Clustal Alignment Research Articles

ViralFP: A Web Application of Viral Fusion Proteins.

Viral fusion proteins are attached to the membrane of enveloped viruses (a group that includes Coronaviruses, Dengue, HIV and Influenza) and catalyze fusion between the viral and host membranes, enabling the virus to insert its genetic material into the host cell. Given the importance of these biomolecules, this work presents a centralized database containing the most relevant information on viral fusion proteins, available through a free-to-use web server accessible through the URL https://viralfp.bio.di.uminho.pt/. This web application contains several bioinformatic tools, such as Clustal sequence alignment and Weblogo, including as well a machine learning-based tool capable of predicting the location of fusion peptides (the component of fusion proteins that inserts into the host's cell membrane) within the fusion protein sequence. Given the crucial role of these proteins in viral infection, their importance as natural targets of our immune system and their potential as therapeutic targets, this web application aims to foster our ability to fight pathogenic viruses.

Molecular characterization of a novel β-defensin isoform from the red-toothed trigger fish, Odonus niger (Ruppel, 1836)

BackgroundThe concern regarding a post-antibiotic era with increasing drug resistance by pathogens imposes the need to discover alternatives for existing antibiotics. Antimicrobial peptides (AMPs) with their versatile therapeutic properties are a group of promising molecules with curative potentials. These evolutionarily conserved molecules play important roles in the innate immune system of several organisms. The β-defensins are a group of cysteine rich cationic antimicrobial peptides that play an important role in the innate immune system by their antimicrobial activity against the invading pathogens. The present study deals with a novel β-defensin isoform from the red-toothed trigger fish, Odonus niger. Total RNA was isolated from the gills, cDNA was synthesized and the β-defensin isoform obtained by polymerase chain reaction was cloned and subjected to structural and functional characterization in silico. ResultsA β-defensin isoform could be detected from the gill mRNA of red-toothed trigger fish, Odonus niger. The cDNA encoded a 63 amino acid peptide, β-defensin, with a 20 amino acid signal sequence followed by 43 amino acid cationic mature peptide (On-Def) having a molecular weight of 5.214 kDa and theoretical pI of 8.89. On-Def possessed six highly conserved cysteine residues forming disulfide bonds between C1–C5, C2–C4, and C3–C6, typical of β-defensins. An anionic pro-region was observed prior to the β-defensin domain within the mature peptide. Clustal alignment and phylogenetic analyses revealed On-Def as a group 2 β-defensin. Furthermore, it shared some structural similarities and functional motifs with β-defensins from other organisms. On-Def was predicted to be non-hemolytic with anti-bacterial, anti-viral, anti-fungal, anti-cancer, and immunomodulatory potential. ConclusionOn-Def is the first report of a β-defensin from the red-toothed trigger fish, Odonus niger. The antimicrobial profile showed the potential for further studies as a suitable candidate for antimicrobial peptide therapeutics.

