shape from rounded to elon-gated, reduced section area, formation of branched mitochondria, as well as the emergence of triangular and rhomboid cristae, densification of a mitochondrial matrix, increase in density of contrasting membranes and their thickness were observed in non-transgenic plants. Transgenic plants without stress applied had mitochondria with rounded and elongated shape, twice as small as in non-transgenic plants, with a dense matrix and sinuous cristae. Surprisingly, the effect of NaCl led to increase in size of mitochondria by 1.5 times, decomposition of matrix and the emergence in organelles of light zones presumably containing mitochondrial DNA strands. Thus, the structural organisation of transgenic plant mitochondria under salinity treatment was comparable to that of non-transgenic plants under native conditions. It was also noted that the transgenic plant peroxisomes differed in non-transgenic tobacco both in normal condition and under the action of NaCl. The observed differences in ultrastructural organisation of mitochondria not only support our earlier notion about successful incorporation of the mature P450scc into this organelle, but for the first time demonstrate that the mammalian CYP11A1 signal peptide sequence could be efficiently used in the formation of targeted mitochondria protection of plants from salinity-induced damage.

BFPI: A Minimal Prothrombinase Inhibitor from the Saliva of Simulium Vittatum

Background: The black fly, Simulium Vittatum, has an anticoagulant protein in its saliva that allows it to feed on mammalian blood (black fly protease inhibitor; BFPI). Remarkably, BFPI is similar to the human anticoagulant tissue factor pathway inhibitor alpha (TFPIα). TFPIα contains three Kunitz-type protease inhibitor domains (K1, K2, K3), which inhibit factor VIIa (FVIIa) and factor Xa (FXa) and bind the co-factor protein S (PS), respectively; BFPI contains a single Kunitz domain that inhibits FXa. In addition, TFPIα and BFPI contain homologous basic regions (BRs) near their C-termini (252LIKTKRKRKK261 in human TFPIα, LIKTRKRKPKK in BFPI). The TFPIα BR binds a regulatory acidic region (AR) in factor Va (FVa). The AR is present in forms of FVa released by collagen-activated platelets and generated through limited proteolysis by FXa (FVaXa), and is removed by thrombin (FVaIIa). We hypothesized that BFPI, through its Kunitz domain and basic C-terminus, inhibits early forms of the prothrombinase complex, but does not possess the other inhibitory functions of TFPIα: (1) K1-dependent inhibition of the tissue factor (TF)-FVIIa complex; and (2) PS/K3-dependent FXa inhibition.Results: Recombinant BFPI inhibited FXa in an amidolytic activity assay, and PS did not promote this inhibition. BFPI did not inhibit TF-FVIIa-mediated FX activation. As described with TFPIα, FV promoted FXa inhibition by BFPI but FVaIIa did not, suggesting that the BFPI BR is capable of binding the FVa AR. In a purified protein assay, BFPI inhibited prothrombinase assembled with FVaXa (IC50=4.9nM), but not FVaIIa. Similarly, 5nM BFPI increased the lag time for FXa-initiated plasma thrombin generation by 10.4±1.5%.We next used BFPI as a backbone to evaluate a reported human mutation in the TFPIα BR, K254E. Every mammalian, avian, or reptilian TFPIα sequence available contains either a Lys or Arg residue at this position, suggesting that this residue is functionally important. In purified protein assays, BFPI-K254E inhibited FXa amidolytic activity identically to BFPI, but FV did not promote this inhibition, suggesting that BFPI-K254E has a specific defect in its interaction with FV. Consistent with this, BFPI-K254E was a weaker inhibitor of prothrombinase assembled with FVaXa (IC50 = 15.8nM) and FXa-initiated plasma thrombin generation.The results obtained with BFPI-K254E were confirmed using peptides and full-length TFPIα proteins. First, a peptide mimicking the wild type TFPIα BR (LIKTKRKRKK) inhibited prothrombinase assembled with FVaXa (IC50 = 1.0 µM), while the substituted peptide (LIETKRKRKK) was substantially weaker (20% inhibition observed with 340 µM peptide). Second, full-length TFPIα-K254E was a weaker inhibitor of prothrombinase containing FXa-activated FVa (IC50 = 14.8 nM, vs. 1.8 nM for TFPIα) and had greatly reduced anticoagulant activity in plasma-based thrombin generation assays.Conclusions: In summary, the anticoagulant effect of BFPI is mediated through inhibition of early forms of prothrombinase, independent of TF-FVIIa inhibition or PS-dependent FXa inhibition. The natural mutation TFPIα K254E disrupts prothrombinase inhibition, despite the presence of six other conserved basic residues, and is thus procoagulant in human plasma. The absolute conservation of the TFPIα BR, and its usurpation to allow feeding by black flies, point to formation of the initial prothrombinase complex as a key regulatory step in blood coagulation. DisclosuresMast:Novo Nordisk: Research Funding.

