Interaction of 18-residue peptides derived from amphipathic helical segments of globular proteins with model membranes

We investigated the interaction of six 18-residue peptides derived from amphipathic helical segments of globular proteins with model membranes. The net charge of the peptides at neutral pH varies from -1 to +6. Circular dichroism spectra indicate that peptides with a high net positive charge tend to fold into a helical conformation in the presence of negatively charged lipid vesicles. In helical conformation, their average hydrophobic moment and hydrophobicity would render them surface-active. The composition of amino acids on the polar face of the helix in the peptides is considerably different. The peptides show variations in their ability to permeabilise zwitterionic and anionic lipid vesicles. Whereas increased net positive charge favours greater permeabilisation, the distribution of charged residues in the polar face also plays a role in determining membrane activity. The distribution of amino acids in the polar face of the helix in the peptides that were investigated do not fall into the canonical classes described. Amphipathic helices, which are part of proteins, with a pattern of amino acid distribution different from those observed in class L, A and others, could help in providing newer insights into peptide-membrane interactions

ELUCIDATION OF GENE REGULATORY NETWORK CONTROLLING EMBRYONIC SKELETAL DEVELOPMENT: FROM THE PERSPECTIVE OF PAX1 & PAX9

Ph.DDOCTOR OF PHILOSOPH

Regulatory Functions of Pax1 and Pax9 in Mammalian Cells

Pax1 and Pax9 are paired-box transcription factors, which play vital roles in axial skeletogenesis, thymus organogenesis, palatogenesis and odontogenesis among others. The importance of these closely related transcription factors can be perceived from the various human anomalies associated with their disruption. Vertebral column abnormalities such as kyphoscoliosis, seen in Jarcho-Levine and Klippel-Feil syndromes, secondary cleft palate, oligodontia/ hypodontia (missing teeth) and thymus developmental defects have all been associated with mutations in PAX1 and/or PAX9. In this chapter, we describe the molecular functions of Pax1 and Pax9 in various tissues during mouse development

Purification and characterization of a novel salivary antimicrobial peptide from the tick, \u3cem\u3eIxodes scapularis\u3c/em\u3e

A novel antimicrobial peptide was isolated from the saliva of the Lyme disease tick vector, Ixodes scapularis, henceforth designated as ISAMP (I. scapularis Antimicrobial Peptide). ISAMP was purified using a sequential method including ultra filtration, gel filtration and reverse-phase high performance liquid chromatography. The purified peak had a molecular weight of 5.3 kDa by MALDI/TOF-MS and its amino acid sequence, determined by Edman degradation was PDxGxPxxVKAGRxPxxSI. A BLASTP search revealed that the protein is a putative 5.3 kDa secreted protein (AAM93656) from I. scapularis. The predicted protein is composed of 69 amino acids with no conserved domain motifs. Purified ISAMP was found to have antimicrobial activities against bacteria. Gene expression studies were carried out to observe ISAMP expression in different tick tissues. RT-PCR results indicated that the gene was expressed in hemocytes, fat body and salivary gland but virtually no expression was observed in the midgut. ISAMP is only similar to other Ixodid tick proteins, thus it is a member of a unique family

Association of estrogen and progesterone with cancer of the uterine cervix in women infected with high-risk human papillomavirus

Background: High-risk human papillomavirus (HR-HPV) infection is essential for the development of dysplasia and cervical cancer. Steroid hormones are implicated as risk factors for cervical carcinogenesis. Thus the aim of the present study is to investigate the association of serum levels of estrogen and progesterone with cervical cancer in HR-HPV infected women. Methods and materials: The present study consisted of 103 subjects infected with HR-HPV from low-grade squamous intraepithelial lesion (LSIL) to cervical cancer. They included 37 premenopausal women (luteal phase) and 43 postmenopausal women as cancer cases (high-grade squamous intraepithelial lesion and cervical cancer). Twelve women with LSIL for premenopausal and 11 women with LSIL for postmenopausal were chosen as controls. The concentration of estradiol and progesterone were estimated using enzyme linked immuno sorbent assay kit. The prevalence of HPV infection was expressed as percentage of HPV positives. The data for estradiol and progesterone were expressed as mean ± SD. Results: The serum levels of estradiol were not significantly altered in premenopausal and postmenopausal cases (p>0.05). However, the serum levels of progesterone were significantly increased in premenopausal cases as compared to premenopausal controls (p0.05). The ratio of estradiol to progesterone was significantly decreased in premenopausal cases (p0.05). Conclusion: A significantly elevated levels of progesterone is associated with cervical cancer in premenopausal women infected with HR-HPV