678. Polymorphic Locus Sequence Typing for Pseudomonas aeruginosa Outbreak Detection and Investigation

Background Pseudomonas aeruginosa is a common agent of healthcare-associated infection (HAI), which can be challenging to treat due to intrinsic or acquired antibiotic resistance. In contrast to most HAI, the source of P. aeruginosa infection is typically environmental; e.g., from the faucets, drains, and showers in patient rooms. To unambiguously identify the source, strain typing of patient and environmental isolates is required. Typing methods include sequence-based MLST and WGS which resolve strains based on relatively rare SNPs, and length-based MLVA which resolves strains based on relatively frequent insertions/deletions within tandem repeats. These and other methods previously applied to P. aeruginosa have one or more limitations relating to cost, technical complexity, reproducibility, and turnaround time that preclude their routine use. Polymorphic locus sequence typing (PLST) is a hybrid approach that employs sequence analysis of the one or two most phylogenetically informative tandem repeat-containing loci within the genome of a microbial species. Strain resolution equals or exceeds MLST and MLVA and approaches that of WGS. Here, the identification and evaluation of candidate PLST loci for P. aeruginosa epidemiology are described.MethodsTandem repeats were bioinformatically identified in representative genome sequences, and screened by BLASTN queries of GenBank genome databases followed by clustal alignments and phylogenetic analysis. Candidate loci were amplified from heat-treated colony lysates and Sanger sequenced.ResultsPLST locus PaMT1 (Mbp 2.568-2.569 in the PA01 genome) resolved 99 alleles from 144 epidemiologically unrelated isolates with GenBank genome sequences, yielding diversity index = 0.991. Nineteen sets of isolates shared healthcare facility and PaMT1 allele, consistent with nosocomial transmission. Examples, confirmed by WGS-SNP analysis, include isolates from 8 VAP patients, 2 sets of cancer patient isolates, and a pair of isolates from patient and hospital sewage. In the laboratory, locus PaMT1 was readily amplified and sequenced from colony lysates, yielding typing data with 1-2 day turnaround.ConclusionPaMT1 PLST provides an affordable, user-friendly, and timely tool for P. aeruginosa outbreak detection and investigaton.Disclosures Tom Edlind, PhD, MicrobiType LLC (Employee, Scientific Research Study Investigator)

Investigation of isoform‐specific interactions between citrate synthase and malate dehydrogenase

Malate dehydrogenase (MDH) and citrate synthase (CS) catalyze subsequent metabolic reactions in the Krebs cycle. MDH and CS interact to form enzyme complexes called metabolons where substrates are directly shuttled from one active site to the other, improving efficiency of the overall metabolic cycle. Prior studies have shown that the mitochondrial isoform (MDH2) has stronger interactions with CS than the cytosolic isoform (MDH1), suggesting that isoformspecific sites of interaction are involved in the formation of the enzyme complex. This study attempts to determine key residues of MDH2 that are responsible for isoform‐specific affinities to CS, allowing for the formation of metabolon complexes. Model metabolon docking complexes between MDH2 and CS were generated computationally in Cluspro, using predicted and published interactions between the two enzymes. Potential interacting residues were identified and then mapped using clustal alignments to identify possible domains of isoformspecific interactions. Previous studies by our lab have shown that CS displays an isoformspecific affinity to MDH. Specifically, CS binds with high affinity to MDH2, moderate affinity to MDH1, and displays undetectable interaction with watermelon glyoxosomal MDH (wgMDH). We identified several regions of residues unique to MDH2 containing shown & predicted interactions with CS. Using docking analysis, we identified five regions of potential isoformspecific interactions. This combined with residue alignment of MDH2, MDH1 and wgMDH isoforms helped to further define potential isoform specific sites of interaction. Mutations exchanging mitochondrial residues for unique cytosolic counterparts or deletion mutations defined by our bioinformatic analysis were designed, generated and expressed. Enzyme kinetic assays were performed to investigate enzymatic activity of mutant constructs for comparison to native MDH1 and MDH2. Secondary structure and thermal stability were investigated using circular dichroism. Interactions between mutant MDH2 constructs and CS were quantified in fluorescence‐based thermal shift assays.Support or Funding InformationNSF EHR‐IUSE Project support NSF‐1726932

Penicillium purpurogenum produces a novel endo-1,5-arabinanase, active on debranched arabinan, short arabinooligosaccharides and on the artificial substrate p-nitrophenyl arabinofuranoside