Genome classification improvements based on k-mer intervals in sequences

Given the vast amount of genomic data, alignment-free sequence comparison methods are required due to their low computational complexity. k-mer based methods can improve comparison accuracy by extracting an effective feature of the genome sequences. The aim of this paper is to extract k-mer intervals of a sequence as a feature of a genome for high comparison accuracy. In the proposed method, we calculated the distance between genome sequences by comparing the distribution of k-mer intervals. Then, we identified the classification results using phylogenetic trees. We used viral, mitochondrial (MT), microbial and mammalian genome sequences to perform classification for various genome sets. We confirmed that the proposed method provides a better classification result than other k-mer based methods. Furthermore, the proposed method could efficiently be applied to long sequences such as human and mouse genomes.

Identification of duck liver-expressed antimicrobial peptide 2 and characterization of its bactericidal activity.

ObjectiveThis study was conducted to identify duck liver-expressed antimicrobial peptide 2 (LEAP-2) and demonstrate its antimicrobial activity against various pathogens.MethodsTissue samples were collected from 6 to 8-week-old Pekin ducks (Anas platyrhynchos domesticus), total RNA was extracted, and cDNA was synthesized. To confirm the duck LEAP-2 transcript expression levels, quantitative real-time polymerase chain reaction was conducted. Two kinds of peptides (a linear peptide and a disulfide-type peptide) were synthesized to compare the antimicrobial activity. Then, antimicrobial activity assay and fluorescence microscopic analysis were conducted to demonstrate duck LEAP-2 bactericidal activity.ResultsThe duck LEAP-2 peptide sequence showed high identity with those of other avian species (>85%), as well as more than 55% of identity with mammalian sequences. LEAP-2 mRNA was highly expressed in the liver with duodenum next, and then followed by lung, spleen, bursa and jejunum and was the lowest in the muscle. Both of LEAP-2 peptides efficiently killed bacteria, although the disulfide-type LEAP-2 showed more powerful bactericidal activity. Also, gram-positive bacteria was more susceptible to duck LEAP-2 than gram-negative bacteria. Using microscopy, we confirmed that LEAP-2 peptides could kill bacteria by disrupting the bacterial cell envelope.ConclusionDuck LEAP-2 showed its antimicrobial activity against both gram-positive and gram-negative bacteria. Disulfide bonds were important for the powerful killing effect by disrupting the bacterial cell envelope. Therefore, duck LEAP-2 can be used for effective antibiotics alternatives.

Characterization, tissue distribution of apela and periprandial, fasting and refeeding changes of apela mRNA in Siberian sturgeon Acipenser baerii.

Apela identified from zebrafish Danio rerio for the first time in 2013 is a novel endogenous peptide ligand for the apelin receptor. To study the role of apela in regulating fish feeding, the complementary (c) DNA sequence of apela of Siberian sturgeon Acipenser baerii was cloned for the first time. The apela cDNA fragment of 836 bp was obtained by cloning. The open reading frame (ORF) of apela was 165 bp encoding a 54 amino acid, including 22 amino acids signal peptide and two proteolytic sites. Phylogenetic tree analysis showed that A. baerii apela was clustered with mammalian and amphibian sequences. A. baerii apela messeger (m)RNA was widely distributed in 11 tissues related to feeding, with high expressions in brain, oesophagus and stomach, especially in the brain. The level of apela mRNA in brain increased significantly after feeding. On the first day of fasting, apela expression in brain was significantly lower than that of the fed group, but after fasting for 3-15 days, the expression of apela in A. baerii brain was significantly higher than that in the fed group. After refeeding apela mRNA expression was obviously reduced. These results suggest that apela plays a bidirectional role in feeding regulation of A. baerii, which may serve as a short-term satiation factor and a long-term hunger factor.