Purification of a serine protease and evidence for a protein C activator from the saliva of the tick, \u3cem\u3eIxodes scapularis\u3c/em\u3e

The saliva of ticks is critical to their survival as parasites and hematophagous animals. In this study, we have purified an enzyme with trypsin-like activity from the saliva of the tick vector of Lyme Disease, Ixodes scapularis. This enzyme, named as IXOSP (I. scapularis salivary serine protease), is a 29.9 kDa molecule with N-terminus FPxMVxLRIKxR. A BLAST search identified IXOSP as a secreted serine protease (AAY66740) with a conserved catalytic triad His, Asp, and Ser. In vitro studies demonstrated that IXOSP cleaves chromogenic substrates with arginine in the P1 position, by a mechanism inhibited by PMSF or aprotinin. Gene expression studies revealed that IXOSP is expressed at different tick developmental stages, including eggs, and unfed or fed adult tick salivary glands, but not in nymphs or in the midgut. While the physiological substrate for IXOSP remains to be identified, we demonstrated that I. scapularis saliva activate protein C (PC) resulting in the production of activated PC, a potent anticoagulant that also regulates a myriad of inflammatory responses through protease activated receptors. In contrast, the salivary glands of Anopheles gambiae, Anopheles stephensi, Anopheles albimanus, Aedes aegypti, Lutzomyia longipalpis, and Phlebotomus ariasi did not activate protein C. These discoveries are discussed in the context of blood coagulation, inflammation and vector–host interactions

Monoamine Neurotransmitters as Substrates for Novel Tick Sulfotransferases, Homology Modeling, Molecular Docking, and Enzyme Kinetics

Blacklegged ticks (Ixodes scapularis) transmit the causative agent of Lyme disease in the Northeastern United States. Current research focuses on elucidating biochemical pathways that may be disrupted to prevent pathogen transmission, thereby preventing disease. Genome screening reported transcripts coding for two putative sulfotransferases in whole tick extracts of the nymphal and larval stages. Sulfotransferases are known to sulfonate phenolic and alcoholic receptor agonists such as 17β-estradiol, thereby inactivating the receptor ligands. We used bioinformatic approaches to predict substrates for Ixosc Sult 1 and Ixosc Sult 2 and tested the predictions with biochemical assays. Homology models of 3D protein structure were prepared, and visualization of the electrostatic surface of the ligand binding cavities showed regions of negative electrostatic charge. Molecular docking identified potential substrates including dopamine, R-octopamine and S-octopamine, which docked into Ixosc Sult 1 with favorable binding affinity and correct conformation for sulfonation. Dopamine, but not R- or S-octopamine, also docked into Ixosc Sult 2 in catalytic binding mode. The predictions were confirmed using cytosolic fractions of whole tick extracts. Dopamine was a good substrate (Km = 0.1−0.4 μM) for the native Ixodes scapularis sulfotransferases from larval and nymphal stages regardless of their fed/unfed status. Octopamine sulfonation was detected only after feeding when gene expression data suggests that Ixosc Sult 1 is present. Because dopamine is known to stimulate salivation in ticks through receptor stimulation, these results imply that the function(s) of Ixosc Sult 1 or 2 may include inactivation of the salivation signal via sulfonation of dopamine and/or octopamine

Molecular characterization of novel sulfotransferases from the tick, Ixodes scapularis