Penicillium purpurogenum secretes numerous lignocellulose-degrading enzymes, including four arabinofuranosidases and an exo-arabinanase. In this work, the biochemical properties of an endo-arabinanase (ABN1) are presented. A gene, coding for a potential ABN was mined from the genome. It includes three introns. The cDNA is 975 bp long and codes for a mature protein of 324 residues. The cDNA was expressed in Pichia pastoris. The enzyme is active on debranched arabinan and arabinooligosaccharides. In contrast to other characterized ABNs, inactive on p-nitrophenyl-α-L-arabinofuranoside (pNPAra), ABN1 is active on this substrate. The enzyme has an optimal pH of 4.5 and an optimal temperature of 30–35 °C. Calcium does not activate ABN1. ABN1 belongs to GH family 43 sub-family 6, and a Clustal alignment with sequences of characterized fungal ABNs shows highest identity (54.6%) with an ABN from Aspergillus aculeatus. A three-dimensional model of ABN1 was constructed and the docking with pNPAra was compared with similar models of an enzyme very active on this substrate and another lacking activity, both from GH family 43. Differences in the number of hydrogen bonds between enzyme and substrate, and distance between the substrate and the catalytic residues may explain the differences in activity shown by these enzymes.

English

Colletotrichum lindemuthianum is the etiological agent of anthracnose, one of the main diseases of bean (Phaseolus vulgaris). In this study, the complete cDNAs of two endo-β-1,4-xylanase genes (xyl1) from non-pathogenic (0) and pathogenic (1472) races of C. lindemuthianum were isolated and characterized. To get an insight into the role of endo-β-1,4-xylanases in their different lifestyles, xyl1 gene expression and enzyme activity in mycelia of both races grown in the presence of xylan or P. vulgaris cell walls were investigated. The xyl1 sequence analysis and Clustal alignment revealed the characteristic elements of genes coding for endo-β-1,4-xylanases of the GH11 family. The growth of the two races with glucose as the sole carbon source showed both basal transcription levels of xyl1 and endoxylanase activity. When glucose was substituted with xylan or plant cell walls, xyl1 transcription, and enzyme activity significantly increased in race 1472 as compared to race 0. The pathogenic race degraded xylan faster and grew better than the non-pathogenic counterpart. Seemingly, the regulation of xylanolytic gene expression, enzyme production and the nature of the assimilatory carbon substrates processed by these organisms play a determinant role in their lifestyle. Phylogenetic analyses of XYL1 and endo-β-1,4-xylanases from other fungi revealed a diversification process and separation of proteins from the same fungal species into different lineages. Key words: Colletotrichum lindemuthianum, Phaseolus vulgaris, endo-β-1,4-xylanase, gene expression, phylogeny.

Abstract B35: Targeting the N-myc oncoprotein using nanobody technology

Abstract Our aim is to raise nanobodies, antigen-binding fragments derived from heavy chain-only antibodies of the Camelidae family, against the N-myc oncoprotein and demonstrate that these can directly and potently cripple the oncogenic activity of N-myc in vitro and in vivo. If successful, this will support the rationale for their use as N-myc-specific inhibitors for the treatment of neuroblastoma, which accounts for 15% of all pediatric cancer-related deaths. Nanobodies, a novel class of therapeutic proteins, have unique and advantageous properties over conventional antibodies. They are small, only ~15 kDa, and have excellent solubility making them ideal for use as an intracellular antibody or ‘intrabody’. Nanobodies have specificities and affinities comparable to that of conventional antibodies and are amenable to high-throughput engineering to modify aspects of their function. MYCN, the gene encoding N-myc, is amplified in ~20% of neuroblastomas and significantly contributes to tumorigenesis.MYCN amplification strongly correlates with poor prognosis. Its inhibition in vitro abrogates oncogenic activity and inhibition in mouse models has resulted in tumor shrinkage and increased survival, making N-myc a rational therapeutic target. However, translating these results into humans has had limited success due to problems such as physically targeting an intracellular transcription factor. We propose that an N-myc-specific nanobody will provide a new, specific way to target N-myc. Established protocols will be used to obtain N-myc-specific nanobodies. To design the immunogen, we have used secondary structure prediction and Clustal alignment software and selected 3 suitable N-myc fragments. These have been successfully expressed as glutathione S-transferase (GST)-fusion proteins, and we are currently optimizing large-scale purification and testing folding. A llama will be immunized with these immunogens, and high-affinity N-myc-specific nanobodies will be selected by a series of methods including phage display technology and their binding and physical properties characterized. Two approaches will be investigated to express nanobodies in N-myc-expressing cancer cells: viral transduction and a novel protein transduction technology known as small molecule carriers (SMoCs). SMoCs are biphenyl mimics of α-helical protein transduction domains which can be coupled to any cargo via a free cysteine residue. They have been shown to enhance cellular uptake of proteins which retain functionality inside the cell. We have demonstrated that a nanobody in development for Parkinson's Disease (NbPD) can be coupled to a SMoC, and demonstrated that this dramatically increases its cellular uptake, providing validation of this proof-of-principle. We will utilize this technology for transporting N-myc-specific nanobodies. We are currently preparing to test SMoCs and SMoC-NbPD in mice. In conclusion, this work provides a basis for the development of N-myc-specific nanobodies for the treatment of MYCN-amplified neuroblastomas and other N-myc-driven cancers. Citation Format: Lisa Kent, Christopher Dobson, Erwin de-Genst, Heike Laman. Targeting the N-myc oncoprotein using nanobody technology. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr B35.