Correlated Selection on Amino Acid Deletion and Replacement in Mammalian Protein Sequences.

A low ratio of nonsynonymous and synonymous substitution rates (dN/dS) at a codon is an indicator of functional constraint caused by purifying selection. Intuitively, the functional constraint would also be expected to prevent such a codon from being deleted. However, to the best of our knowledge, the correlation between the rates of deletion and substitution has never actually been estimated. Here, we use 8595 protein-coding region sequences from nine mammalian species to examine the relationship between deletion rate and dN/dS. We find significant positive correlations at the levels of both sites and genes. We compared our data against controls consisting of simulated coding sequences evolving along identical phylogenetic trees, where deletions occur independently of substitutions. A much weaker correlation was found in the corresponding simulated sequences, probably caused by alignment errors. In the real data, the correlations cannot be explained by alignment errors. Separate investigations on nonsynonymous (dN) and synonymous (dS) substitution rates indicate that the correlation is most likely due to a similarity in patterns of selection rather than in mutation rates.

Cone Snail Glutaminyl Cyclase Sequences from Transcriptomic Analysis and Mass Spectrometric Characterization of Two Pyroglutamyl Conotoxins.

The post-translational modification of N-terminal glutamine (Q) to a pyroglutamyl (Z) residue is observed in the conotoxins produced by marine cone snails. This conversion requires the action of the enzyme glutaminyl cyclase (QC). Four complete QC sequences from the species C. araneosus, C. frigidus, C. litteratus, and C. monile and two partial sequences from C. amadis and C. miles have been obtained by analysis of transcriptomic data. Comparisons with mammalian enzyme sequences establish a high level of identity and complete conservation of functional active site residues, including a cluster of hydrogen-bonded acidic side chains. Mass spectrometric analysis of crude venom samples coupled to conotoxin precursor protein sequences obtained from transcriptomic data establishes the presence of pyroglutamyl conotoxins in the venom of C. frigidus and C. amadis. The C. frigidus peptide belongs to the M superfamily, with cysteine framework III, whereas the C. amadis peptide belongs to the divergent superfamily with cysteine framework VI/VII. Additionally, gamma carboxylation of glutamic acid and hydroxylation of proline are observed in the C. frigidus peptide. Mass spectral data are available via ProteomeXchange with identifier PXD009006.

Lineage-associated underrepresented permutations (LAUPs) of mammalian genomic sequences based on a Jellyfish-based LAUPs analysis application (JBLA).

This study addresses several important questions related to naturally underrepresented sequences: (i) are there permutations of real genomic DNA sequences in a defined length (k-mer) and a given lineage that do not actually exist or underrepresented? (ii) If there are such sequences, what are their characteristics in terms of k-mer length and base composition? (iii) Are they related to CpG or TpA underrepresentation known for human sequences? We propose that the answers to these questions are of great significance for the study of sequence-associated regulatory mechanisms, such cytosine methylation and chromosomal structures in physiological or pathological conditions such as cancer. We empirically defined sequences that were not included in any well-known public databases as lineage-associated underrepresented permutations (LAUPs). Then, we developed a Jellyfish-based LAUPs analysis application (JBLA) to investigate LAUPs for 24 representative species. The present discoveries include: (i) lengths for the shortest LAUPs, ranging from 10 to 14, which collectively constitute a low proportion of the genome. (ii) Common LAUPs showing higher CG content over the analysed mammalian genome and possessing distinct CG*CG motifs. (iii) Neither CpG-containing LAUPs nor CpG island sequences are randomly structured and distributed over the genomes; some LAUPs and most CpG-containing sequences exhibit an opposite trend within the same k and n variants. In addition, we demonstrate that the JBLA algorithm is more efficient than the original Jellyfish for computing LAUPs. We developed a Jellyfish-based LAUP analysis (JBLA) application by integrating Jellyfish (Marçais and Kingsford, 2011), MEME (Bailey, et al., 2009) and the NCBI genome database (Pruitt, et al., 2007) applications, which are listed as Supplementary Material. Supplementary data are available at Bioinformatics online.