<p>Abstract</p> <p>Background</p> <p><it>Ixodes scapularis</it>, commonly known as the blacklegged or deer tick, is the main vector of Lyme disease in the United States. Recent progress in transcriptome research has uncovered hundreds of different proteins expressed in the salivary glands of hard ticks, the majority of which have no known function, and include many novel protein families. We recently identified transcripts coding for two putative cytosolic sulfotransferases in these ticks which recognized phenolic monoamines as their substrates. In this current study, we characterize the genetic expression of these two cytosolic sulfotransferases throughout the tick life cycle as well as the enzymatic properties of the corresponding recombinant proteins. Interestingly, the resultant recombinant proteins showed sulfotransferase activity against both neurotransmitters dopamine and octopamine.</p> <p>Results</p> <p>The two sulfotransferase genes were coded as <it>Ixosc </it>SULT 1 & 2 and corresponding proteins were referred as <it>Ixosc </it>Sult 1 and 2. Using gene-specific primers, the sulfotransferase transcripts were detected throughout the blacklegged tick life cycle, including eggs, larvae, nymphs, adult salivary glands and adult midgut. Notably, the mRNA and protein levels were altered upon feeding during both the larval and nymphal life stages. Quantitative PCR results confirm that <it>Ixosc </it>SULT1 was statistically increased upon blood feeding while <it>Ixosc </it>SULT 2 was decreased. This altered expression led us to further characterize the function of these proteins in the Ixodid tick. The sulfotransferase genes were cloned and expressed in a bacterial expression system, and purified recombinant proteins <it>Ixosc </it>Sult 1(R) and 2(R) showed sulfotransferase activity against neurotransmitters dopamine and octopamine as well as the common sulfotransferase substrate <it>p-</it>nitrophenol. Thus, dopamine- or octopamine-sulfonation may be involved in altering the biological signal for salivary secretion in <it>I. scapularis.</it></p> <p>Conclusions</p> <p>Collectively, these results suggest that a function of <it>Ixosc </it>Sult 1 and Sult 2 in <it>Ixodid </it>tick salivary glands may include inactivation of the salivation signal via sulfonation of dopamine or octopamine.</p

Genome wide binding (ChIP-Seq) of murine Bapx1 and Sox9 proteins in vivo and in vitro

AbstractThis work pertains to GEO submission GSE36672, in vivo and in vitro genome wide binding (ChIP-Seq) of Bapx1/Nkx3.2 and Sox9 proteins. We have previously shown that data from a genome wide binding assay combined with transcriptional profiling is an insightful means to divulge the mechanisms directing cell type specification and the generation of tissues and subsequent organs [1]. Our earlier work identified the role of the DNA-binding homeodomain containing protein Bapx1/Nkx3.2 in midgestation murine embryos. Microarray analysis of EGFP-tagged cells (both wildtype and null) was integrated using ChIP-Seq analysis of Bapx1/Nkx3.2 and Sox9 DNA-binding proteins in living tissue

Comparison of IRES and F2A-Based Locus-Specific Multicistronic Expression in Stable Mouse Lines

Efficient and stoichiometric expression of genes concatenated by bi- or multi-cistronic vectors has become an invaluable tool not only in basic biology to track and visualize proteins in vivo, but also for vaccine development and in the clinics for gene therapy. To adequately compare, in vivo, the effectiveness of two of the currently popular co-expression strategies - the internal ribosome entry site (IRES) derived from the picornavirus and the 2A peptide from the foot-and-mouth disease virus (FDMV) (F2A), we analyzed two locus-specific knock-in mouse lines co-expressing SRY-box containing gene 9 (Sox9) and enhanced green fluorescent protein (EGFP) linked by the IRES (Sox9IRES-EGFP) or the F2A (Sox9F2A-EGFP) sequence. Both the constructs expressed Sox9 and EGFP proteins in the appropriate Sox9 expression domains, with the IRES construct expressing reduced levels of EGFP compared to that of the F2A. The latter, on the other hand, produced about 42.2% Sox9-EGFP fusion protein, reflecting an inefficient ribosome ‘skipping’ mechanism. To investigate if the discrepancy in the ‘skipping’ process was locus-dependent, we further analyzed the FLAG3-Bapx1F2A-EGFP mouse line and found similar levels of fusion protein being produced. To assess if EGFP was hindering the ‘skipping’ mechanism, we examined another mouse line co-expressing Bagpipe homeobox gene 1 homolog (Bapx1), Cre recombinase and EGFP (Bapx1F2A-Cre-F2A-EGFP). While the ‘skipping’ was highly efficient between Bapx1 and Cre, the ‘skipping’ between Cre and EGFP was highly inefficient. We have thus demonstrated in our comparison study that the efficient and close to equivalent expression of genes linked by F2A is achievable in stable mouse lines, but the EGFP reporter may cause undesirable inhibition of the ‘skipping’ at the F2A sequence. Hence, the use of other reporter genes should be explored when utilizing F2A peptides