Development of a pan-Babesia FRET-qPCR and a survey of livestock from five Caribbean islands.

BackgroundBabesia spp. are tick-borne protozoan hemoparasites and the second most common blood-borne parasites of mammals, in particular domestic animals. We used the Clustal Multiple Alignment program and 18S rRNA gene sequences of 22 Babesia species from GenBank to develop a PCR that could detect a wide variety of Babesia spp. in a single reaction. The pan-Babesia FRET-qPCR we developed reliably detected B. gibsoni, B. canis, B. vogeli, B. microti, B. bovis, and B. divergens under controlled conditions but did not react with closely related species, mainly Hepatozoon americanum, Theileria equi, and Toxoplasma gondii.ResultsWhen we tested the pan-Babesia FRET-qPCR on DNA of whole blood from 752 cattle, sheep, goats, donkeys and horses from five Caribbean islands, we detected Babesia spp. expected to be present in the animals, mainly B. bovis and B. bigemina in cattle and B. caballi in horses and donkeys. Further, we found that animals were not uncommonly infected with species of Babesia usually associated with other hosts, mainly B. vogeli and B. gibsoni in cattle, sheep and goats, B. rossi in goats, and B. caballi in goats and sheep. Finally, the pan-Babesia FRET-qPCR enabled us to identify unknown species of Babesia in cattle, goats, sheep and donkeys.ConclusionsOverall, 70 % (525/752) of the animals we tested were positive confirming earlier limited studies that infections with Babesia spp. are common in livestock in the Caribbean.

YZGD, a Two‐Domain Enzyme, as a Teaching Tool in a Biochemistry Laboratory

YZGD is a two domain enzyme that is both a member of the HAD superfamily with pyridoxal phosphatase activity and a member of the Nudix Hydrolase superfamily with comparable activity on CDP alcohols, ADP sugars and ADP coenzymes, TDP sugars, and some activity on UDP and GDP sugars. Due to its two‐domain nature, I have found it to be a useful enzyme to use in the teaching laboratory. We have created a mutant (YZGDD12N) that has no pyridoxal phosphatase activity but full Nudix activity and a mutant (YZGDE324Q) that has no Nudix activity but full pyridoxal phosphatase activity, demonstrating the independent nature of enzyme domains. We also have cloned both a his‐tagged YZGD and a non‐his‐tagged YZGD, thus demonstrating the power of affinity chromatography in enzyme purification. The students express the various YZGDs (mutants, wildtype, and his‐tagged), purify these enzymes, and characterize them (Bradford assays, gel electrophoresis, and enzyme assays). We then work as a group to compare and contrast the results that each student gets; it is this comparison that especially shows the students the power of site‐directed mutagenesis and affinity chromatography purification. In addition, the students do a protein structure ‐ function project where they use the structural database SCOP, the visualization program CN3D, BLAST searches and CLUSTAL alignments, and the scientific literature on any HAD or Nudix superfamily member that has been structurally determined.