Characterization of the Pyrimidine Catabolic Pathway of Oryza sativa

ObjectiveTo characterize plant pyrimidine catabolic enzymes and to identify potential candidate for additional enzymes participating in the pathwayRice is a primary global food source and is an important crop in Colombia. Its production is affected by abiotic stress, caused by climate change and other factors. Recently, genes of the pyrimidine catabolic pathway have emerged as potential participants in the abiotic stress response of plants.In mammals, three enzymes are involved in the degradation of uracil or thymine. The first is dihydropyrimidine dehydrogenase (DHPD), the second is dihydropyrimidinase (DHP) and the last is β‐ureidopropionase (β‐UP). Liu and coworkers (2009) found that overexpression of DHPD in rice plants (OsDHPD) results in tolerance to salt and drought stress, however, in that study OsDHPD was misidentified as a type I dihydroorotate dehydrogenase. DHP appears to be involved in cold stress‐response in grass (Xuan et al., 2013). In addition, the overexpression of β‐UP in transgenic tomatoes plants made them resistant to heat during anthesis (Shen et al., 2014). The role of pyrimidine catabolism in stress tolerance could be related to this pathway's production of precursors for proline biosynthesis. In plants, a complete characterization of the pathway is lacking. Comparison of plant and mammalian DHPD sequences showed that the plant protein lacks a domain needed for binding NADH(P), FAD, and Fe/S clusters, suggesting that another, unidentified, partner protein should be interacting with OsDHPD.ResultsWe cloned and expressed three enzymes of the pathway, OsDHPD, OsDHP and Osβ‐UP. The third enzyme, Osβ‐UP, was soluble, and preliminary kinetics revealed a Km 0.85 ± 0.11 mM for 3‐ureidopropionic acid, and a specific activity apparently 10‐fold faster (Vmax 384.3 ± 12.0 nmol/min/mg) than the Arabidopsis thaliana recombinant enzyme (30 nmol/min/mg) (Walsh et al., 2001). Truncations lacking chloroplast localization signals were required to produce soluble recombinant proteins of the first two enzymes. Truncated OsDHPD, was soluble, but inactive, presumably because it requires a partner protein for catalysis. By screening the rice database for proteins with the appropriate combination of cofactors we identified two potential candidates that may act as partners of OsDHPD; the first was a salt‐induced chloroplast ferredoxin, and the second was a rice chloroplast “fruit protein”. Both were cloned and expressed, and testing of interactions with OsDHPD on native gels are in progress. In addition, we produced polyclonal antibodies to OsDHPD to permit pull‐down assays of chloroplast extracts.ConclusionsThree rice pyrimidine catabolic enzymes were cloned, expressed and purified. Potential candidates for the missing protein in the pathway were identified using bioinformatics tools, and antibodies to OsDHPD were generated. Together these approaches may allow identification of the missing participant in pyrimidine catabolism in plants.Support or Funding InformationThe research was supported by funding from Colciencias project 120471250531, and from the Facultad de Ciencias and the Vicerrectoria de Investigaciones (Universidad de los Andes, Colombia).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Alternative miRNAs? Human sequences misidentified as plant miRNAs in plant studies and in human plasma

Background: A 2017 study reported that “Plant miRNAs found in human circulating system provide evidences of cross kingdom RNAi”. Analysis of two human blood plasma sequencing datasets was said to provide evidence for uptake of plant miRNAs into human plasma. The results were also purportedly inconsistent with contamination. Methods: Sequences from public datasets and miRNA databases were compared with results downloaded from the website of the reporting journal. Results: Only one putative plant miRNA (“peu-MIR2910) mapped consistently above background, and this sequence is found with 100% identity in a human rRNA. Several other rarer but consistently mapped putative plant miRNAs also have 100% or near 100% matches to human transcripts or genomic sequences, and some do not appear to map to plant genomes at all. Conclusions: Reanalysis of public data suggests that dietary plant xenomiR uptake is not supported, but instead confirms previous findings that detection of rare plant miRNAs in mammalian sequencing datasets is artifactual. Some putative plant miRNAs, including MIR2910 and MIR2911, may represent human sequence contamination or other artifacts in plant studies, emphasizing the need for rigorous controls and data filtering strategies when assessing possible xenomiRNAs.

Alternative miRNAs? Human sequences misidentified as plant miRNAs in plant studies and in human plasma.

Background: A 2017 study reported that "Plant miRNAs found in human circulating system provide evidences of cross kingdom RNAi". Analysis of two human blood plasma sequencing datasets was said to provide evidence for uptake of plant miRNAs into human plasma. The results were also purportedly inconsistent with contamination. Methods: Sequences from public datasets and miRNA databases were compared with results downloaded from the website of the reporting journal. Results: Only one putative plant miRNA ("peu-MIR2910) mapped consistently above background, and this sequence is found with 100% identity in a human rRNA. Several other rarer but consistently mapped putative plant miRNAs also have 100% or near 100% matches to human transcripts or genomic sequences, and some do not appear to map to plant genomes at all. Conclusions: Reanalysis of public data suggests that dietary plant xenomiR uptake is not supported, but instead confirms previous findings that detection of rare plant miRNAs in mammalian sequencing datasets is artifactual. Some putative plant miRNAs, including MIR2910 and MIR2911, may represent human sequence contamination or other artifacts in plant studies, emphasizing the need for rigorous controls and data filtering strategies when assessing possible xenomiRNAs.

TFClass: expanding the classification of human transcription factors to their mammalian orthologs.

TFClass is a resource that classifies eukaryotic transcription factors (TFs) according to their DNA-binding domains (DBDs), available online at http://tfclass.bioinf.med.uni-goettingen.de. The classification scheme of TFClass was originally derived for human TFs and is expanded here to the whole taxonomic class of mammalia. Combining information from different resources, checking manually the retrieved mammalian TFs sequences and applying extensive phylogenetic analyses, >39 000 TFs from up to 41 mammalian species were assigned to the Superclasses, Classes, Families and Subfamilies of TFClass. As a result, TFClass now provides the corresponding sequence collection in FASTA format, sequence logos and phylogenetic trees at different classification levels, predicted TF binding sites for human, mouse, dog and cow genomes as well as links to several external databases. In particular, all those TFs that are also documented in the TRANSFAC® database (FACTOR table) have been linked and can be freely accessed. TRANSFAC® FACTOR can also be queried through an own search interface.

Role of TRP Channels in Dinoflagellate Mechanotransduction.

Transient receptor potential (TRP) ion channels are common components of mechanosensing pathways, mainly described in mammals and other multicellular organisms. To gain insight into the evolutionary origins of eukaryotic mechanosensory proteins, we investigated the involvement of TRP channels in mechanosensing in a unicellular eukaryotic protist, the dinoflagellate Lingulodinium polyedra. BLASTP analysis of the protein sequences predicted from the L. polyedra transcriptome revealed six sequences with high similarity to human TRPM2, TRPM8, TRPML2, TRPP1, and TRPP2; and characteristic TRP domains were identified in all sequences. In a phylogenetic tree including all mammalian TRP subfamilies and TRP channel sequences from unicellular and multicellular organisms, the L. polyedra sequences grouped with the TRPM, TPPML, and TRPP clades. In pharmacological experiments, we used the intrinsic bioluminescence of L. polyedra as a reporter of mechanoresponsivity. Capsaicin and RN1734, agonists of mammalian TRPV, and arachidonic acid, an agonist of mammalian TRPV, TRPA, TRPM, and Drosophila TRP, all stimulated bioluminescence in L. polyedra. Mechanical stimulation of bioluminescence, but not capsaicin-stimulated bioluminescence, was inhibited by gadolinium (Gd3+), a general inhibitor of mechanosensitive ion channels, and the phospholipase C (PLC) inhibitor U73122. These pharmacological results are consistent with the involvement of TRP-like channels in mechanosensing by L. polyedra. The TRP channels do not appear to be mechanoreceptors but rather are components of the mechanotransduction signaling pathway and may be activated via a PLC-dependent mechanism. The presence and function of TRP channels in a dinoflagellate emphasize the evolutionary conservation of both the channel structures and their functions.