The exoribonuclease Dis3L2 defines a novel eukaryotic RNA degradation pathway.

The final step of cytoplasmic mRNA degradation proceeds in either a 5'-3' direction catalysed by Xrn1 or in a 3'-5' direction catalysed by the exosome. Dis3/Rrp44, an RNase II family protein, is the catalytic subunit of the exosome. In humans, there are three paralogues of this enzyme: DIS3, DIS3L, and DIS3L2. In this work, we identified a novel Schizosaccharomyces pombe exonuclease belonging to the conserved family of human DIS3L2 and plant SOV. Dis3L2 does not interact with the exosome components and localizes in the cytoplasm and in cytoplasmic foci, which are docked to P-bodies. Deletion of dis3l2(+) is synthetically lethal with xrn1Δ, while deletion of dis3l2(+) in an lsm1Δ background results in the accumulation of transcripts and slower mRNA degradation rates. Accumulated transcripts show enhanced uridylation and in vitro Dis3L2 displays a preference for uridylated substrates. Altogether, our results suggest that in S. pombe, and possibly in most other eukaryotes, Dis3L2 is an important factor in mRNA degradation. Therefore, this novel 3'-5' RNA decay pathway represents an alternative to degradation by Xrn1 and the exosome.

Molecular Characterization of Nucleopolyhedrovirus of Three Lepidopteran Pests Using Late Expression Factor-8 Gene

A PCR based method for detection of viral DNA in nucleopolyhedrovirus of three lepidopterans, Spodoptera litura, Amsacta albistriga and Helicoverpa armigera, was developed by employing the late expression factor-8 (lef-8) gene of three NPV using specific primers. The amplicons of 689, 699 and 665bp were amplified, respectively, and the nucleotide sequences were submitted to GenBank and the accession numbers were obtained. The sequences of lef-8 gene of S. litura NPV and H. armigera NPV matched with those of their respective references in the GenBank database, thereby confirming their identity, however, the sequence of A. albistriga NPV was the first sequence submitted to the GenBank database. The sequence similarity analysis between the three lef-8 gene of NPV sequenced in the present study revealed that there was no significant similarity between them, however A. albistriga NPV and S. litura NPV were found to be closely related. CLUSTAL alignment of the sequences generated revealed general relatedness among NPVs lef-8 gene. The study confirmed that lef-8 gene can be used for quick and correct discriminatory identification of insect viruses.

Molecular phylogeny of the family Bacillariaceae based on 18S rDNA sequences: focus on freshwater Nitzschia of the section Lanceolatae

Sequence analysis of the 18S rDNA gene from 93 taxa belonging to the pennate diatoms plus one centric, Cyclotella meneghiniana Kützing, were made using two different alignments (Clustal and secondary structure) and two different types of algorithms (neighbour-joining and maximum likelihood). The monophyly of the bacillariacean taxa depends on the type of alignment used for the 18S gene. A secondary structure alignment does not support its monophyly, whereas a Clustal alignment does, but only in the maximum likelihood analysis. The Eunotiales were basal to all other raphid diatoms if a maximum likelihood analysis was used, regardless of the alignment, whereas a neighbour-joining analysis, regardless of the alignment, pulled the Eunotiales inside the raphid diatom sister to one of the bacillariophycean clades in the secondary structure alignment and sister to a monophyletic bacillariophycean clade in the Clustal alignment. The classification of the Bacillariaceae by Krammer & Lange-Bertalot and Round, Crawford & Mann was not supported by the 18S phylogeny. Taxa of the section Lanceolatae Grunow were present in different clades, but sister relationships between well-supported clades were not supported. Multiseriate striae, which are often considered an important feature, were not supported as being clade-defining features. The two groups, A and B of Krammer & Lange-Bertalot in the section Lanceolatae were not supported by the phylogenetic analyses.