Characterization and distribution of GHRH, PACAP, TRH, SST and IGF1 mRNAs in the green iguana

The somatotropic axis (SA) regulates numerous aspects of vertebrate physiology such as development, growth, and metabolism and has influence on several tissues including neural, immune, reproductive and gastric tract. Growth hormone (GH) is a key component of SA, it is synthesized and released mainly by pituitary somatotrophs, although now it is known that virtually all tissues can express GH, which, in addition to its well-described endocrine roles, also has autocrine/paracrine/intracrine actions. In the pituitary, GH expression is regulated by several hypothalamic neuropeptides including GHRH, PACAP, TRH and SST. GH, in turn, regulates IGF1 synthesis in several target tissues, adding complexity to the system since GH effects can be exerted either directly or mediated by IGF1. In reptiles, little is known about the SA components and their functional interactions. The aim of this work was to characterize the mRNAs of the principal SA components in the green iguana and to develop the tools that allow the study of the structural and functional evolution of this system in reptiles. By employing RT-PCR and RACE, the cDNAs encoding for GHRH, PACAP, TRH, SST and IGF1 were amplified and sequenced. Results showed that these cDNAs coded for the corresponding protein precursors of 154, 170, 243, 113, and 131 amino acids, respectively. Of these, GHRH, PACAP, SST and IGF1 precursors exhibited a high structural conservation with respect to its counterparts in other vertebrates. On the other hand, iguana's TRH precursor showed 7 functional copies of mature TRH (pyr-QHP-NH2), as compared to 4 and 6 copies of TRH in avian and mammalian proTRH sequences, respectively. It was found that in addition to its primary production site (brain for GHRH, PACAP, TRH and SST, and liver for IGF1), they were also expressed in other peripheral tissues, i.e. testes and ovaries expressed all the studied mRNAs, whereas TRH and IGF1 mRNAs were observed ubiquitously in all tissues considered. These results show that the main SA components in reptiles of the Squamata Order maintain a good structural conservation among vertebrate phylogeny, and suggest important physiological interactions (endocrine, autocrine and/or paracrine) between them due to their wide peripheral tissue expression.

A prokaryotic viral sequence is expressed and conserved in mammalian brain

A natural and permanent transfer of prokaryotic viral sequences to mammals has not been reported by others. Circular "SPHINX" DNAs <5 kb were previously isolated from nuclease-protected cytoplasmic particles in rodent neuronal cell lines and brain. Two of these DNAs were sequenced after Φ29 polymerase amplification, and they revealed significant but imperfect homology to segments of commensal Acinetobacter phage viruses. These findings were surprising because the brain is isolated from environmental microorganisms. The 1.76-kb DNA sequence (SPHINX 1.8), with an iteron before its ORF, was evaluated here for its expression in neural cells and brain. A rabbit affinity purified antibody generated against a peptide without homology to mammalian sequences labeled a nonglycosylated ∼41-kDa protein (spx1) on Western blots, and the signal was efficiently blocked by the competing peptide. Spx1 was resistant to limited proteinase K digestion, but was unrelated to the expression of host prion protein or its pathologic amyloid form. Remarkably, spx1 concentrated in selected brain synapses, such as those on anterior motor horn neurons that integrate many complex neural inputs. SPHINX 1.8 appears to be involved in tissue-specific differentiation, including essential functions that preserve its propagation during mammalian evolution, possibly via maternal inheritance. The data here indicate that mammals can share and exchange a larger world of prokaryotic viruses than previously envisioned.

Evolutionary Analysis of the Mammalian Tuftelin Sequence Reveals Features of Functional Importance

Tuftelin (TUFT1) is an acidic, phosphorylated glycoprotein, initially discovered in developing enamel matrix. TUFT1 is expressed in many mineralized and non-mineralized tissues. We performed an evolutionary analysis of 82 mammalian TUFT1 sequences to identify residues and motifs that were conserved during 220million years (Ma) of evolution. We showed that 168 residues (out of the 390 residues composing the human TUFT1 sequence) are under purifying selection. Our analyses identified several, new, putatively functional domains and confirmed previously described functional domains, such as the TIP39 interaction domain, which correlates with nuclear localization of the TUFT1 protein, that was demonstrated in several tissues. We also identified several sites under positive selection, which could indicate evolutionary changes possibly related to the functional diversification of TUFT1 during evolution in some lineages. We discovered that TUFT1 and MYZAP (myocardial zonula adherens protein) share a common ancestor that was duplicated circa 500million years ago. Taken together, these findings expand our knowledge of TUFT1 evolution and provide new information that will be useful for further investigation of TUFT1 functions.