Abstract LB-177: HPV16 CpG and de novo-cytosine methylation is differentially associated with low-grade versus high-grade anal intraepithelial neoplasia in HIV-infected men

Abstract Objective: To determine if cytosine methylation in CpG and de novo sites is differentially associated with high- or low-grade anal intraepithelial neoplasias (HG-, LG-AIN), HPV16 DNA from182 clinical specimens. Background: Intra-anal cancer (IAC) is an emerging health crisis for gay, bisexual, transgender and other men who have sex with men (MSM) and rates have risen sharply among HIV-infected MSM despite introduction of HAART. High-risk HPVs are a causal risk factor for IAC and are especially common where MSM show HIV-coinfection. Further, cytosine methylation has been posited as an epigenetic transcription regulator that is poorly described in intra-anal dysplasias and cancers. Methods: HPV16 DNA was extracted from anal swab specimens and tested using bisulfite modification, PCR, cloning and sequencing of ∼10 clones/sample. Cervical cancer cell lines, CaSki and SiHa, and paraffin-embedded anal cancer specimens were similarly characterized as controls. Genomic sequences were evaluated using CLUSTAL and BiQ Sequence Alignment analysis software. Descriptive, tabular and multivariate analyses were performed using SAS (Version 9.2). Graphical representations were compiled using SIGMAPLOT (Version 9.0). Findings: Overall, methylation was relatively low in clinical AIN specimens. For 25,085 cytosines, the mean prevalence of methylation was 2.7% (SEM=0.4%) across 3′ L1 and LCR; the prevalence me-CpG, -CpA, -CpT, and -CpC was 3.2% (0.2%), 2.6% (0.1%), 2.5% (0.1%), and 2.7% (0.1%), respectively. However, some variation in HPV16 genomes was observed across specific functional gene sequences for HG- and LG-AIN specimens. Specifically, LG-AINs showed 1.30–2.62 times greater mean prevalence of methylation when compared to HG-AINs, across 24 of the 31 cytosines between nt7428–7564 (p-values<0.05) in the 5′ LCR and enhancer. In total, across the 148 cytosine positions, 37 sites showed statistically significantly greater prevalence of meC in LG-AINs and none showed higher methylation in HG-AINs, yielding a false discovery rate of 0.2 (7.4/37). The nt7428–7564 contains binding sites for E2–1, TEF-1, NF-1, YY1, OCT-1, GRE and potential deamination targets for ApoBec3G. Analyses are ongoing, nonetheless, multivariate analyses show risk for HG-AIN decreases by 27% for each cumulative increase of 100% cytosine-methylation across nt7220–7558, even after we controlled for age, CD4+ T-lymphocyte count, and repeated measurements. Conclusions: Data suggest the HPV16 5′LCR/enhancer region contains a large concentration of cytosine-containing transcription regulatory elements where binding interference by methylation might alter expression of the p97 promoter. Methylation in the HPV16 5′LCR/enhancer may decrease risk for HG-AIN. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-177. doi:10.1158/1538-7445.AM2011-LB-177

Amplified Fragment Length Polymorphism Reveals Specific Epigenetic Distinctions between Mycobacterium avium Subspecies paratuberculosis Isolates of Various Isolation Types

Amplified fragment length polymorphism (AFLP) was employed as a genetic analysis tool for the study of the genetic relatedness of Mycobacterium avium subsp. paratuberculosis isolates harvested from bovine fecal samples and from bovine or human tissues. This analysis revealed genetic differences between these two isolate types that were confirmed through cluster analysis. Dendrogram analysis separated these two isolate types based on the isolation scheme (tissue-associated versus fecal M. avium subsp. paratuberculosis isolates). Further sequence analysis of unique genetic regions from each isolation type revealed no genetic sequence differences. However, Clustal DNA alignments identified AFLP restriction enzyme sites that were undigested in the tissue-associated isolates. AFLP analysis also disclosed that the same AFLP restriction sites were digested in all of the fecal isolates. Sequence analysis further revealed a consensus sequence upstream of the undigested restriction sites for possible methyltransferase recognition in the tissue-associated M. avium subsp. paratuberculosis isolates.

Distinctive Properties of the Hyaluronan-binding Domain in the Lymphatic Endothelial Receptor Lyve-1 and Their Implications for Receptor Function

The lymphatic endothelial hyaluronan (HA) receptor Lyve-1 is a member of the Link protein superfamily most similar to the leukocyte HA receptor CD44. However, the structure of Lyve-1 and the nature of its interaction with ligand are obscure. Here we present new evidence that Lyve-1 is functionally distinct from CD44. Using truncation mutagenesis we confirm that Lyve-1 in common with CD44 contains an extended HA-binding unit, comprising elements flanking the N and C termini of the consensus lectin-like Link module, bridged by a third conserved disulfide linkage that is critical for HA binding. In addition, we identify six essential residues Tyr-87, Ile-97, Arg-99, Asn-103, Lys-105, and Lys-108 that define a compact HA-binding surface on Lyve-1, encompassing the epitope for an adhesion-blocking monoclonal antibody 3A, in an analogous position to the HA-binding surface in CD44. The overtly electrostatic character of HA binding in Lyve-1 and its sensitivity to ionic strength (IC(50) of 150 mm NaCl) contrast markedly with CD44 (IC(50) > 2 m NaCl) in which HA binding is mediated by hydrogen bonding and hydrophobic interactions. In addition, unlike the extended Link module in CD44, which binds HA efficiently when expressed as a soluble monomer (K(d) = 65.7 mum), that of Lyve-1 requires artificial dimerization, although the full ectodomain is active as a monomer (K(d) = 35.6 mum). Finally, full-length Lyve-1 did not form stable dimers in binding-competent 293T transfectants when assessed using bioluminescent resonance energy transfer. These results reveal that elements additional to the extended Link module are required to stabilize HA binding in Lyve-1 and indicate important structural and functional differences with CD44.

Cysteine Peptidase Expression in Trichomonas vaginalis Isolates Displaying High- and Low-Virulence Phenotypes

In the present study, we identified and characterized the cysteine peptidase (CP) profiles of Trichomonas vaginalis isolates exhibiting high- and low-virulence phenotypes using a combination of two-dimensional SDS-PAGE (2DE), tandem mass spectrometry (MS/MS), and data mining. Seven of the eight CPs identified belong to Clan CA, family C1, cathepsin L-like CP, and one belongs to Clan CD, family C13, asparaginyl endopeptidase-like CP. Quantitative and qualitative differences in CP expression were detected between the isolates. BLAST analysis followed by CLUSTAL alignment of amino acid sequences of differentially expressed CPs showed identity or high homology to previously described CP cDNA clones CP1, CP3, CP4, and to a secreted CP fraction of 30 kDa involved in apoptosis of vaginal epithelial cells. One- and two-dimensional-substrate gel analyses revealed the differential CP profiles between the isolates, indicating that the combination of zymography with 2DE and MS/MS might be a powerful experimental approach to map and identify active peptidases in T. vaginalis. Toxicity exerted upon HeLa cells by high- and low-virulence isolates was 98.3% and 31%, respectively. Pretreatment of parasites with specific Clan CA papain-like CP inhibitor l-3-carboxy-2,3-trans-epoxypropionyl-leucylamido(4-guanidino)butane (E-64) drastically reduced the cytotoxic effect to 21.7% and 0.8%, respectively, suggesting that T. vaginalis papain-like CPs are the main factors involved in the cellular damage.