Lactoferrin: A Powerful Antimicrobial Protein Present in Milk

Lactoferrin (Lf) is an iron-chelating glycoprotein present in milk and mucosal secretions, a component of the mammalian innate immune system. Lf is microbiostatic and microbicidal. Lf can reduce the bacterial expression of virulence factors, such as those involved in biofilm production and protease secretion. The high identity among mammalian Lf sequences facilitates its use in human and veterinary medicine. Lf of bovine origin is the principal Lf used due to its commercial availability through purification from milk whey; recombinant Lfs (bovine, human, and porcine) have been used as well. Lf is a stable protein that retains its physicochemical characteristics under gastric pH conditions, and in most cases it is bioactive even after digestion; thus, the incorporation of Lf into diets facilitates its administration to animals. The aim of this review is to examine original research in which the effects of bovine and porcine Lf on pathogens of domestic animals have been demonstrated through in vitro and in vivo assays, with the purpose of ascertaining the benefits that Lf provides in the treatment of infectious diseases.

Mammalian Glutamyl Aminopeptidase Genes (ENPEP) and Proteins: Comparative Studies of a Major Contributor to Arterial Hypertension.

Glutamyl aminopeptidase (ENPEP) is a member of the M1 family of endopeptidases which are mammalian type II integral membrane zinc-containing endopeptidases. ENPEP is involved in the catabolic pathway of the renin-angiotensin system forming angiotensin III, which participates in blood pressure regulation and blood vessel formation. Comparative ENPEP amino acid sequences and structures and ENPEP gene locations were examined using data from several mammalian genome projects. Mammalian ENPEP sequences shared 71-98% identities. Five N-glycosylation sites were conserved for all mammalian ENPEP proteins examined although 9-18 sites were observed, in each case. Sequence alignments, key amino acid residues and predicted secondary and tertiary structures were also studied, including transmembrane and cytoplasmic sequences and active site residues. Highest levels of human ENPEP expression were observed in the terminal ileum of the small intestine and in the kidney cortex. Mammalian ENPEP genes contained 20 coding exons. The human ENPEP gene promoter and first coding exon contained a CpG island (CpG27) and at least 6 transcription factor binding sites, whereas the 3′-UTR region contained 7 miRNA target sites, which may contribute to the regulation of ENPEP gene expression in tissues of the body. Phylogenetic analyses examined the relationships of mammalian ENPEP genes and proteins, including primate, other eutherian, marsupial and monotreme sources, using chicken ENPEP as a primordial sequence for comparative purposes.

Prediction of human miRNA target genes using computationally reconstructed ancestral mammalian sequences.

MicroRNAs (miRNA) are short single-stranded RNA molecules derived from hairpin-forming precursors that play a crucial role as post-transcriptional regulators in eukaryotes and viruses. In the past years, many microRNA target genes (MTGs) have been identified experimentally. However, because of the high costs of experimental approaches, target genes databases remain incomplete. Although several target prediction programs have been developed in the recent years to identify MTGs in silico, their specificity and sensitivity remain low. Here, we propose a new approach called MirAncesTar, which uses ancestral genome reconstruction to boost the accuracy of existing MTGs prediction tools for human miRNAs. For each miRNA and each putative human target UTR, our algorithm makes uses of existing prediction tools to identify putative target sites in the human UTR, as well as in its mammalian orthologs and inferred ancestral sequences. It then evaluates evidence in support of selective pressure to maintain target site counts (rather than sequences), accounting for the possibility of target site turnover. It finally integrates this measure with several simpler ones using a logistic regression predictor. MirAncesTar improves the accuracy of existing MTG predictors by 26% to 157%. Source code and prediction results for human miRNAs, as well as supporting evolutionary data are available at http://cs.mcgill.ca/∼blanchem/mirancestar.