SLC24A5 Encodes a trans-Golgi Network Protein with Potassium-dependent Sodium-Calcium Exchange Activity That Regulates Human Epidermal Melanogenesis

A non-synonymous single nucleotide polymorphism in the human SLC24A5 gene is associated with natural human skin color variation. Multiple sequence alignments predict that this gene encodes a member of the potassium-dependent sodium-calcium exchanger family denoted NCKX5. In cultured human epidermal melanocytes we show using affinity-purified antisera that native human NCKX5 runs as a triplet of approximately 43 kDa on SDS-PAGE and is partially localized to the trans-Golgi network. Removal of the NCKX5 protein through small interfering RNA-mediated knockdown disrupts melanogenesis in human and murine melanocytes, causing a significant reduction in melanin pigment production. Using a heterologous expression system, we confirm for the first time that NCKX5 possesses the predicted exchanger activity. Site-directed mutagenesis of NCKX5 and NCKX2 in this system reveals that the non-synonymous single nucleotide polymorphism in SLC24A5 alters a residue that is important for NCKX5 and NCKX2 activity. We suggest that NCKX5 directly regulates human epidermal melanogenesis and natural skin color through its intracellular potassium-dependent exchanger activity.

Alignment and Topological Accuracy of the Direct Optimization approach via POY and Traditional Phylogenetics via ClustalW + PAUP*

Direct optimization frameworks for simultaneously estimating alignments and phylogenies have recently been developed. One such method, implemented in the program POY, is becoming more common for analyses of variable length sequences (e.g., analyses using ribosomal genes) and for combined evidence analyses (morphology + multiple genes). Simulation of sequences containing insertion and deletion events was performed in order to directly compare a widely used method of multiple sequence alignment (ClustalW) and subsequent parsimony analysis in PAUP* with direct optimization via POY. Data sets were simulated for pectinate, balanced, and random tree shapes under different conditions (clocklike, non-clocklike, and ultrametric). Alignment accuracy scores for the implied alignments from POY and the multiple sequence alignments from ClustalW were calculated and compared. In almost all cases (99.95%), ClustalW produced more accurate alignments than POY-implied alignments, judged by the proportion of correctly identified homologous sites. Topological accuracy (distance to the true tree) for POY topologies and topologies generated under parsimony in PAUP* from the ClustalW alignments were also compared. In 44.94% of the cases, Clustal alignment tree reconstructions via PAUP* were more accurate than POY, whereas in 16.71% of the cases POY reconstructions were more topologically accurate (38.38% of the time they were equally accurate). Comparisons between POY hypothesized alignments and the true alignments indicated that, on average, as alignment error increased, topological accuracy decreased.

Sensitivity analysis, molecular systematics and natural history evolution of Scathophagidae (Diptera: Cyclorrhapha: Calyptratae).

The 60 000 described species of Cyclorrhapha are characterized by an unusual diversity in larval life-history traits, which range from saprophagy over phytophagy to parasitism and predation. However, the direction of evolutionary change between the different modes remains unclear. Here, we use the Scathophagidae (Diptera) for reconstructing the direction of change in this relatively small family (≈ 250 spp.) whose larval habits mirror the diversity in natural history found in Cyclorrhapha. We subjected a molecular data set for 63 species (22 genera) and DNA sequences from seven genes (12S, 16S, Cytb, COI, 28S, Ef1-alfa, Pol II) to an extensive sensitivity analysis and compare the performance of three different alignment strategies (manual, Clustal, POY). We find that the default Clustal alignment performs worst as judged by character incongruence, topological congruence and branch support. For this alignment, scoring indels as a fifth character state worsens character incongruence and topological congruence. However, manual alignment and direct optimization perform similarly well and yield near-identical trees, although branch support is lower for the direct-optimization trees. All three alignment techniques favor the upweighting of transversion. We furthermore confirm the independence of the concepts "node support" and "node stability" by documenting several cases of poorly supported nodes being very stable and cases of well supported nodes being unstable. We confirm the monophyly of the Scathophagidae, its two constituent subfamilies, and most genera. We demonstrate that phytophagy in the form of leaf mining is the ancestral larval feeding habit for Scathophagidae. From phytophagy, two shifts to saprophagy and one shift to predation has occurred while a second origin of predation is from a saprophagous ancestor.

Protein Phosphatase 6 Subunit with Conserved Sit4-associated Protein Domain Targets IκBϵ

Protein Phosphatase 6 Subunit with Conserved Sit4-associated Protein Domain Targets IκBϵ